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2018
A. Batista, HG Breunig, A. König, A. Schindele, T. Hager, B. Seitz, AM Morgado, K. König. Assessment of human corneas prior to transplantation using high-resolution two-photon imaging. Invest Ophthalmol Vis Sci. 59 (2018) 176-184. Doi:10.1167/iovs.17-22002. Abstract The purpose of this study was to evaluate the feasibility of using two-photon imaging (TPI) to assess the condition of human corneas for transplantation. Human corneas were imaged after different storage times: short-term (STS), mediumterm (MTS), and long-term (LTS) storage. A high-resolution, custom-built 5-dimensional multiphoton microscope with 12-fs pulsed laser excitation was used for image acquisition. S. Springer, M. Zieger, UC hippler, K. König, J. Lademann, M. Kaatz, MJ Köhler. Non-invasive evaluation of human mucosal structures by multiphoton laser scanning tomography in vitro. Skin Res. Tech 1-5 (2018). Doi: 10.1111/srt.12451. Abstract Background: Mucous membranes may be affected by various diseases and the diagnostic accessibility is limited. Multiphoton laser tomography (MPT) is a useful tool for in vivo evaluation of superficial skin structures and might also be useful for the imaging of mucosa. Objectives: In order to investigate the suitability of MPT for the evaluation of mucous membranes, tissue samples of different donors and anatomical localizations have been imaged. Methods: Human mucosa samples from the urinary bladder, palatine tonsil and ocular conjunctiva were investigated by MPT and subsequently compared with conventional histology. A. Batista, HG Breunig, A. König, A. Schindele, T. Hager, B. Seitz, K. König. High-resolution, label-free two-photon imaging of diseased human corneas. J Biomed Opt 23 (3), 036002 (2018). Doi: 10.1117/1JBO.23.3.036002. Abstract The diagnosis of corneal diseases may be improved by monitoring the metabolism of cells and the structural organization of the stroma using two-photon imaging (TPI). We used TPI to assess the differences between nonpathological (NP) human corneas and corneas diagnosed with either keratoconus, Acanthamoeba keratitis, or stromal corneal scars. Images were acquired using a custom-built five-dimensional laser-scanning microscope with a broadband sub-15 femtosecond near-infrared pulsed excitation laser and a 16-channel photomultiplier tube detector in combination with a time-correlated single photon counting module. Morphological alterations of epithelial cells were observed for all pathologies. Moreover, diseased corneas showed alterations to the cells’ metabolism that were revealed using the NAD(P)H free to protein-bound ratios. The mean autofluorescence lifetime of the stroma and the organization of the collagen fibers were also significantly altered due to the pathologies. We demonstrate that TPI can be used to distinguish between NP and diseased human corneas, based not only on alterations of the cells’ morphology, which can also be evaluated using current clinical devices, but on additional morphological and functional features such as the organization of the stroma and the cells’ metabolism. Therefore, TPI could become an efficient tool for diagnosing corneal diseases and better understanding the biological processes of the diseases.
2017
K. Reinhardt, H.G. Breunig, K. König. Autofluorescence lifetime variation in the cutucle of the bedbug Cimex lectularius. Arthropod Strucure & Development 46 (2017) 56-62. Abstract The decay time of the fluorescence of excited molecules, called fluorescence lifetime, can provide information about the cuticle composition additionally to widely used spectral characteristics. We compared autofluorescence lifetimes of different cuticle regions in the copulatory organ of females of the bedbug, Cimex lectularius. After two-photon excitation at 720 nm, regions recently characterised as being rich in resilin showed a longer bimodal distribution of the mean autofluorescence lifetime tm (tau-m) at 0.4 ns and 1.0e1.5 ns, while resilin-poor sites exhibited a unimodal pattern with a peak around 0.8 ns. The mean lifetime, and particularly its second component, can be useful to distinguish resilin-rich from resilin-poor parts of the cuticle. The few existing literature data suggest that chitin is unlikely responsible for the main autofluorescent component observed in the resilin-poor areas in our study and that melanin requires further scrutiny. Autofluorescence lifetime measurements can help to characterise properties of the arthropod cuticle, especially when coupled with multiphoton excitation to allow for deeper tissue penetration. M. Balu, G. Lentsch, D.Z. Korta, K. König, K.M. Kelly, B.J. Tromberg, C.B. Zachary. In vivo multiphoton-microscopy of picosecond-laser-induced optical breakdown in human skin. Lasers in Surgery and Medicine (2017). Doi:10.1002/lsm.22655. Abstract The purpose of this study was to demonstrate the capability of multiphoton microscopy (MPM), a highresolution, label-free imaging technique, to characterize in vivo the skin response to a fractionated non-ablative picosecond-laser treatment.
2016
M. Klemp, M. Meinke, M. Weinigel, HJ. Roewert-Huber, K. König, M. Ulrich, J. Lademann, M. Darvin. Comparison of morphologic criteria for actinic keratosis and squamous cell carcinoma using in vivo multiphoton tomography. Exp Dermatol 25 (2016) 218-222. doi: 10.1111/exd.12912. Abstract The routine diagnostic procedure of actinic keratosis (AK) and invasive squamous cell carcinoma (SCC) is a histological examination after taking a biopsy. In the past decades, non-invasive optical methods for skin examination have been developed. Patients with clinical diagnosis of AK or SCC were examined. The morphological criteria were determined for healthy, AK and SCC skin and compared for statistically significant differences. In this study, the applicability of multiphoton tomography (MPT) as an in vivo diagnostic tool for AK and SCC was evaluated. Changes in the morphology of the keratinocytes such as broadened epidermis, large intercellular spaces, enlarged nucleus and a large variance in cell shape could easily be recognized. The cell nuclei of AK and SCC were significantly larger compared to healthy skin cells in all cell layers. The nucleus–cytoplasm ratio was also significantly higher for AK and SCC than for the healthy skin cells. It was even higher in SCC compared to spinous and basal cell layer of AK. The cell density in AK and SCC was significantly lower than in the basal and spinous cell layers of healthy skin. In SCC, the cell density was significantly lower than in AK. Concerning the intercellular spaces, significant differences were found for AK and healthy skin in spinous and basal cell layer and for SCC compared to AK and healthy skin. In this study, MPT proved to be a valuable noninvasive imaging method for in vivo detection and discrimination of AK and SCC from healthy skin. A. Batista, HG. Breunig, A. Uchugonova, AM. Morgado, K. König. Two-photon spectral fluorescence lifetime and second harmonic generation imaging of the porcine eye with a 12 femtosecond laser microscope. JBO 21(3), 036002 (2016). Abstract Five dimensional microscopy with a 12-fs laser scanning microscope based on spectrally resolved two-photon autofluorescence lifetime and second-harmonic generation (SHG) imaging was used to characterize all layers of the porcine cornea. This setup allowed the simultaneous excitation of both metabolic cofactors, NAD(P)H and flavins, and their discrimination based on their spectral emission properties and fluorescence decay characteristics. Furthermore, the architecture of the stromal collagen fibrils was assessed by SHG imaging in both forward and backward directions. Information on the metabolic state and the tissue architecture of the porcine cornea were obtained with subcellular resolution, and high temporal and spectral resolutions. S. Kantelhardt, D. Kalasauskas, K. König, E. Kim, M. Weinigel, A. Uchugonova, A. Giese. In vivo multiphoton tomography and fluorescence lifetime imaging of human brain tumor tissue. J Neurooncol online 30. Januar 2016. DOI 10.1007/s11060-016-2062-8. Abstract High resolution multiphoton tomography and fluorescence lifetime imaging differentiates glioma from adjacent brain in native tissue samples ex vivo. Presently, multiphoton tomography is applied in clinical dermatology and experimentally. We here present the first application of multiphoton and fluorescence lifetime imaging for in vivo imaging on humans during a neurosurgical procedure. We used a MPTflexTM Multiphoton Laser Tomograph (JenLab, Germany). We examined cultured glioma cells in an orthotopic mouse tumor model and native human tissue samples. Finally the multiphoton tomograph was applied to provide optical biopsies during resection of a clinical case of glioblastoma. All tissues imaged by multiphoton tomography were sampled and processed for conventional histopathology. The multiphoton tomograph allowed fluorescence intensity- and fluorescence lifetime imaging with submicron spatial resolution and 200 picosecond temporal resolution. Morphological fluorescence intensity imaging and fluorescence lifetime imaging of tumor-bearing mouse brains and native human tissue samples clearly differentiated tumor and adjacent brain tissue. Intraoperative imaging was found to be technically feasible. Intraoperative image quality was comparable to ex vivo examinations. To our knowledge we here present the first intraoperative application of high resolution multiphoton tomography and fluorescence lifetime imaging of human brain tumors in situ. It allowed in vivo identification and determination of cell density of tumor tissue on a cellular and subcellular level within seconds. The technology shows the potential of rapid intraoperative identification of native glioma tissue without need for tissue processing or staining. V. Huck, C. Gorzelanny, K. Thomas, V. Getova, V. Niemeyer, K. Zens, TR. Unnerstall, JS. Feger, MA. Fallah, D. Metze, S. Ständer, TA. Luger, K. König, C. Mess, SW. Schneider. From morphology to biochemical state-intravital multiphoton fluorescence lifetime imaging of inflamed human skin. Sci. Rep. 6(2016)22789, doi: 10.1038/srep22789 (2016). Abstract The application of multiphoton microscopy in the field of biomedical research and advanced diagnostics promises unique insights into the pathophysiology of inflammatory skin diseases. In the present study, we combined multiphoton-based intravital tomography (MPT) and fluorescence lifetime imaging (MPTFLIM) within the scope of a clinical trial of atopic dermatitis with the aim of providing personalised data on the aetiopathology of inflammation in a non-invasive manner at patients’ bedsides. These ‘optical biopsies’ generated via MPT were morphologically analysed and aligned with classical skin histology. Because of its subcellular resolution, MPT provided evidence of a redistribution of mitochondria in keratinocytes, indicating an altered cellular metabolism. Two independent morphometric algorithms reliably showed an even distribution in healthy skin and a perinuclear accumulation in inflamed skin. Moreover, using MPT-FLIM, detection of the onset and progression of inflammatory processes could be achieved. In conclusion, the change in the distribution of mitochondria upon inflammation and the verification of an altered cellular metabolism facilitate a better understanding of inflammatory skin diseases and may permit early diagnosis and therapy. M. Afshar, M. Leber, W. Poppendieck, K. König, H. Seidel, D. Feili. On-chip nanostructuring and impedance trimming of transparent and flexible ITO electrodes by laser induced coherent sub-20 nm cuts. Applied Surface Science 360 (216) 494-501. Abstract In this work, the effect of laser-induced nanostructuring of transparent indium tin oxide (ITO) electrodeson flexible glass is investigated. Multi-electrode arrays (MEA) for electrical and optical characterizationof biological cells were fabricated using standard MEMS technologies. Optimal sputter parameters con-cerning oxygen flow, sputter power and ambient pressure for ITO layers with both good optical andelectrical properties were determined. Afterwards, coherent sub-20 nm wide and 150 nm deep nanocutsof many micrometers in length were generated within the ITO electrodes by a sub-15 femtosecond (fs)pulsed laser. The influence of laser processing on the electrical and optical properties of electrodes wasinvestigated. The electrochemical impedance of the manufactured electrodes was measured before andafter laser modification using electrochemical impedance spectroscopy. A small reduction in electrodeimpedance was observed. These nanostructured electrodes show also polarizing effects by the visiblespectrum. HG Breunig, A. Batista, A. Uchugonova, K. König. Cell optoporation with a sub-15 fs and a 250-fs laser. JBO 21(6), 060501 (2016), doi: 10.1117/1.JBO.21.6.060501. Abstract We employed two commercially available femtosecond lasers, a Ti:sapphire and a ytterbium-based oscillator, to directly compare from a user’s practical point-of-view in one common experimental setup the efficiencies of transient laser-induced cell membrane permeabilization, i.e., of so-called optoporation. The experimental setup consisted of a modified multiphoton laser-scanning microscope employing high-NA focusing optics. An automatic cell irradiation procedure was realized with custom-made software that identified cell positions and controlled relevant hardware components. The Ti:sapphire and ytterbium-based oscillators generated broadband sub-15-fs pulses around 800 nm and 250-fs pulses at 1044 nm, respectively. A higher optoporation rate and posttreatment viability were observed for the shorter fs pulses, confirming the importance of multiphoton effects for efficient optoporation. K. König, A. Kasenbacher. Thermal damage behaviour of human dental pulp stem cells. ZWP Online 4 (2016). Abstract This study was designed to examine the influence of temperatures ranging from 37 to 65 °C on the cell morphology of DPSC stem cells via light and electron microscopy, a synthesis of Heat Shock Proteins (HSP) with fluorescence-marked antibodies and vitality via the Life/Dead Fluorescence Kit.
2015
HG. Breunig, M. Weinigel, K. König. In vivo imaging of ZnO Nanoparticles from sunscreen on human skin with a mobile multiphoton tomograph. BioNanoScience 5(2014)42-47. DOI: 10.1007/s12668-014-0155-4. Abstract Reports on the toxicity of inorganic nanoparticles used in sunscreen, in particular of zinc oxide and titanium dioxide, have raised concerns about a possible particle penetration through the skin barrier. We used two-photon imaging to visualize the distribution of zinc-oxide nanoparticles after topical application of a commercially available sunscreen on human skin in vivo and to investigate a possible penetration of nanoparticles beyond the stratum corneum. Two-photon imaging is in particular suitable for these investigations since the excitation and the nonlinear signal light from zinc-oxide nanoparticles as well as the endogenous skin autofluorescence are all spectrally well-separated which allows a clear identification of the signal origin by detection in two-spectral channels. Furthermore,microscopic modifications in the cutaneous structure like skin wrinkles which exhibit different thicknesses of the dermal layers and at the same time are regions where nanoparticle accumulation can be specifically investigated. The results indicate no penetration of nanoparticle through the barrier of the stratum corneum of healthy skin even in microscopic wrinkles. M. Balu, C.B. Zachary, R.M. Harris, T.B. Krasieva, K. König, B.J. Tromberg, K.M. Kelly. In vivo multiphoton microsocpy of basal cell carcinoma. JAMA Dermatol. published online April 24, 2015, doi:10.1001/jamadermatol.2015.0453. Abstract Basal cell carcinomas (BCCs) are diagnosed by clinical evaluation, which can include dermoscopic evaluation, biopsy, and histopathologic examination. Recent translation of multiphoton microscopy (MPM) to clinical practice raises the possibility of noninvasive, label-free in vivo imaging of BCCs that could reduce the time from consultation to treatment. K. König, H.G. Breunig. Two-photon imaging of intact living plants during freezing with a flexible multiphoton tomograph. Laser Phys. Lett. 12 (2015) 025601. Abstract We describe the combination of a flexible multiphoton tomograph (MPTflex) with a heating and cooling stage. The stage allows temperature control in the range of -196°C (77K) to +600°C (873K) with selectable heating/Freezing rates between 0.01 K min-1 and 150 K min-1. To illustrate the imaging capabilities of the combined system, fluorescence intensity and lifetime of intrinsic molecules from aplant leaf were imaged with submicron resolution during freezing in vivo without detaching the leaf from the plant. An increase of fluorescence intensitiy and decay times with decreasing temperature was abserved. The measurements illustrate the usefulness of multiphoton imaging as a non-invasive online tool to investigate temperature-induced effects. The flexible multiphoton tomograph with its adjustable mechano-optical arm and scan head allows imaging at otherwise hardly accessible sample regions. K. König, T. Liu, P. Anderson, G. Breunig. Multiphoton imaging with a novel compact diode-pumped Ti:sapphire oscillator. Microsc Res Technol 78(2015)1154-1158. Abstract Multiphoton laser scanning microscopy commonly relies on bulky and expensive femtosecond lasers. We integrated a novel minimal-footprint Ti:sapphire oscillator, pumped by a frequency-doubled distributed Bragg reflector tapered diode laser, into a clinical multiphoton tomograph and evaluated its imaging capability using different biological samples, i.e. cell monolayers, corneal tissue, and human skin. With the novel laser, the realization of very compact Ti:sapphire-based systems for high-quality multiphoton imaging at a significantly size and weight compared to current systems will become possible. A. Uchugonova, HG. Breunig, A. Batista, K. König. Optical reprogramming of human somatic cells using ultrashort Bessel-shaped near-infrared femtosecond laser pulses. JBO 20(2015)11, 115008. Abstract We report a virus-free optical approach to human cell reprogramming into induced pluripotent stem cells with low-power nanoporation using ultrashort Bessel-shaped laser pulses. Picojoule near-infrared sub-20 fs laser pulses at a high 85 MHz repetition frequency are employed to generate transient nanopores in the membrane of dermal fibroblasts for the introduction of four transcription factors to induce the reprogramming process. In contrast to conventional approaches which utilize retro- or lentiviruses to deliver genes or transcription factors into the host genome, the laser method is virus-free; hence, the risk of virus-induced cancer generation limiting clinical application is avoided. A. Uchugonova. Optical reprogramming of human cells in an ultrashort femtosecond laser microfluidic transfection platform. J. Biophoton 1-6(2015). DOI 10.1002/jbio.201500240. Abstract Induced pluripotent stem cell (iPS cell) technology can be used to produce unlimited numbers of functional cells for both research and therapeutic purposes without ethical controversy. Typically, viruses are applied for efficient intracellular delivery of genes/transcription factors to generate iPS cells. However, the viral genomic integration may cause a risk of mutation as well as tumor formation therefore limits its clinical application. Here we demonstrate that spatially shaped extreme ultrashort laser pulses of sub-20 femtoseconds induce transient membrane permeabilisation which enables contamination-free transfection of cells in a microfluidic tube with multiple genes at the individual cell level in order to achieve optical reprogramming of large cell populations. We found that the ultrashort femtosecond laser-microfluidic cell transfection platform enhanced the efficacy of iPS-like colony-forming following merely a single transfection. M. Weinigel, HG. Breunig, A. Uchugonova, K. König. Multipurpose nonlinear optical imaging system for in vivo and ex vivo multimodal histology. J Medical Imaging 2(2015)0160031-016003110. Abstract We report on a flexible multipurpose nonlinear microscopic imaging system based on a femtosecond excitation source and a photonic crystal fiber with multiple miniaturized time-correlated single-photon counting detectors. The system provides the simultaneous acquisition of e.g., two-photon autofluorescence, secondharmonic generation, and coherent anti-Stokes Raman scattering images. Its flexible scan head permits ex vivo biological imaging with subcellular resolution such as rapid biopsy examination during surgery as well as imaging on small as well as large animals. Above all, such an arrangement perfectly matches the needs for the clinical investigation of human skin in vivo where knowledge about the distribution of endogenous fluorophores, second-harmonic generation–active collagen as well as nonfluorescent lipids is of high interest. M. Afshar, EM. Preiss, T. Sauerwald, M. Rodner, D. Feili, M. Straub, K. König, A. Schütze, H. Seidel. Indium-tin-oxide single-nanowire gas sensor fabricated via laser writing and subsequent etching. Sensors and Actuators B215(2015)525-535. Abstract We report on the design and nanofabrication of a single nanowire (NW) indium-tin-oxide (ITO) gassensor and on test results obtained with an oxidizing and a reducing gas. As a novel fabrication approach,direct laser writing and a subsequent etching process on sputtered ITO thin-film layers is applied. Forthis technique a near-infrared Ti:sapphire laser with sub-15 fs pulses and a repetition rate of 85 MHz isused. NWs for gas sensors are realized in two versions with a thickness of 125 ± _25 nm; one with 350 nmin width and 90 _m in length the other with 700 nm in width and 200 _m in length. The sensors areexposed to nitrogen dioxide (NO2) in synthetic air with concentrations from 1 ppm to 50 ppm showing asignificant change in resistance (up to 15.8%), whereas the reaction to 2000 ppm carbon monoxide (CO)turns out to be negligible (0.05%). At ambient temperature, the sensor exhibits integrating dosimeter-like behavior with relaxation times of more than 20 h. By self-heating, the NW can be reset to its initialcondition, thus enabling a new dosimeter run at room-temperature. When the sensors are operated inself-heating mode, a conventional behavior is observed, enabling the detection of NO2concentrationsdown to about 1 ppm at a stationary temperature below 200◦C. HG. Breunig, A. Uchugonova, A. Batista, K. König. Software-aided automatic laser optoporation and transfection of cells. Scientific Reports 5(2015)11185. DOI 10.1038/srep11185. Abstract Optoporation, the permeabilization of a cell membrane by laser pulses, has emerged as a powerful non-invasive and highly efficient technique to induce transfection of cells. However, the usual tedious manual targeting of individual cells significantly limits the addressable cell number. To overcome this limitation, we present an experimental setup with custom-made software control, for computer-automated cell optoporation. The software evaluates the image contrast of cell contours, automatically designates cell locations for laser illumination, centres those locations in the laser focus, and executes the illumination. By software-controlled meandering of the sample stage, in principle all cells in a typical cell culture dish can be targeted without further user interaction. The automation allows for a significant increase in the number of treatable cells compared to a manual approach. For a laser illumination duration of 100 ms, 7-8 positions on different cells can be targeted every second inside the area of the microscope field of view. The experimental capabilities of the setup are illustrated in experiments with Chinese hamster ovary cells. Furthermore, the influence of laser power is discussed, with mention on post-treatment cell survival and optoporation-efficiency rates. M. Weinigel, HG. Breunig, ME. Darvin, M. Klemp, J. Röwert-Huber, J. Lademann, K. König. Impact of refractive index mismatches on coherent anti-Stokes Raman scattering and multiphoton autofluorescence tomography of human skin in vivo. Physics in Medicine & Biology 60(2015)6881-6899, Doi:10.1088/0031-9155/60/17/6881. Abstract Optical non-linear multimodal tomography is a powerful diagnostic imaging tool to analyse human skin based on its autofluorescence and second-harmonic generation signals. Recently, the field of clinical non-linear imaging has been extended by adding coherent anti-Stokes Raman scattering (CARS)— a further optical sectioning method for the detection of non-fluorescent molecules. However, the heterogeneity of refractive indices of different substances in complex tissues like human skin can have a strong influence on CARS image formation and requires careful clinical interpretation of the detected signals. Interestingly, very regular patterns are present in the CARS images, which have no correspondence to the morphology revealed by autofluorescence at the same depth. The purpose of this paper is to clarify this phenomenon and to sensitize users for possible artefacts. A further part of this paper is the detailed comparison of CARS and autofluorescence images of healthy human skin in vivo covering the complete epidermis and part of the upper dermis by employing the flexible medical non-linear tomograph MPTflex CARS. K. Reinhardt, H.G. Breunig, A. Uchugonova, K. König. Sperm metabolism is altered during storage by females: evidence from fluorescence lifetime measurements in bedbugs. J. R. Soc. Interface 12 (2015), doi:10.1098/rsif.2015.0609. Abstract We explore the possibility of characterizing sperm cells without the need to stain them using spectral and fluorescence lifetime analyses after multiphoton excitation in an insect model. The autofluorescence emission spectrum of sperm of the common bedbug, Cimex lectularius,was consistent with the presence of flavins andNAD(P)H. The mean fluorescence lifetimes showed smaller variation in sperm extracted from the male (tau m, tm . 1.54–1.84 ns) than in that extracted from the female sperm storage organ (tau m, tm . 1.26–2.00 ns). The fluorescence lifetime histograms revealed four peaks. These peaks (0.18, 0.92, 2.50 and 3.80 ns) suggest the presence ofNAD(P)Hand flavins and show that sperm metabolism can be characterized using fluorescence lifetime imaging. The difference in fluorescence lifetime variation between the sexes is consistent with the notion that female animals alter the metabolism of sperm cells during storage. It is not consistent, however, with the idea that sperm metabolism represents a sexually selected character that provides females with information about the male genotype. S. Springer, M. Zieger, K. König, M. Kaatz, J. Lademann, ME Darvin. Optimization of the measurement procedure during multiphoton tomography of human skin in vivo. Skin Res Tech 0(2015) 1-7, DOI: 10.1111/srt.12273. Abstract The in vivo multiphoton tomography has evolved into a useful tool for the non-invasive investigation of morphological and biophysical characteristics of human skin. Until now, changes of skin have been evaluated mainly by clinical and histological techniques. The current study addresses the effects of a changed acquisition time for single scans in a Zstack on the directly related qualitative and quantitative interpretability of the data. A. Uchugonova, W. Cao, R.M. Hoffman, K. König. Comparison of label-free and GFP multiphoton imaging of hair-follicle-associated pluripotent (HAP) stem cells in mouse whiskers. Cell Cycle 14(2015) 3430-3433. doi:10.1080/15384101.2015.1090064. Abstract Hair-follicle-associated pluripotent (HAP) stem cells can differentiate into many cell types, including neurons and heart muscle cells, and have been shown to repair peripheral nerves and the spinal cord in mice. HAP stem cells can be obtained from each individual patient for regenerative medicine which overcomes problems with immune rejection. Previously, we have demonstrated that genetically-encoded protein markers such as GFP in transgenic mice can be used to visualize HAP stem cells in vivo by multiphoton tomography. Detection and visualization of stem cells in vivo without exogenous labels such as GFP would be important for human application. In the present report, we demonstrate label-free visualization of hair follicle stem cells in mouse whiskers by multiphoton tomography due to the intrinsic fluorophores such as NAD(P)H/flavins. We compared multiphoton tomography of GFP-labeled HAP stem cells and unlabeled stem cells in isolated mouse whiskers. We show that observation of HAP stem cells by label-free multiphoton tomography is comparable to detection using GFP-labeled stem cells. The results described here have important implications for detection and isolation of human HAP stem cells for regenerative medicine.
2014
K. König, A. Uchugonova, HG Breunig. High-resolution multiphoton crymicroscopy. Methods, online July 16,2013, doi: 10.1016. Abstract An ultracompact high-resolution multiphoton cryomicroscope with a femtosecond near infrared fiber laser has been utilized to study the cellular autofluorescence during freezing and thawing of cells. Cooling resulted in an increase of the intracellular fluorescence intensity followed by morphological modifications at temperatures below _10 _C, depending on the application of the cryoprotectant DMSO and the cooling rate. Furthermore, fluorescence lifetime imaging revealed an increase of the mean lifetime with a decrease in temperature. Non-destructive, label-free optical biopsies of biomaterial in ice can be obtained with sub-20mW mean powers. M. Weinigel, H.G.Breunig, J. Lademann, K. König. In vivo histology: optical biopsies with chemical contrast using multiphoton/CARS tomography. Laser Phys Lett, 11(2014)055601. Abstract The majority of existing coherent anti-Stokes Raman scattering (CARS) imaging systems are still huge and complicated laboratory systems and neither compact nor user-friendly nor mobile medically certified CARS systems. We have developed a new flexible multiphoton/CARS tomograph for imaging in a clinical environment. The system offers exceptional 360° flexibility with a very stable setup and enables label free ‘in vivo histology’ with chemical contrast within seconds. It can be completely operated by briefly trained non-laser experts. The imaging capability and flexibility of the novel in vivo tomograph are shown on optical biopsies with subcellular resolution and chemical contrast of patients suffering from psoriasis and squamous cell carcinoma. M. Straub, M. Schüle, M. Afshar, D. Feili, H. Seidel, K. König. Sub-15fs laser-induced nanostructures emerging in Si(100)surfaces immersed in water: analysis of structural phases. Appl. Phys. A 115(2014)221-228. Abstract Nanoscale periodic rifts and subwavelength ripples as well as randomly nanoporous surface structures were generated on Si(100) surfaces immersed in water by tightly focused high-repetition rate sub-15 femtosecond subnanojoule pulsed Ti:sapphire laser light. Subsequent to laser processing, silicon oxide nanoparticles, which originated from a reaction of ablated silicon with water and aggregated on the exposed areas, were etched off by hydrofluoric acid. The structural phases of the three types of silicon nanostructures were investigated by transmission electron microscopy diffraction images recorded on focused ion beam sections. On nanorift patterns, which were produced at radiant exposure extremely close to the ablation threshold, only the ideal Si-I phase at its original bulk orientation was observed. Electron diffraction micrographs of periodic ripples, which were generated at slightly higher radiant exposure, revealed a compression of Si-I in the vertical direction by 6 %, which is attributed to recoil pressure acting during ablation. However, transitions to the high-pressure phase Si-II, which implies compression in the same direction at pressures in excess of 10 GPa, to the metastable phases Si-III or Si-IV that arise from Si-II on pressure relief or to other high-pressure phases (Si-V–Si-XII) were not observed. The nanoporous surfaces featured Si-I material with grains of resolidified silicon occurring at lattice orientations different from the bulk. Characteristic orientational relationships as well as smallangle grain boundaries reflected the rapid crystal growth on the substrate. M. Schüle, M. Afshar, D. Feili, H. Seidel, K. König, M. Straub. Incubation and nanostructure formation on n- and p-type Si(100) and Si(111) at various doping levels induced by sub-nanojoule femto- and picosecond near-infrared laser pulses. Applied Surface Science 314(2014)21-29. Abstract N- and p-doped Si(1 0 0) and Si(1 1 1) surfaces with dopant concentrations of 2 × 1014–1 × 1019cm−3were irradiated by tightly focused 85-MHz repetition rate Ti:sapphire laser light (central wavelength 800 nm, bandwidth 120 nm) at pulse durations of 12 fs to 1.6 ps. Dependent on pulse peak intensity and exposure time nanorifts, ripples of period 130 nm as well as sponge-like randomly nanoporous surface structures were generated with water immersion and, thereafter, laid bare by etching off aggregated oxide nanoparticles. The same structure types emerged in air or water with transform-limited 100-fs pulses. At apulse length of 12 fs pronounced incubation occurred with incubation coefficients S = 0.66–0.85, where as incubation was diminished for picosecond pulses (S > 0.95). The ablation threshold strongly rose with dopant concentration. At similar doping level it was higher for n-type than for p-type samples and forSi(1 0 0) compared to Si(1 1 1) surfaces. These observations are attributed to laser-induced defect statesin the bandgap which participate in photoexcitation, deactivation of dopants by complex formation, and different densities of interface states at the boundary with the ultrathin native silicon dioxide surfacelayer. The threshold increase with pulse length revealed predominant single-photon excitation as well as multiphoton absorption.
2013
M. Manfredini, F. Arginelli, C. Dunsby, P. French, C. Talbot, K. König, G. Pellacani, G. Ponti, S. Seidenari. High-resolution imaging of basal cell carcinoma: a comparison between multiphoton microscopy with fluorescence lifetime imaging and reflectance confocal microscopy Skin Research and Technology 2013; 19: e433–e443 Abstract Aims: The aim of this study was to compare morphological aspects of basal cell carcinoma (BCC) as assessed by two different imaging methods: in vivo reflectance confocal microscopy (RCM) and multiphoton tomography with fluorescence lifetime imaging implementation (MPT-FLIM). Methods: The study comprised 16 BCCs for which a complete set of RCM and MPT-FLIM images were available. The presence of seven MPT-FLIM descriptors was evaluated. The presence of seven RCM equivalent parameters was scored in accordance to their extension. Chi-squared test with Fisher’s exact test and Spearman’s rank correlation coefficient were determined between MPT-FLIM scores and adjusted-RCM scores. Results: MPT-FLIM and RCM descriptors of BCC were coupled to match the descriptors that define the same pathological structures. The comparison included: Streaming and Aligned elongated cells, Streaming with multiple directions and Double alignment, Palisading (RCM) and Palisading (MPTFLIM), Typical tumor islands, and Cell islands surrounded by fibers, Dark silhouettes and Phantom islands, Plump bright cells and Melanophages, Vessels (RCM), and Vessels (MPTFLIM). The parameters that were significantly correlated were Melanophages/Plump Bright Cells, Aligned elongated cells/ Streaming, Double alignment/Streaming with multiple directions, and Palisading (MPT-FLIM)/Palisading (RCM). Conclusion: According to our data, both methods are suitable to image BCC’s features. The concordance between MPT-FLIM and RCM is high, with some limitations due to the technical differences between the two devices. The hardest difficulty when comparing the images generated by the two imaging modalities is represented by their different field of view. A. Alex, J. Weingast, M. Weinigel, M. Kellner-Höfer, R. Nemecek, M. Binder, H. Pehamberger, K. König, W. Drexler. Three-dimensional multiphoton/optical coherence tomography for diagnostic applications in dermatology J. Biophotonics 6, No. 4, 352–362 (2013) Abstract A preliminary clinical trial using state-of-the-art multiphoton tomography (MPT) and optical coherence tomography (OCT) for three-dimensional (3D) multimodal in vivo imaging of normal skin, nevi, scars and pathologic skin lesions has been conducted. MPT enabled visualization of sub-cellular details with axial and transverse resolutions of <2 mm and <0.5 mm, respectively, from a volume of 0.35 _ 0.35 _ 0.2 mm3 at a frame rate of 0.14 Hz (512 _ 512 pixels). State-of-the-art OCT, operating at a center wavelength of 1300 nm, was capable of acquiring 3D images depicting the layered architecture of skin with axial and transverse resolutions _8 mm and _20 mm, respectively, from a volume of 7 _ 3.5 _ 1.5 mm3 at a frame rate of 46 Hz (1024 _ 1024 pixels). This study demonstrates the clinical diagnostic potential of MPT/OCT for pre-screening relatively large areas of skin using 3D OCT to identify suspicious regions at microscopic level and subsequently using high resolution MPT to obtain zoomed in, sub-cellular level information of the respective regions H. G. Breunig, M. Weinigel, R. Bückle, M. Kellner-Höfer, J. Lademann, M. E. Darvin, W. STerry, K. König. Clinical coherent anti-Stokes Raman scattering and multiphoton tomography of human skin with a femtosecond laser and photonic crystal fiber Laser Phys. Lett. 10 (2013) Abstract We report on in vivo coherent anti-Stokes Raman scattering spectroscopy (CARS), two-photon fluorescence and second-harmonic-generation imaging of human skin with a novel multimodal clinical CARS / multiphoton tomograph. CARS imaging is realized by a combination of femtosecond pulses with broadband continuum pulses generated by a photonic crystal fiber. The images reveal the microscopic distribution of non-fluorescent lipids, endogenous fluorophores and the collagen network inside the human skin in vivo with subcellular resolution. Examples of healthy as well as cancer-affected skin are presented. M. Balu, A. Mazhar, C. K. Hayakawa, R. Mittal, T. B. Krasieva, K. König, V. Venugopalan, B. J. Tromberg. In Vivo Multiphoton NADH Fluorescence Reveals Depth-Dependent Keratinocyte Metabolism in Human Skin Abstract We employ a clinical multiphoton microscope to monitor in vivo and noninvasively the changes in reduced nicotinamide adenine dinucleotide (NADH) fluorescence of human epidermal cells during arterial occlusion. We correlate these results with measurements of tissue oxy- and deoxyhemoglobin concentration during oxygen deprivation using spatial frequency domain imaging. During arterial occlusion, a decrease in oxyhemoglobin corresponds to an increase in NADH fluorescence in the basal epidermal cells, implying a reduction in basal cell oxidative phosphorylation. The ischemia-induced oxygen deprivation is associated with a strong increase in NADH fluorescence of keratinocytes in layers close to the stratum basale, whereas keratinocytes from epidermal layers closer to the skin surface are not affected. Spatial frequency domain imaging optical property measurements, combined with a multilayer Monte Carlo-based radiative transport model of multiphoton microscopy signal collection in skin, establish that localized tissue optical property changes during occlusion do not impact the observed NADH signal increase. This outcome supports the hypothesis that the vascular contribution to the basal layer oxygen supply is significant and these cells engage in oxidative metabolism. Keratinocytes in the more superficial stratum granulosum are either supplied by atmospheric oxygen or are functionally anaerobic. Based on combined hemodynamic and two-photon excited fluorescence data, the oxygen consumption rate in the stratum basale is estimated to be ~0.035 mmoles/106 cells/h. M. Ulrich, M. Klemp, M. E. Darvin, K. König, J. Lademann, M. C. Meinke. In vivo detection of basal cell carcinoma: comparison of a reflectance confocal microscope and a multiphoton tomograph Journal of Biomedical Optics 18(6), 061229 (June 2013) Abstract The standard diagnostic procedure for basal cell carcinoma (BCC) is invasive tissue biopsy with timeconsuming histological examination. To reduce the number of biopsies, noninvasive optical methods have been developed providing high-resolution skin examination. We present direct comparison of a reflectance confocal microscope (RLSM) and a multiphoton tomograph (MPT) for BCC diagnosis. Both systems are applied to nine patients prior to surgery, and the results are analyzed, including histological results. Both systems prove suitable for detecting typical characteristics of BCC in various stages. The RLSM allows large horizontal overview images to be obtained, enabling the investigator to find the regions of interest quickly, e.g., BCC nests. Elongated cells and palisading structures are easily recognized using both methods. Due to the higher resolution, changes in nucleus diameter or cytoplasm could be visualized with the MPT. Therefore, the nucleus diameter, nucleus/cytoplasm ratio, and cell density are estimated for normal and BCC cells using the MPT. The nucleus of elongated BCC cells is significantly longer than other measured normal skin cells, whereas the cell density and nucleus/cytoplasm ratio of BCC cannot be significantly distinguished from granular cells. A. Uchugonova, M. Zhao, M. Weinigel, Y. Zhang, M. Bouvet, R. M. Hoffman, K. König. Multiphoton Tomography Visualizes Collagen Fibers in the Tumor Microenvironment That Maintain Cancer-Cell Anchorage and Shape Journal of Cellular Biochemistry 114:99–102 (2013) Abstract Second harmonic generation (SHG) multiphoton imaging can visualize fibrillar collagen in tissues. SHG has previously shown that fibrillar collagen is altered in various types of cancer. In the present study, in vivo high resolution SHG multi-photon tomography in living mice was used to study the relationship between cancer cells and intratumor collagen fibrils. Using green fluorescent protein (GFP) to visualize cancer cells and SHG to image collagen, we demonstrated that collagen fibrils provide a scaffold for cancer cells to align themselves and acquire optimal shape. These results suggest a new paradigm for a stromal element of tumors: their role in maintaining anchorage and shape of cancer cells that may enable them to proliferate. K. König, A. Ostendorf. Optically generated sub-100 nm structures for biomedical and technical applications Lasers in Manufacturing Conference 2013 Abstract Near infrared femtosecond laser - material interactions such as multiphoton ionization and plasma formation provide the possibility to perform 3D nanoprocessing in a variety of organic and non-organic materials. Sub-100 nm structures that are more than one order smaller than the laser wavelength and therefore far below Abbes diffraction limit can be generated on the surface and inside the bulk. Compared to conventional extreme ultraviolet lithography, multiphoton technology provides the chance to produce 3D nanofeatures much simpler, less expensive, more flexible, and even inside transparent bulk material. Rapid prototyping and low power nanosurgery are two examples of applications of this novel nonlinear nanotechnology tool. This paper provides an overview of projects within Germany regarding to multiphoton generation of sub-100 nm structures. M. Straub, B. Weigand, M. Afshar, D. Feili, H. Seidel, K. König. Periodic subwavelength ripples on lithium niobate surfaces generated by tightly focused sub-15 femtosecond sub-nanojoule pulsed near-infrared laser light J. Opt. 15 (2013) Abstract On exposure to 85 MHz sub-15 fs pulsed 800 nm Ti:sapphire laser light in the focal spot of a high-numerical aperture oil immersion objective, characteristic periodic nanostructures emerged on the surface of z-cut congruent LiNbO3 crystals. Close to the ablation threshold shallow ripples oriented parallel to the laser polarization appeared on the surface at a periodicity 3k _ 200 nm as well as ripples running perpendicular to the laser polarization at almost the same period (3? _ 190 nm). Nanostructure formation involved melting and resolidification of LiNbO3 as evidenced by scanning electron microscopy images of structural peculiarities. Micro-Raman spectroscopy demonstrated that the resolidified material crystallized in the original structural phase. Ripple formation is attributed to plasma wave interference patterns that arise in the electron–hole plasma generated by multiphoton and avalanche collisional excitation. K. König, A. Uchugonova, H. G. Breunig. High-resolution multiphoton cryomicroscopy Abstract An ultracompact high-resolution multiphoton cryomicroscope with a femtosecond near infrared fiber laser has been utilized to study the cellular autofluorescence during freezing and thawing of cells. Cooling resulted in an increase of the intracellular fluorescence intensity followed by morphological modifications at temperatures below _10 _C, depending on the application of the cryoprotectant DMSO and the cooling rate. Furthermore, fluorescence lifetime imaging revealed an increase of the mean lifetime with a decrease in temperature. Non-destructive, label-free optical biopsies of biomaterial in ice can be obtained with sub-20mW mean powers.
2012
H. Zhang, M. Afshar, D. Feili, H. Seidel, K. König. Submicron-dot chains at and beneath surfaces of glasses written by a picojoule 12-fs laser scanning microscope Abstract A near-infrared 12-fs laser scanning microscope was employed for structuring glass surfaces. A Ti–sapphire laser operated at 85 MHz and emitted light pulses at wavelengths around 800 nm. A focused laser beam with a mean power of less than 27mW, corresponding to amaximal pulse energy of 318 pJ, was applied for line scanning at and beneath the surfaces of cover slips as well as of a filter glass BG39. Periodic arrangements of dots along the processed lines were produced through digital control of the scanner. Depending on the pulse energy and the scan speed, the diameter of the dots ranged from 550 nm down to 100 nm. For the cover slips, the dots occur as cavities after wet chemical etching. For BG39, which exhibits strong near-infrared absorption, both chains of cavities and bumps can be generated without any etching process. The result shows that structures with a size down to 1/8λ can be generated, probably through nonlinear single-photon processes confined within the focal volume. M. Straub, M. Afshar, D. Feili, H. Seidel, K. König. Periodic nanostructures on Si(100) surfaces generated by high-repetition rate sub-15 fs pulsed near-infrared laser light OPTICS LETTERS / Vol. 37, No. 2 / January 15, 2012 Abstract Nanoscale rifts and ripples at a periodicity of 130 nm were generated on Si(100) surfaces immersed in water using tightly focused 800 nm 12 fs pulsed 85 MHz laser light at subnanojoule pulse energies. At radiant exposure close to the ablation threshold rifts were typically 20–50 nm in width and 70 nm in depth running perpendicular to the laser polarization. On increase of the irradiance, the rifts broadened and formed periodic ripples, whereas at highest exposure, a random nanoporous surface topology emerged. Rift and ripple formation is explained by laser-induced standing surface plasma waves, which result in periodic variation of dissipation and ablation. M. Afshar, M. Straub, H. Voellm, D. Feili, K. Koenig, H. Seidel. Sub-100 nm structuring of indium-tin-oxide thin films by sub-15 femtosecond pulsed near-infrared laser light February 15, 2012 / Vol. 37, No. 4 / OPTICS LETTERS Abstract In magnetron sputtered indium-tin-oxide thin films of varying oxygen content, nanostructures were formed using tightly focused high-repetition rate near-infrared sub-15 femtosecond pulsed laser light. At radiant exposure well beyond the ablation threshold, cuts of 280–350 nm in width were generated. Illumination close to the ablation threshold resulted in periodic cuts of typically 20 nm in width at periodicities between 50 nm and 180 nm, as well as single sub-20 nm cuts. Subthreshold exposure, in combination with hydrochloric acid etching, yielded nanowires of 50 nm minimum lateral dimensions. K. König, A. Uchugonova, M. Straub, H. Zhang, M. Licht, M. Afshar, D. Feili, H. Seidel. Sub-100 nm material processing and imaging with a sub-15 femtosecond laser scanning microscope JOURNAL OF LASER APPLICATIONS, JULY 2012 Abstract Low mean powers of 1–10 mW are sufficient for material nanoprocessing when using femtosecond laser microscopes. In particular, near infrared 12 fs laser pulses at peak TW/cm2 intensities, picojoule pulse energies, and 85 MHz repetition rate have been employed. Threedimensional two-photon lithography as well as direct multiphoton ablation have been performed. Subwavelength sub-100 nm cuts have been realized in photoresists, silicon wafers, glass, polymers, metals, and biological targets. When reducing the mean power to the microwatt range, nondestructive two-photon imaging was performed with the same setup taking advantage of the broad laser emission spectrum. Multiphoton microscopes based on low-cost ultracompact sub-20 fs laser sources may become novel nonlinear optical tools for highly precise nanoprocessing and two-photon imaging. S. Seidenari, F. Arginelli, S. Bassoli, J. Cautela, P.M.W. French, M. Guanti, D. Guardoli, K. König, C. Talbot, C. Dunsby. Multiphoton LaserMicroscopy and Fluorescence Lifetime Imaging for the Evaluation of the Skin Dermatology Research and Practice, Volume 2012, Article ID 810749 Abstract Multiphoton laser microscopy is a new, non-invasive technique providing access to the skin at a cellular and subcellular level, which is based both on autofluorescence and fluorescence lifetime imaging.Whereas the former considers fluorescence intensity emitted by epidermal and dermal fluorophores and by the extra-cellular matrix, fluorescence lifetime imaging (FLIM), is generated by the fluorescence decay rate. This innovative technique can be applied to the study of living skin, cell cultures and ex vivo samples. Although still limited to the clinical research field, the development ofmultiphoton lasermicroscopy is thought to become suitable for a practical application in the next few years: in this paper, we performed an accurate review of the studies published so far, considering the possible fields of application of this imaging method and providing high quality images acquired in the Department of Dermatology of the University of Modena. H. G. Breunig, R. Bückle, M. Kellner-Höfer, M. Weinigel, J. Lademann , W. Sterry, K. König. Combined In Vivo Multiphoton and CARS Imaging of Healthy and Disease-Affected Human Skin Microscopy Research and Technique 75:492–498 (2012) Abstract We present combined epi-coherent anti-Stokes Raman scattering (CARS) and multiphoton imaging with both chemical discrimination and subcellular resolution on human skin in vivo. The combination of both image modalities enables label-free imaging of the autofluorescence of endogenous fluorophores by two-photon excited fluorescence, as well as imaging of the distribution of intercellular lipids, topically applied substances and water by CARS. As an example for medical imaging, we investigated healthy and psoriasis-affected human skin with both image modalities in vivo and found indications for different lipid distributions on the cellular level. M. Licht, A. Uchugonova, K. König, M. Straub. Sub-15 fs multiphoton lithography of three-dimensional structures for live cell applications J. Opt. 14 (2012) 065601 Abstract Development, morphology and intratissue location of cells are influenced by the 3D nano- and microenvironment. In this paper we demonstrate multiphoton photopolymerization to generate three-dimensional structures for cell culture applications with micro- and nanotopographic features using SU-8 photoresist and mr-NIL 6000 nanoimprint resist. Moving the focal spot of high-repetition rate near-infrared sub-15 fs pulsed laser light by a galvanometric beam scanner in combination with a piezoelectric vertical stage, nearly arbitrary trajectories of polymerized photoresist were generated. This technique can be used to generate cage structures with submicron interior features for live cell applications. Preliminary experiments with PC-3 and HT-1080 cells indicate the influence of the structures on cell behavior. A. Uchugonova, M. Lessel, S. Nietzsche, C. Zeitz, K. Jacobs, C. Lemke, K. König. Nanosurgery of cells and chromosomes using near-infrared twelve-femtosecond laser pulses Journal of Biomedical Optics 17(10), 101502 (October 2012) Abstract Laser-assisted surgery based on multiphoton absorption of near-infrared laser light has great potential for high precision surgery at various depths within the cells and tissues. Clinical applications include refractive surgery (fs-LASIK). The non-contact laser method also supports contamination-free cell nanosurgery. In this paper we describe usage of an ultrashort femtosecond laser scanning microscope for sub-100 nm surgery of human cells and metaphase chromosomes. A mode-locked 85 MHz Ti:Sapphire laser with an M-shaped ultrabroad band spectrum (maxima∶ 770 nm∕830 nm) and an in situ pulse duration at the target ranging from 12 fs up to 3 ps was employed. The effects of laser nanoprocessing in cells and chromosomes have been quantified by atomic force microscopy. These studies demonstrate the potential of extreme ultrashort femtosecond laser pulses at low mean milliwatt powers for sub-100 nm surgery of cells and cellular organelles. M.E. Darvin, K. König, M. Kellner-Höfer, H.G. Breunig, W. Werncke, M.C. Meinke, A. Patzelt, W. Sterry, J. Lademann. Safety Assessment by Multiphoton Fluorescence/ Second Harmonic Generation/Hyper-Rayleigh Scattering Tomography of ZnO Nanoparticles Used in Cosmetic Products Skin Pharmacol Physiol 2012;25:219–226 Abstract Zinc oxide nanoparticles (ZnO NPs) are commonly used as UV filters in commercial sunscreen products. Their penetration into the skin is intensively discussed in the literature. In the present in vivo study, penetration of ZnO NPs (30 nm in size) into human skin was investigated by multiphoton tomography. Based on the non-linear effects of a second harmonic generation and hyper-Rayleigh scattering, the distribution of ZnO NPs in the horny layers of the epidermis, as well as the furrows, wrinkles and orifice of the hair follicles was analyzed. This method permitted distinguishing between the particulate and dissolved forms of Zn. A detection limit of 0.08 fg/ _ m 3 was estimated. Taking advantage of this sensitivity, it was clearly shown that ZnO NPs penetrate only into the outermost layers of stratum corneum, furrows and into the orifices of the hair follicles and do not reach the viable epidermis. M. Straub, M. Afshar, D. Feili, H. Seidel, K. König. Surface plasmon polariton model of high-spatial frequency laser-induced periodic surface structure generation in silicon J. Appl. Phys. 111, 124315 (2012) Abstract In recent years, high-spatial frequency laser-induced surfaces structures have been generated in a large variety of dielectrics. In silicon subwavelength ripples, some of which featured periodicities below 100 nm, were formed using ultrafast lasers. We demonstrate for Si(100) surfaces that generation of a dense electron-hole plasma in the focal spot of ultrashort-pulsed laser light followed by massive excitation of plasma waves provides an explanation for the formation of such high-spatial frequency surface structures. The applied Drude-like model includes carrier-carrier collisions and is in excellent agreement with the experimentally observed ripple period. C. Huss, M. Krause, U. Löw, I. Riemann, F. Stracke, P. Mestres, B. Seitz, K. König. Experimentelle Hornhautbildgebung und Hornhautchirurgie mit nicht verstärkten Femtosekundenlaserpulsen Ophthalmologe 2012 · 109:995–1000 Abstract Hintergrund. Ziel war, mit einem unverstärkten Femtosekundenlaser Multiphotoneneffekte zu induzieren und für Hornhautbildgebung und Gewebeabtrag zu nutzen.Material und Methoden. Unverstärkte Titan-Saphir-Laser wurden zur Untersuchung von menschlichen Hornhäuten und Schweinehornhäuten an Laserscanningmikroskope gekoppelt. Bildgebung sowie Erzeugung von Gewebeläsionen erfolgten im gleichen Strahlengang mit Pulsenergien <2 nJ.Ergebnisse. Zelluläre Bestandteile und extrazelluläre Matrix wurden selektiv mittels Autofluoreszenz und Frequenzverdopplung in Submikrometerauflösung dargestellt. Nach intrastromaler linearer Laserabtastung mit höheren Leistungen entstand ein lumineszentes Plasma. Die Läsionsbreite nahm mit zunehmender Gewebetiefe ab und mit zunehmender Laserleistung am Objektiv zu. In der Lichtmikroskopie zeigte sich nach dem Laserabtrag in der Umgebung der Läsion intaktes Stroma.Schlussfolgerung. Mit niederenergetischen Femtosekundenlaserpulsen wurden hochauflösende Bilder sowie präzise Gewebeläsionen in der Kornea erzeugt. Der einfache Wechsel zwischen Bildgebung und Gewebeabtrag lässt diagnostische und therapeutische Anwendungen möglich erscheinen. M. J. Koehler, A. Preller, P. Elsner, K. König, U. C. Hipler, M. Kaatz. Non-invasive evaluation of dermal elastosis by in vivo multiphoton tomography with autofluorescence lifetime measurements Abstract The non-invasive differentiation of dermal elastic fibres from solar elastosis in vivo is of great interest in dermatologic research, especially for efficacy testing of antiageing products. To date, no studies on multiphoton excited fluorescence lifetime characteristics of human elastic fibres and solar elastosis are reported. The goal of the present work was the identification of differential criteria for elastic fibres and solar elastosis by the analysis of fluorescence decay curves acquired by time-correlated single photon counting in vivo multiphoton tomography. For this purpose, fluorescence lifetime measurements (FLIM) were performed with 47 volunteers of different age groups at sun-protected and sun-exposed localizations. Bi-exponential curve fitting was applied to the FLIM data, and characteristic differences between age groups and localizations were found in both relevant fit parameters describing the decay slope. The FLIM analyses have shown that dermal autofluorescence has different lifetimes depending on age and in part on localization. R. Patalay, C. Talbot, Y. Alexandrov, M.O. Lenz, S. Kumar, S. Warren, I. Munro, M. A. A. Neil, k. König, P. M. W. French, A. Chu, G. W. H. Stamp, C. Dunsby. Multiphoton Multispectral Fluorescence Lifetime Tomography for the Evaluation of Basal Cell Carcinomas Abstract We present the first detailed study using multispectral multiphoton fluorescence lifetime imaging to differentiate basal cell carcinoma cells (BCCs) from normal keratinocytes. Images were acquired from 19 freshly excised BCCs and 27 samples of normal skin (in & ex vivo). Features from fluorescence lifetime images were used to discriminate BCCs with a sensitivity/ specificity of 79%/93% respectively. A mosaic of BCC fluorescence lifetime images covering .1 mm2 is also presented, demonstrating the potential for tumour margin delineation. Using 10,462 manually segmented cells from the image data, we quantify the cellular morphology and spectroscopic differences between BCCs and normal skin for the first time. Statistically significant increases were found in the fluorescence lifetimes of cells from BCCs in all spectral channels, ranging from 19.9% (425–515 nm spectral emission) to 39.8% (620–655 nm emission). A discriminant analysis based diagnostic algorithm allowed the fraction of cells classified as malignant to be calculated for each patient. This yielded a receiver operator characteristic area under the curve for the detection of BCC of 0.83. We have used both morphological and spectroscopic parameters to discriminate BCC from normal skin, and provide a comprehensive base for how this technique could be used for BCC assessment in clinical practice. Karsten König. Hybrid multiphoton multimodal tomography of in vivo human skin IntraVital 1:1, 11–26; July/August/September 2012 Abstract Clinical multiphoton tomography based on femtosecond near infrared laser pulses for in vivo high-resolution skin imaging has been employed to thousands of volunteers and patients. Two-photon cellular autofluorescence and second harmonic generation of collagen can be detected with single-photon sensitivity and submicron spatial resolution. Also in vivo clinical CARS has been realized to image intratissue lipids and water. Novel developments focus on multimodal hybrid imaging to generate optical tissue biopsies with subcellular resolution, deep-tissue information, and chemical fingerprints. Widefield imaging tools such as dermoscopes, optical coherence tomographs as well as ultrasound and photoacoustic devices can be integrated. The hybrid tomographs have the potential to trace cosmetics and pharmaceutical components such as sunscreen nanoparticles and anti-aging products in humans. Skin cancer such as malignant melanoma and basal cell carcinoma as well as dermatitis can be detected at an early stage and the efficiency of the treatment can be monitored. These novel hybrid multimodal multiphoton tomographs may become important biopsy-free and label-free imaging tools in personalized medicine, pharmacy, biotechnology as well as cosmetic research. S. Seidenari, F. Arginelli, C. Dunsby, P. French, K. König, C. Magnoni, M. Manfredini, C. Talbot, G. Ponti. Multiphoton laser tomography and fluorescence lifetime imaging of basal cell carcinoma: morphologic features for non-invasive diagnostics Experimental Dermatology, 2012, 21, 831–836 Abstract Multiphoton laser tomography (MPT) combined with fluorescence lifetime imaging (FLIM) is a non-invasive imaging technique, which gives access to the cellular and extracellular morphology of the skin. The aim of our study was to assess the sensitivity and specificity of MPT/FLIM descriptors for basal cell carcinoma (BCC), to improve BCC diagnosis and the identification of tumor margins. In the preliminary study, FLIM images referring to 35 BCCs and 35 healthy skin samples were evaluated for the identification of morphologic descriptors characteristic of BCC. In the main study, the selected parameters were blindly evaluated on a test set comprising 63 BCCs, 63 healthy skin samples and 66 skin lesions. Moreover, FLIM values inside a region of interest were calculated on 98 healthy skin and 98 BCC samples. In the preliminary study, three epidermal descriptors and 7 BCC descriptors were identified. The specificity of the diagnostic criteria versus ‘other lesions’ was extremely high, indicating that the presence of at least one BCC descriptor makes the diagnosis of ‘other lesion’ extremely unlikely. FLIM values referring to BCC cells significantly differed from those of healthy skin. In this study, we identified morphological and numerical descriptors enabling the differentiation of BCC from other skin disorders and its distinction from healthy skin in ex vivo samples. In future, MPT/FLIM may be applied to skin lesions to provide direct clinical guidance before biopsy and histological examination and for the identification of tumor margins allowing a complete surgical removal.
2011
K. König, A. Uchugonova, E. Gorjup. Multiphoton fluorescence lifetime imaging of 3D-stem cell spheroids during differentiation. Microscopy Research and Technique 74 (2011) 9–17 / DOI 10.1002/jemt.20866 Abstract Long-term high-resolution multiphoton imaging of nonlabeled human salivary gland stem cell spheroids has been performed with submicron spatial resolution, 10.5-nm spectral resolution, and picosecond temporal resolution. In particular, the two-photon-excited coenzyme NAD(P)H and flavins have been detected by time-correlated single photon counting (TCSPC). Stem cells increased their autofluorescence lifetimes and decreased their total fluorescence intensity during the adipogenic-differentiation process. In addition, the onset of the biosynthesis of lipid vacuoles was monitored over a period of several weeks in stem-cell spheroids. Time-resolved multiphoton autofluorescence imaging microscopes may become a promising tool for marker-free stemcell characterization and cell sorting.
A. Uchugonova, J. Duong, N. Zhang, K. König, R.M. Hoffman. The bulge area is the origin of nestin-expressing pluripotent stem cells of the hair follicle. J Cell Biochem. (2011) Abstract Nestin-expressing pluripotent stem cells have been found both in the bulge area (BA) as well as the dermal papilla (DP). Nestin-expressing stem cells of both the BA and DP have been previously shown to be able to form neurons and other non-follicle cell types. The nestinexpressing stem cells from the DP have been termed skin precursor or SKP cells. Both nestinexpressing DP and BA cells have been previously shown to effect repair of the injured spinal cord and peripheral nerve, with the BA being the greater and more constant source of the stem cells. The BA contains nestin-expressing stem cells throughout the hair cycle, whereas nestinexpressing dermal papillae stem cells were found in early and mid-anagen only. Our previous studies have shown that the nestin-expressing stem cells in the BA and DP have similar morphological features. The cells from both regions have a small body diameter of approximately 7 μm with long extrusions, as shown by 2-photon confocal microscopy. In the present study, using 2-photon confocal microscopy of whisker follicles from transgenic mice expressing nestin-driven green fluorescent protein (ND-GFP), we demonstrate that the BA is the source of the nestin-expressing stem cells of the hair follicle. The nestin-expressing stem cells migrate from the BA to the DP as well as into the surrounding skin tissues including the epidermis, including during wound healing, suggesting that the BA maybe the source of the stem cells of the skin itself.
F. Liu, A. Uchugonova, H. Kimura, C. Zhang, M. Zhao, L. Zhang, K. König, J. Duong, R. Aki, N. Saito, S. Mii, Y. Amoh, K. Katsuoka, R. M. Hoffman. The bulge area is the major hair follicle source of nestin-expressing pluripotent stem cells which can repair the spinal cord compared to the dermal papilla Cell Cycle 5 (2011), 830-9 Abstract Nestin has been shown to be expressed in the hair follicle, both in the bulge area (BA) as well as the dermal papilla (DP). Nestin-expressing stem cells of both the BA and DP have been previously shown to be pluripotent and be able to form neurons and other non-follicle cell types. The nestin-expressing pluripotent stem cells from the DP have been termed skin precursor or SKP cells. The objective of the present study was to determine the major source of nestin-expressing pluripotent stem cells in the hair follicle and to compare the ability of the nestin-expressing pluripotent stem cells from the BA and DP to repair spinal cord injury. Transgenic mice in which the nestin promoter drives GFP (ND-GFP) were used in order to observe nestin expression in the BA and DP. Nestin-expressing DP cells were found in early and middle anagen. The BA had nestin expression throughout the hair cycle and to a greater extent than the DP. The cells from both regions had very long processes extending from them as shown by two-photon confocal microscopy. Nestin-expressing stem cells from both areas differentiated into neuronal cells at high frequency in vitro. Both nestin-expressing DP and BA cells differentiated into neuronal and glial cells after transplantation to the injured spinal cord and enhanced injury repair and locomotor recovery within four weeks. Nestin-expressing pluripotent stem cells from both the BA and DP have potential for spinal cord regeneration, with the BA being the greater and more constant source.
J. Köhler, K. König, M. Speicher, S. Astner, E. Stockfleth, P. Elsner, M. Kaatz. Clinical application of multiphoton tomography in combination with confocal laser scannig microscopy for evaluation of skin diseases. Skin Research and Technology. Abstract The first-ever application of high-frequency ultrasound combined with multiphoton tomography (MPT) and dermoscopy in a clinical trial is reported. 47 patients with different dermatoses such as benign and malign skin cancers, connective tissue diseases, inflammatory skin diseases, and autoimmune bullous skin diseaseshave been investigated with (i) state-of-the-art and highly sophisticated ultrasound systems for dermatology, (ii) the femtosecond laser multiphoton tomograph and (iii) dermoscopes. Dermoscopy provides two-dimensional color images of the skin surface with a magnification up to 70 . Depending on the ultrasonic frequencies from 7.5 MHz to 100 MHz, the signal depth varies from about 1 mm to 80 mm. Vertical ultrasound wide-field images provide fast information on depth and volume of the lesion. The 100 MHz ultrasound allows imaging with resolutions down to 16 mm (axial) and 32 mm (lateral). Multiphoton tomography provides 0.36 0.36 0.001 mm3 horizontal optical sections of a particular region of interest with submicron resolution down to 200 mm tissue depth. The autofluorescence of mitochondrial coenzymes, keratin, melanin, and elastin as well as the network of collagen structures can be imaged. The combination of ultrasound and MPT opens novel synergistic possibilities in diagnostics of skin diseases with a special focus on the early detection of skin cancer as well as the evaluation of treatments.
M. Koehler, S. Zimmerman, S. Springer, P. Elsner, K. König, M. Kaatz. Keratinocyte morphology of human skin evaluated by in vivo multiphoton laser tomography. Skin Research and Technology 17 (2011) / DOI: 10.1111/j.1600-0846.2011.00522.x Abstract Multiphoton tomography (MPT) is a novel noninvasive imaging method in dermatology allowing the depiction of the epidermis with sub-cellular resolution. Here, we present a descriptive characterization of unaffected human epidermis, morphometric data on human keratinocytes and some epidermal parameters in vivo and a morphological characterization of keratinocyte changes in actinic keratoses. Methods: In a clinical setting, 57 volunteers of different age groups were examined using MPT. Results: The morphological appearance of keratinocytes showed polygonal cells in the horny layer, a granular cytoplasm in the stratum granulosum, smaller prickle cells in the stratum spinosum and hyperpigmented small round basal cells. Actinic keratoses presented remarkable differences including widened inter-cellular spaces, heterogeneity in cellular fluorescence and shape as well as an increased ratio of nuclear to cellular size. Finally, the thickness of the epidermis was significantly increased in actinic keratoses compared with the control.
M. S. Roberts, Y. Dancik, T. W. Prow, C. A. Thorling, L. L. Lin, J. E. Grice, T. A. Robertson, K. König, W. Becker. Non-invasive imaging of skin physiology and percutaneous penetration using fluorescence spectral and lifetime imaging with multiphoton and confocal microscopy European Journal of Pharmaceutics and Biopharmaceutics 77 (2011) 469-488 / DOI: 10.1016/j.ejpb.2010.12.023. Abstract New multiphoton and confocal microscope technologies and fluorescence lifetime imaging techniques are now being used to non-invasively image, in space (three dimensions),in time, in spectra, in lifetime and in fluorescence anisotropy (total of 7 dimensions), fluorescent molecules in in situ and in vivo biological tissue, including skin. The process involves scanning a 2D area and measuring fluorescence at a given tissue depth below the surface after excitation by a laser beam with a wavelength within the one-photon or two-photon absorption band of the fluorophores followed by the stacking together of a series of 2D images from different depths to reconstruct the full spatial structure of the sample. Our aim in this work is to describe the principles, opportunities, limitations and applications of this new technology and its application in defining skin morphology, disease and skin penetration in vitro and in vivo by drugs, chemicals and nanoparticles. A key emphasis is in the use of fluorescence lifetime imaging to add additional specificity and quantitation to the detection of the various exogenous chemicals and nanoparticles that may be applied to the skin as well as endogenous fluorescent species in the skin. Examples given include equipment configuration; components in skin autofluorescence in various skin strata; imaging and quantification of coexisting drugs and their metabolites; skin pH; nanoparticle zinc oxide skin penetration; liposome delivery of drugs to deeper tissues; and observations in skin ageing and in various skin diseases.
H. Studier, H.G. Breunig, K. König. Comparison of broadband and ultrabroadband pulses at MHz and GHz pulse-repetition rates for nonlinear fs-laser scanning microscopy Journal of Biophotonics 4 (2011) 84–91. Abstract Nonlinear optical imaging of human skin and of polychromatic microspheres was carried out to compare and evaluate the imaging properties of three different excitation femtosecond lasers: a spectrally tunable 80 MHz Ti : sapphire oscillator that produced 100 fs pulses (spectral width 10 nm) and two ultrabroadband Ti : sapphire oscillators with repetition rates of 85 MHz and 1 GHz. The latter of these two and the 100 fs laser were combined with a laser scanning microscope (TauMap). The intensities of images of the polychromatic microsphere samples obtained with both lasers are in accordance with the usual dependence of two-photon processes on laser pulse parameters, i.e. the intensity is proportional to the square of the mean laser power and the reciprocal pulse duration. In contrast to that, skin images measured with all three different excitation sources with mean powers of each laser adjusted to the particular pulse length and repetition rate exhibited discrepancies from this relation. For characterization of the ultrabroadband GHz laser, the measurements are supplemented by spectra of second-harmonic-generation signals of urea and collagen.
K. König, H.G. Breunig, R. Bückle, M. Kellner-Höfer, M. Weinigel, E. Büttner, W. Sterry, J. Lademann Optical skin biopsies by clinical CARS and multiphoton fluorescence/SHG tomography Laser Physics Letters 8 (2011) 1-4 / DOI: 10.1002/lapl.201110014 Abstract The ultimate challenge for early diagnostics is labelfree high-resolution intratissue imaging without taking physical biopsies. A novel hybrid femtosecond laser tomograph provides in vivo optical biopsies of human skin based on non-linear excitation of autofluorescence and the detection of lipids and water by CARS. Applications include skin cancer detection, biosafety tests of intradermal nanoparticles, and the testing of anti-aging products.
2010
D. Bruneel, E. Audouard, K. König, R. Le Harzic. Flexible tool for two-photon laser nanoprocessing and large area mapping with high resolution. Optics and Lasers in Engineering In press / DOI 10.1016/j.optlaseng.2010.06.003 Abstract A high accurate, compact and flexible laser nanoprocessing and large area mapping with high-resolution apparatus is presented in this paper. Judicious hardware and software developments of the device are presented, which allow a high range of applications and performances. Coupled to a low average and low cost femtosecond laser system, results on accurate nanoprocessing on biological and biotechnological samples demonstrate the potential of the machine. Nanostructuring of different type of materials with this device is conceivable for a wide spectrum of technological applications in material science, nanobiotechnology and nanomedicine.
D. Bruneel, G. Matras, R. Le Harzic, N. Huot, K. König, E. Audouard. Micromachining of metals with ultra-short Ti-Sapphire lasers: Prediction and optimization of the processing time. Optics and Lasers in Engineering 48 (2010) 268-271 / DOI 10.1016/j.optlaseng.2009.10.010 Abstract We investigate the processing times of ultrafast laser machining in the case of metals (copper and stainless steel). At a fluence of 2.5 J/cm2, measurements of processing times are in good agreement with the calculations based on the ablation rates. We study the influence of laser repetition rates for 1, 5, 10 and 15 kHz. A linear reduction of the processing time can be expected with an increase of the repetition rate.
H. Studier, H. G. Breunig, K. König. Comparison of broadband and ultrabroadband pulses at MHz and GHz pulse-repetition rates for nonlinear femtosecond-laser scanning microscopy. Journal of Biophotonics (2010) DOI 10.1002/jbio.201000010 Abstract Nonlinear optical imaging of human skin and of polychromatic microspheres was carried out to compare and evaluate the imaging properties of three different excitation femtosecond lasers: a spectrally tunable 80 MHz Ti: sapphire oscillator that produced 100 fs pulses (spectral width 10 nm) and two ultrabroadband Ti: sapphire oscillators with repetition rates of 85 MHz and 1 GHz. The latter of these two and the 100 fs laser were combined with a laser scanning microscope (TauMap). The intensities of images of the polychromatic microsphere samples obtained with both lasers are in accordance with the usual dependence of two-photon processes on laser pulse parameters, i.e. the intensity is proportional to the square of the mean laser power and the reciprocal pulse duration. In contrast to that, skin images measured with all three different excitation sources with mean powers of each laser adjusted to the particular pulse length and repetition rate exhibited discrepancies from this relation. For characterization of the ultrabroadband GHz laser, the measurements are supplemented by spectra of second-harmonic-generation signals of urea and collagen. (© 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim) Link zum Artikel
M.J. Koehler, T. Vogel, P. Elsner, K. König, R. Bückle, M. Kaatz. In vivo measurement of the human epidermal thickness in different localizations by multiphoton laser tomography. Skin Research and Technology 16 (2010) 259-264 / DOI 10.1111/j.1600-0846.2010.00437.x Abstract Background: The in vivo measurement of epidermal thickness is still challenging. While ultrasound, optical coherence tomography and confocal laser microscopy are used with moderate success, this issue has not been addressed by multiphoton laser tomography. Objectives: In the present study, an in vivo measurement of four different morphometric epidermal parameters is performed. Methods: Thirty healthy volunteers aged 21–82 years were included in the study after informed consent and approval of the local ethics committee. At the dorsal forearm and the dorsum of the hand, the thicknesses of the total epidermis, viable epidermis and stratum corneum and the depth of the papillary dermis were calculated from depth-resolved intensity curves after correlation with multiphoton images. Results: We have shown consistently that in all age groups, the four morphometric parameters are significantly higher at the hand compared with the forearm, while there were no differences between age groups. This is consistent with most previous findings. Conclusion: The method presented here provides a novel in vivo investigation tool for the measurement of epidermal morphometric parameters that may be useful for the observation of epidermal changes over time in skin disorders, therapy side effects or in cosmetic science. Link zum Artikel
R. Bazin, F. Flament, A. Colonna, R. Le Harzic, R. Bückle, B. Piot, F. Laizé, M. Kaatz, K. König, J. W. Fluhr: Clinical study on the effects of a cosmetic product on dermal extracellular matrix components using a high-resolution multiphoton tomograph. Skin Research and Technology 16 (2010) 305-310 / DOI 10.1111/j.1600-0846.2010.00432.x Abstract: Background/purpose: The aim of this study was to demonstrate the effects of selected plant extracts in a cosmetic cream on the dermal network components after a 3-month treatment using an in vivo multiphoton tomographic device. Methods: Twenty-four Caucasian women aged between 45 and 65 applied randomly a cosmetic emulsion B containing active ingredients (soy and jasmine) twice a day on one arm and its vehicle A (without active ingredients) on the other arm during 3 months. Measurements were performed on the internal side of the forearm before starting the treatment (T0), after 4 week (T4) and 12 weeks (T12) treatment. Measurements consisted in a multi-layers acquisitions using a multiphoton tomograph with subcellular resolution. Optical sections (about 6 μm thick) were recorded from 0 to about 200 μm using two different wavelengths: 760 and 820 nm. To compare the series of images and obtain an objective quantification of the signal of second harmonic generation (SHG) and autofluorescence, the method used consisted in taking the integrated brightness of an image (same rectangular area for all images) as a measure of the signal. Following this step a ratio between brightness of images from the area treated with cream A or B and brightness of untreated area was calculated and used as an assessment of treatment efficacy. The parameter used for statistical analysis (variance analysis) is the difference before and after 12 weeks of treatment by either cream A or B of the signal ratios calculated in the upper dermis (118–130 μm) and those from a deeper region of the upper dermis (165–178 μm). Results: Signals (autofluorescence+SHG) of extracellular matrix do not change significantly with time (weeks 0, 4 and 12) when cream A (vehicle with no active ingredient) is applied. Treatment with cream B results in an enhancement in the signal level of extracellular matrix at week 12. The comparison of signals, in both areas (118–130 μm and 160–178 μm), show an higher increase in the deeper region than in the more superficial one for product B while we do not notice any change with product A. Conclusion: The multiphoton tomograph provided excellent high-resolution images, which describe clearly the different skin layers, single cells and extracellular matrix components in all the 24 volunteers. Statistic analyses reveal a real effect for product B with selected plant extracts, known to increase collagen synthesis. Changes observed are characteristics of modifications in dermal collagen and elastin content. To our knowledge, it is the first time that it was possible to demonstrate in vivo the effect of a cosmetic product on the superficial dermal layer, in a non-invasive and non-destructive process, i.e. without cutting the skin. Link zum Artikel
M. Kaatz, A. Sturm, P. Elsner, K. König, R. Bückle, M.J. Koehler. Depth-resolved measurement of the dermal matrix composition by multiphoton laser tomography. Skin Research and Technology 16 (2010) 131-136 / DOI 10.1111/j.1600-0846.2009.00423.x Abstract Background: In the last years, multiphoton laser tomography (MLT) has emerged as a promising tool for non-invasive diagnostics in dermatology and other medical specialties. The present work is dedicated to the question to what degree the measurement depth and the thickness of the epidermis influence the evaluation of dermal matrix composition and if recommendations for future measurement procedures can be given. Methods: In a study group of 30 healthy volunteers aged 21–82 years multiphoton depth-resolved measurements of autofluorescence and second harmonics have been performed in order to evaluate the dermal matrix composition. Results: Characteristic intensity curves depending on the penetration depth were derived and differences between age groups were found. Conclusion: With the present work we provide evidence for the accuracy of the measurement of dermal matrix composition by MLT and give detailed advice for the measurement procedure. Furthermore, we propose the use of depth-dependent emission intensity curves for monitoring of anti-aging treatment. Link zum Artikel
M. Kaatz, K. König. Multiphotonenmikroskopie und In-vivo-Multiphotonentomographie in der dermatologischen Bildgebung. Der Hautarzt 61(2010)397-409 / DOI 10.1007/s00105-009-1880-4 Abstract Multiphoton microscopy (MPM) and in vivo multiphoton tomography (MPT) are non-invasive examination techniques that allow for the evaluation of cellular as well as extra-cellular structures by working at a subcellular resolution level. These techniques are thus appropriate not only for clinical diagnostics but also for scientific issues in basic and applied research. MPM and MPT are based on the stimulation of biogenic fluorophores by two or more long-wave, low-energy photons and the evocation of second harmonic generation (SHG). Thus, the evaluation quality of cell clusters and tissues is similar to histological sections. At the same time the dermal fiber network can be assessed. MPT was developed further for the application in non-invasive in vivo diagnostics of skin diseases. This review presents the capabilities of multiphoton-based diagnostics in the evaluation of transcutaneous metabolism. In addition, the multiphoton techniques employed for the evaluation of physiologic and pathologic changes of the dermal fiber network as well as in the diagnosis of dermal and epidermal disorders by visual biopsy. Besides the morphological classification of benign and malignant skin tumors or allergic or inflammatory skin lesions, the techniques also allow for recording metabolic processes. Link zum Artikel
H. G. Breunig, H. Studier, K. König. Multiphoton excitation characteristics of cellular fluorophores of human skin in vivo. Optics Express 18(2010)7857-7871 / DOI 10.1364/OE.18.007857 Abstract In vivo multiphoton tomography with a wavelength-tunable femtosecond laser has been performed to investigate the autofluorescence intensity of major endogenous fluorophores of human skin in dependence on the excitation wavelength. In high-resolution multiphoton images of different skin layers, clear trends were found for fluorophores like keratin, NAD(P)H, melanin as well as for the elastin and collagen networks. The analysis of the measurements is supplemented by additional measurements of fluorescence lifetime imaging and signal-decay curves by time-correlated single-photon counting. © 2010 OSA Link zum Artikel
K. König, A. Uchugonova, E. Gorjup. Multiphoton fluorescence lifetime imaging of 3D-stem cell spheroids during differentiation. Microscopy Research and Technique DOI 10.1002/jemt.20866 Abstract Long-term high-resolution multiphoton imaging of nonlabeled human salivary gland stem cell spheroids has been performed with submicron spatial resolution, 10.5-nm spectral resolution, and picosecond temporal resolution. In particular, the two-photon-excited coenzyme NAD(P)H and flavins have been detected by time-correlated single photon counting (TCSPC). Stem cells increased their autofluorescence lifetimes and decreased their total fluorescence intensity during the adipogenic-differentiation process. In addition, the onset of the biosynthesis of lipid vacuoles was monitored over a period of several weeks in stem-cell spheroids. Time-resolved multiphoton autofluorescence imaging microscopes may become a promising tool for marker-free stem-cell characterization and cell sorting. Microsc. Res. Tech., 2010. © 2010 Wiley-Liss, Inc. Link zum Artikel
2009
Z. Földes-Papp, K. König, H. Studier, R. Bückle, H.G. Breunig, A. Uchugonova, G.M. Kostner. Trafficking of mature miRNA-122 into the nucleus of live liver cells. Current Pharmaceutical Biotechnology 10(2009)569-578 / DOI 10.2174/138920109789069332 Abstract The binding of superquencher molecular beacon (SQMB) probes to human single-stranded cellular miRNA- 122 targets was detected in various single live cells with femtosecond laser microscopy. For delivery of the SQMBprobes, 3D-nanoprocessing of single cells with sub-15 femtosecond 85 MHz near-infrared laser pulses was applied. Transient nanopores were formed by focusing the laser beam for some milliseconds on the membrane of a single cell in order to import of SQMB-probes into the cells. In single cells of the human liver cell lines Huh-7D12 and IHH that expressed miRNA-122, we measured target binding in the cytoplasm by two-photon fluorescence imaging. We found increased fluorescence with time in a nonlinear manner up to the point where steady state saturation was reached. We also studied the intracellular distribution of target SQMB and provide for the first time strong experimental evidence that cytoplasmic miRNA travels into the cell nucleus. To interpret nonlinear binding, a number of individual miRNA-122 positive cells (Huh-7D12 and IHH) and negative control cells, human VA13 fibroblasts and Caco-2 cells were analyzed. Our experimental data are consistent with the cytoplasmic assembly of nuclear miRNA and provide further mechanistic insight in the regulatory function of miRNAs in cellular physiology. An open issue in the regulation of gene expression by miRNA is whether miRNA can activate gene expression in addition to the well-known inhibitory effect. A first step for such a regulatory role could be the travelling of miRNA-RISC into the nucleus. Link zum Artikel
K. König, M. Speicher, R. Bückle, J. Reckfort, G. McKenzie, J. Welzel, M. J. Koehler, P. Elsner, M. Kaatz. Clinical optical coherence tomography combined with multiphoton tomography of patients with skin diseases. Journal of Biophotonics 2(2009)389-397 / DOI 10.1002/jbio.200910013 Abstract We report on the first clinical study based on optical coherence tomography (OCT) in combination with multiphoton tomography (MPT) and dermoscopy. 47 patients with a variety of skin diseases and disorders such as skin cancer, psoriasis, hemangioma, connective tissue diseases, pigmented lesions, and autoimmune bullous skin diseases have been investigated with (i) state-of-the-art OCT systems for dermatology including multibeam swept source OCT, (ii) the femtosecond laser multiphoton tomograph, and (iii) dermoscopes. Dermoscopy provides two-dimensional color images of the skin surface. OCT images reflect modifications of the intratissue refractive index whereas MPT is based on nonlinear excitation of endogenous fluorophores and second harmonic generation. A stack of cross-sectional OCT wide field images with a typical field of view of 5 × 2 mm2 gave fast information on the depth and the volume of the lesion. Multiphoton tomography provided 0.36 × 0.36 mm2 horizontal/diagonal optical sections within seconds of a particular region of interest with superior submicron resolution down to a tissue depth of 200 m. The combination of OCT and MPT provides a unique powerful optical imaging modality for early detection of skin cancer and other skin diseases as well as for the evaluation of the efficiency of treatments. (© 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim) Link zum Artikel
M. J. Köhler, A Preller, P. Elsner, N. Kindler, K. König, Karsten, R. Bückle, M. Kaatz. Intrinsic, solar and sunbed-induced skin aging measured in vivo by multiphoton laser tomography and biophysical methods. Skin Research and Technology 15(2009)357-363 / DOI 10.1111/j.1600-0846.2009.00372.x Abstract Background: Skin aging is accelerated by extrinsic factors, particularly actinic damage. Over the last decades, both clinical and pathological differences between intrinsic and actinic aging have been characterized. In this work, we aimed at quantifying skin aging by non-invasive in vivo methods. Methods: Young healthy volunteers using indoor tanning facilities and aged people were compared with appropriate controls by measurements of skin elasticity with the Cutometer and the Reviscometer and by semi-quantitative evaluation of the dermal matrix composition by the multiphoton laser tomograph DermaInspect. Results: We found differences between the sun-protected volar forearm and the dorsal side as well as between young and old test persons with all three methods. No significant differences were found between the skin of indoor-tanned test persons and control. Also, gender had no influence on the severity of skin aging. Conclusion: The most consistent results were obtained with the DermaInspect. The considerable inter-individual variation due to the cross-sectional design of the study may have disguised the factual skin damage caused by tanning beds. Link zum Artikel
E. Dimitrow, M. Ziemer, M.J. Koehler, J. Norgauer, K. König, P. Elsner, M. Kaatz. Sensitivity and specificity of multiphoton laser tomography for in vivo and ex vivo diagnosis of malignant melanoma. Journal of Investigative Dermatology 129(2009)1752-1758 / DOI 10.1038/jid.2008.439 Abstract The incidence of malignant melanoma has shown a dramatic increase over the past three decades. Patient outcome and curability depend on early diagnosis. In vivo multiphoton laser tomography represents a recently developed diagnostic tool that allows non-invasive tissue imaging. We aim to demonstrate the application of multiphoton laser tomography for the in vivo differentiation and diagnosis of melanoma. Laser radiation in the near infrared spectrum was used to image endogenous fluorophores by multiphoton excitation. Eighty-three melanocytic skin lesions have been investigated. The results showed distinct morphological differences in melanoma compared with melanocytic nevi. In particular, six characteristic features of malignant melanoma were specified and statistically evaluated. Sensitivity values up to 95% (range: 71–95%) and specificity values up to 97% (range: 69–97%) were achieved for diagnostic classification. Logistic regression analysis was performed to identify the most significant diagnostic criteria. We found that architectural disarray of the epidermis, poorly defined keratinocyte cell borders as well as the presence of pleomorphic or dendritic cells were of prime importance. By means of this procedure accuracy values up to 97% were reached. These findings underline the potential applicability of multiphoton laser tomography in melanoma diagnosis of melanocytic skin lesions. Link zum Artikel
E. Dimitrow, I. Riemann, A. Ehlers, M.J. Köhler, J. Norgauer, P. Elsner, K. König, M. Kaatz. Spectral fluorescence lifetime detection and selective melanin imaging by multiphoton laser tomography for melanoma diagnosis. Experimental Dermatology 18(2009)509 - 515 / DOI 10.1111/j.1600-0625.2008.00815.x Abstract Abstract: Multiphoton excited tissue fluorescence summarises the emission of all naturally occurring endogenous fluorescent bio-molecules with their often overlapping fluorescence spectra. Common fluorescence intensity measurements could not be utilised to distinguish between different fluorophores or metabolic states. To overcome this limitation, we investigated new procedures of selective melanin imaging and spectral fluorescence lifetime imaging in combination with high resolution multiphoton laser tomography. Overall 46 melanocytic lesions of human skin were analysed. We suggested that fluorescence light, detected in such a way, may yield additional information for melanoma diagnostics. Remarkable differences in lifetime behaviour of keratinocytes in contrast to melanocytes were observed. Fluorescence lifetime distribution was found in correlation with the intracellular amount of melanin. Spectral analysis of melanoma revealed a main fluorescence peak around 470 nm in combination with an additional peak close to 550 nm throughout all epidermal layers. Excitation at 800 nm shows a selectively observable fluorescence of melanin containing cells and offers the possibility of cell classification. Procedures of selective imaging as well as spectral fluorescence lifetime imaging by means of multiphoton laser tomography support diagnostic decisions and may improve the process of non-invasive early detection of melanoma. Link zum Artikel
R. Le Harzic, I. Riemann, M. Weinigel, K. König, B. Messerschmidt. Rigid and high-numerical-aperture two-photon fluorescence endoscope. Applied Optics 48(2009)3396-3400 / DOI 10.1364/AO.48.003396 Abstract We present a rigid miniaturized optical system block fiber-optic two-photon endoscope based on a compact two-axis piezo scanner system and a miniature high (0.65) NA GRIN lens objective. The optical system is scanned as a whole by a piezo scanner allowing always an on-axis beam irradiation of the optical system. A photonic crystal fiber is used for excitation and ultrashort laser pulses can be delivered with typical power up to 100 mW at 800 nm. Two-photon fluorescence signal is collected by the use of a multimode fiber. Lateral resolution values for the system were experimentally measured to be 0.67 μm vertically and 1.08 μm horizontally. Axial resolution was found to be 5.8 μm. The endoscope is highly flexible and controllable in terms of time acquisition, resolution, and magnification. Fluorescence images were acquired over a 420 μm×420 μm field of view. Results presented here demonstrate the ability of the system to resolve subcellular details and the potential of the technology for in vivo applications. © 2009 Optical Society of America Link zum Artikel
R. Le Harzic, K. König, C. Wüllner, K. Vogler, C. Donitzky. Ultraviolet femtosecond laser creation of corneal flap. Journal of Refractive Surgery 25(2009) Abstract Purpose: To study parameters for ocular femtosecond laser surgery in terms of process efficiency and safety aspects using ultraviolet (UV) femtosecond laser pulses. Methods: Studies on corneal surgery and flap processing on enucleated porcine eyes were performed using a newly developed ytterbium-doped gain media laser source. Ultraviolet femtosecond laser pulses centered at a wavelength of 345 nm and working at a repetition rate of 100 kHz were generated by the third harmonics of the 1035-nm fundamental wavelength. Results: Flaps with a diameter of 6 mm and a thickness of 100 µm were created in less than 2 minutes with low energy pulses. Transmissions and spectral measurements were performed during flap processing. Less than 2% UV radiation reaches the retina during corneal flap processing. A detectable transmittance towards the retina of visible light centered on 440 nm was found for UV pulses. Conclusions: Ultraviolet corneal refractive surgery is a novel procedure and has the potential to be an alternative to infrared refractive surgery considering safety aspects. [J Refract Surg. 2009;25:383-389.] Link zum Artikel
2008
B.G. Wang, P.C. Lohmann, I. Riemann, H. Schubert, K.J. Halbhuber, K. König. Multiphoton-mediated Corneal Flap Generation Using the 80 MHz Nanojoule Femtosecond Near-Infrared Laser. Journal of Refractive Surgery 4(2008) Abstract Purpose: To evaluate whether corneal flaps can be generated by the 80 MHz near-infrared, intense nanojoule femtosecond laser based on multiphoton absorption. Methods: A solid-state Ti:Sapphire femtosecond laser system was integrated in an inverted JenLab Femt-O-Cut laser scanning microscope. A diffraction-limited 40x objective was used to induce multiphoton ionization and plasma production. New Zealand albino rabbits and porcine eyes were used. Surgical outcomes were determined using frame and line scans with nanojoule pulses at a wavelength of 800 nm. Results: Surgical performance was assessed by optical imaging, histology, and electron microscopy. No significant corneal turbidity was observed. Optical imaging and histological examinations detected virtually no perturbation in the surrounding tissue. Corneal flaps and stromal lenticules were generated. Wound repair of the unlifted flaps was observed up to 90 days postoperatively. Conclusions: Surgical results and follow-up studies confirm that this femtosecond laser at nanojoule pulse energy is able to generate corneal flaps precisely, without causing visible collateral damage to the surrounding tissue or overlying epithelium. Link zum Artikel
M.J. Koehler, S. Hahn, A. Preller, P. Elsner, M. Ziemer, A. Bauer, K König, R. Bückle, J.W. Fluhr, M. Kaatz. Morphological skin ageing criteria by multiphoton laser scanning tomography : non-invasive in vivo scoring of the dermal fibre network. Experimental Dermatology 17(2008)519-523 / DOI 10.1111/j.1600-0625.2007.00669.x Abstract Background: Morphological changes in the dermal collagen and elastin fibre network are characteristic for skin ageing and for pathological skin conditions of the dermis. Objectives: To characterize pathological and physiological conditions by multiphoton laser scanning tomography (MLT) in vivo, it is necessary to investigate and identify morphological alterations related to ageing. Methods: In vivo MLT was used to image two-photon excited autofluorescence (AF) and second harmonics generation (SHG) in human dermis of 18 volunteers of different ages. Criteria for the evaluation of age-dependent morphological changes in MLT images were fibre tension and morphology, network pattern, clot formation and image homogeneity. These criteria were weighted and a score was calculated. Results: The resulting MLT-based Dermis Morphology Score is correlated with age (R2 = −0.90) and with the previously published SHG to AF Ageing Index of Dermis (R2 = 0.66). The two groups of young (age 21–38) and old (age 66–84) volunteers showed a significant difference in MLT score values (P < 0.001). Conclusions: We could demonstrate an in vivo relationship between morphological characteristics of human dermis assessed by MLT and age. The present score allows the semi-quantitative evaluation of specific morphological changes of the dermal fibre network in ageing skin by in vivo AF and SHG imaging. This method will be useful for diagnostics of pathological conditions and their differentiation from ageing effects. Link zum Artikel
E. Dimitrow, I. Riemann, A. Ehlers, M.J. Köhler, J. Norgauer, P. Elsner, K. König, M. Kaatz. Spectral fluorescence lifetime detection and selective melanin imaging by multiphoton laser tomography for melanoma diagnosis. Experimental Dermatology 18(2008)509-515 / DOI 10.1111/j.1600-0625.2008.00815.x Abstract Multiphoton excited tissue fluorescence summarises the emission of all naturally occurring endogenous fluorescent bio-molecules with their often overlapping fluorescence spectra. Common fluorescence intensity measurements could not be utilised to distinguish between different fluorophores or metabolic states. To overcome this limitation, we investigated new procedures of selective melanin imaging and spectral fluorescence lifetime imaging in combination with high resolution multiphoton laser tomography. Overall 46 melanocytic lesions of human skin were analysed. We suggested that fluorescence light, detected in such a way, may yield additional information for melanoma diagnostics. Remarkable differences in lifetime behaviour of keratinocytes in contrast to melanocytes were observed. Fluorescence lifetime distribution was found in correlation with the intracellular amount of melanin. Spectral analysis of melanoma revealed a main fluorescence peak around 470 nm in combination with an additional peak close to 550 nm throughout all epidermal layers. Excitation at 800 nm shows a selectively observable fluorescence of melanin containing cells and offers the possibility of cell classification. Procedures of selective imaging as well as spectral fluorescence lifetime imaging by means of multiphoton laser tomography support diagnostic decisions and may improve the process of non-invasive early detection of melanoma. Link zum Artikel
A. Uchugonova, A. Isemann, E. Gorjup, G. Tempea, R. Bückle, W. Watanabe, K. König. Optical knock out of stem cells with extremely ultrashort femtosecond laser pulses. Journal of Biophotonics 1(2008)463-469 / DOI 10.1002/jbio.200810047 Abstract Novel ultracompact multiphoton sub-20 femtosecond near infrared 85 MHz laser scanning microscopes and conventional 250 fs laser microscopes have been used to perform high spatial resolution two-photon imaging of stem cell clusters as well as selective intracellular nanoprocessing and knock out of living single stem cells within an 3D microenvironment without any collateral damage. Also lethal cell exposure of large parts of cell clusters was successfully probed while maintaining single cells of interest alive. The mean power could be kept in the milliwatt range for 3D nanoprocessing and even in the microwatt range for two-photon imaging. Ultracompact low power sub-20 fs laser systems may become interesting tools for optical nanobiotechnology such as optical cleaning of stem cell clusters as well as optical transfection. (© 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim) Link zum Artikel
K. König, M. Weinigel, D. Hoppert, R. Bückle, H. Schubert, M.J. Köhler, M. Kaatz, P. Elsner. Multiphoton tissue imaging using high-NA microendoscopes and flexible scan heads for clinical studies and small animal research. Journal of Biophotonics 1(2008)506-513 / DOI 10.1002/jbio.200810049 Abstract Multiphoton tomographs based on femtosecond laser and GRIN lens technology in combination with flexible scan heads have been developed for clinical high-resolution tissue imaging and small animal research. The novel tissue tomograph possesses a 0.5 m long flexible mirror arm in combination with piezodriven focusing optics and multiple single photon counting PMT detectors. The photodetectors are in particular useful to obtain information on the extracellular matrix by the simultaneous measurement of the two-photon autofluorescence of elastin as well as the second harmonic generation of collagen. A major application is the in vivo determination of the skin age index. The rigid two-photon microendoscope with a high numerical aperture of 0.8 is based on a combination of silver-doped 1.0 mm rod-shaped GRIN lens with a hemispheric front optics. It was used in combination with the multiphoton tomograph for clinical studies as well as for inner organ and eye imaging of small animals. (© 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim) Link zum Artikel
K. König. Clinical multiphoton tomography. Journal of Biophotonics 1(2008)13-23 / DOI 10.1002/jbio.200710022 Abstract Clinical multiphoton tomography and two-photon microendoscopy provide clinicians and researchers with high-resolution in vivo optical biopsies based on two-photon autofluorescence, second harmonic generation, and fluorescence lifetime imaging. This review reflects state of the art technology and reports on applications in the fields of early stage melanoma detection, skin aging, nanoparticle imaging, tissue engineering, and in situ screening of pharmaceutical and cosmetical products. So far, more than 500 patients and volunteers in Europe, Asia, and Australia have been investigated with these novel molecular imaging tools. (© 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim) Link zum Artikel
R. Le Harzic, M. Weinigel, I. Riemann, K. König, B. Messerschmidt. Nonlinear optical endoscope based on a compact two axes piezo scanner and a miniature objective lens. Optics Express 16(2008)20588-20596 / DOI 10.1364/OE.16.020588 Abstract We report on a nonlinear optical endoscope that adopts a hollow core photonic crystal fiber for single-mode illumination delivery and a multimode one for signal collection. Femtosecond laser pulses up to 100 mW can be delivered at a centered wavelength of 800 nm. The two-photon fluorescence response of our system is shown to have axial and lateral resolutions of 5.8um and 0.6um respectively. Fluorescence detection was obtained at different wavelengths between 790 and 840 nm which could allow SHG detection for example. The maximal field-of-view of the acquired images is 420 µm x 420 µm. Detection efficiency is greater by using an avalanche photodiode in comparison to a photo multiplier tube. Results presented here demonstrate the ability of the system to resolve cellular details and the potential of the device for future in vivo imaging diagnosis © 2008 Optical Society of America Link zum Artikel
S.G. Toropygin, M. Krause, I. Riemann, B. Seitz, P. Mestres-Ventura, K.W. Ruprecht, K. König. In vitro femtosecond laser-assisted nanosurgery of porcine posterior capsule. Journal of Cataract & Refractive Surgery 34(2008)2128-2132 DOI 10.1016/j.jcrs.2008.08.038 Abstract Purpose: To investigate femtosecond laser–assisted nanosurgery of the posterior capsule in a prospective in vitro animal study. Setting: Department of Ophthalmology and Eye Hospital, University of Saarland, Homburg, and Fraunhofer Institute for Biomedical Engineering, St. Ingbert, Germany. Methods: The posterior capsules of 12 porcine eyes were irradiated with a nonamplified 90 MHz near-infrared 750 nm titanium:sapphire femtosecond laser. Intratissue and superficial laser cuts of laser-ablated (5 capsules) and control (1 capsule) specimens were examined by femtosecond multiphoton laser scanning microscopy (MLSM) and transmission electron microscopy (TEM). Results: Laser exposure time and pulse power determined the width of the lesions, which ranged from 0.69 mm G 0.19 (SD) to 2.81 G 0.5 mm. Both MLSM and TEM revealed minimal collateral alterations in the tissue surrounding the laser cuts. Conclusions: Nonamplified near-infrared femtosecond laser pulses at low pulse energies may be a promising strategy for precise lamellar noncontact nanosurgery of the posterior capsule, with minimal structural collateral damage to surrounding tissue. High-resolution MLSM offers 3-dimensional, noninvasive, nondestructive imaging at submicrometer resolution within seconds before and after ablation. Link zum Artikel
S.G. Toropygin, M. Krause, I. Riemann, M. Hild, P. Mestres-Ventura, B. Seitz, E. Khurieva, K.W. Ruprecht, U. Löw, Z. Gatzioufas, K. König. In Vitro Noncontact Intravascular Femtosecond Laser Surgery in Models of Branch Retinal Vein. Current Eye Research 33(2008)277-283 / DOI 10.1080/02713680701875299 Abstract Purpose: To investigate intravenous femtosecond laser surgery in models of branch retinal vein occlusion. Materials and Methods: Non-amplified near infrared femtosecond laser was used to ablate polyamide sutures and human hairs inserted into the vascular lumina of porcine retinal veins in vitro. Specimens were subjected to multiphoton laser scanning microscopy and electron microscopy. Results: Regular laser cuts within sutures and hairs were detected with laser microscopy and electron microscopy. Neither laser microscopy nor histology revealed collateral damage of the vascular wall. Conclusions: Non-amplified femtosecond lasers may allow precise atraumatic non-contact intravenous retinal surgery controlled by high-resolution imaging of the target. Link zum Artikel
M. Hild, M. Krause, I. Riemann, P. Mestres-Ventura, S.G. Toropygin, U. Löw, K. Brückner, B. Seitz, C. Jonescu-Cuypers, K. König. Femtosecond laser-assisted retinal imaging and ablation : experimental pilot study. Current Eye Research 33(2008)351-363 / DOI 10.1080/02713680801956452 Abstract Purpose: To investigate retinal imaging and ablation using femtosecond laser pulses. Materials and Methods: Two non-amplified near-infrared femtosecond lasers were used to irradiate porcine retinal specimens in vitro. The lasers were used for tissue removal as well as multiphoton laser scanning microscopy. Results: Ablation of the nerve fiber layer was performed at pulse energies of 1.0 nJ to 3.9 nJ. Control laser scanning images were acquired within seconds after irradiation. Specimens were additionally investigated with electron microscopy. Conclusions: Non-amplified femtosecond lasers may allow precise surgery controlled by fast high-resolution imaging of the target. Link zum Artikel
R. Le Harzic, M. Stark, P. Becker, E. Lai, D. Bruneel, F. Bauerfeld, D. Sauer, T. Velten, K. König. Nanostructuring with nanojoule femtosecond laser pulses. Journal of Laser Micro/Nanoengineering 3(2008)106-113 Abstract Sub-wavelength multiphoton nanoprocessing of silicon wafers, direct nanowriting of metals, boro-silicate glass or polymers, laser processing for nanofluidics applications as well as 3D maskless li-thography by two-photon polymerization have been performed using several compact near infrared MHz femtosecond lasers as tuneable turn-key, one-box Chameleon (coherent) and Mai-Tai (spectra physics) oscillators as well as a special femtoTrain system (High Q laser production GmbH). The lasers were coupled with the scanning microscopes FemtOcut (JenLab GmbH) and a modified ZEISS LSM510-NLO system. MHz femtosecond laser pulses with nanojoule photon energies can be considered as novel tools for nanoprocessing in material science, nanobiotechnology and nanomedicine. Link zum Artikel
A. Uchugonova, K. König. Two-photon autofluorescence and second-harmonic imaging of adult stem cells. Journal of Biomedical Optics 13(2008)054068 / DOI 10.1117/1.3002370 Abstract Human and animal stem cells (rat and human adult pancreatic stem cells, salivary gland stem cells, and human dental pulp stem cells) are investigated by femtosecond laser 5-D two-photon microscopy. Autofluorescence and second-harmonic generation (SHG) are imaged with submicron spatial resolution, 270 ps temporal resolution, and 10 nm spectral resolution. In particular, the reduced coenzyme nicotinamide adenine (phosphorylated) dinucleotide [NAD(P)H] and flavoprotein fluorescence is detected in stem cell monolayers and stem cell spheroids. Major emission peaks at 460 and 530 nm with typical long fluorescence lifetimes (2) of 1.8 and 2.0 ns, respectively, are measured using spectral imaging and time-correlated single photon counting. Differentiated stem cells produce the extra cellular matrix (ECM) protein collagen, detected by SHG signals at 435 nm. Multiphoton microscopes may become novel noninvasive tools for marker-free optical stem cell characterization and for on-line monitoring of differentiation within a 3-D microenvironment. ©2008 Society of Photo-Optical Instrumentation Engineers Link zum Artikel
A. Uchugonova, K. König, R. Bückle, A. Isemann, G. Tempea. Targeted transfection of stem cells with sub-20 femtosecond laser pulses. Optics Express 16(2008)9357-9364 / DOI 10.1364/OE.16.009357 Abstract
F. Garbe, U. Bauerschäfer, A. Csaki, A. Steinbrück, K. Ritter, A. Bochmann, J. Bergmann, A. Weise, D. Akimov, G. Maubach, K. König, G. Hüttmann, W. Paa, J. Popp, W. Fritzsche. Optically controlled thermal management on the nanometer length scale. Nanotechnology 19(2008) 055207 / DOI 10.1088/0957-4484/19/05/055207 Abstract

eine Liste aller Publikationen finden sie hier.

Originalarbeiten

2018

A. Batista, HG Breunig, A. König, A. Schindele, T. Hager, B. Seitz, AM Morgado, K. König.

Assessment of human corneas prior to transplantation using high-resolution two-photon imaging.

Invest Ophthalmol Vis Sci. 59 (2018) 176-184. Doi:10.1167/iovs.17-22002.

Abstract

The purpose of this study was to evaluate the feasibility of using two-photon imaging (TPI) to assess the condition of human corneas for transplantation. Human corneas were imaged after different storage times: short-term (STS), mediumterm (MTS), and long-term (LTS) storage. A high-resolution, custom-built 5-dimensional multiphoton microscope with 12-fs pulsed laser excitation was used for image acquisition.

S. Springer, M. Zieger, UC hippler, K. König, J. Lademann, M. Kaatz, MJ Köhler.

Non-invasive evaluation of human mucosal structures by multiphoton laser scanning tomography in vitro.

Skin Res. Tech 1-5 (2018). Doi: 10.1111/srt.12451.

Abstract

Background: Mucous membranes may be affected by various diseases and the diagnostic

accessibility is limited. Multiphoton laser tomography (MPT) is a useful tool for in vivo evaluation of superficial skin structures and might also be useful for the imaging of mucosa.

Objectives: In order to investigate the suitability of MPT for the evaluation of mucous membranes, tissue samples of different donors and anatomical localizations have been imaged.

Methods: Human mucosa samples from the urinary bladder, palatine tonsil and ocular conjunctiva were investigated by MPT and subsequently compared with conventional histology.

A. Batista, HG Breunig, A. König, A. Schindele, T. Hager, B. Seitz, K. König.

High-resolution, label-free two-photon imaging of diseased human corneas.

J Biomed Opt 23 (3), 036002 (2018). Doi: 10.1117/1JBO.23.3.036002.

Abstract

The diagnosis of corneal diseases may be improved by monitoring the metabolism of cells and the structural organization of the stroma using two-photon imaging (TPI). We used TPI to assess the differences between nonpathological (NP) human corneas and corneas diagnosed with either keratoconus, Acanthamoeba keratitis, or stromal corneal scars. Images were acquired using a custom-built five-dimensional laser-scanning microscope with a broadband sub-15 femtosecond near-infrared pulsed excitation laser and a 16-channel photomultiplier tube detector in combination with a time-correlated single photon counting module. Morphological alterations of epithelial cells were observed for all pathologies. Moreover, diseased corneas showed alterations to the cells’ metabolism that were revealed using the NAD(P)H free to protein-bound ratios. The mean autofluorescence lifetime of the stroma and the organization of the collagen fibers were also significantly altered due to the pathologies. We demonstrate that TPI can be used to distinguish between NP and diseased human corneas, based not only on alterations of the cells’ morphology, which can also be evaluated using current clinical devices, but on additional morphological and functional features such as the organization of the stroma and the cells’ metabolism. Therefore, TPI could become an efficient tool for diagnosing corneal diseases and better understanding the biological processes of the diseases.

2017

K. Reinhardt, H.G. Breunig, K. König.

Autofluorescence lifetime variation in the cutucle of the bedbug Cimex lectularius.

Arthropod Strucure & Development 46 (2017) 56-62.

Abstract

The decay time of the fluorescence of excited molecules, called fluorescence lifetime, can provide information about the cuticle composition additionally to widely used spectral characteristics. We compared autofluorescence lifetimes of different cuticle regions in the copulatory organ of females of the bedbug, Cimex lectularius. After two-photon excitation at 720 nm, regions recently characterised as being rich in resilin showed a longer bimodal distribution of the mean autofluorescence lifetime tm (tau-m) at 0.4 ns and 1.0e1.5 ns, while resilin-poor sites exhibited a unimodal pattern with a peak around 0.8 ns. The mean lifetime, and particularly its second component, can be useful to distinguish resilin-rich from resilin-poor parts of the cuticle. The few existing literature data suggest that chitin is unlikely responsible for the main autofluorescent component observed in the resilin-poor areas in our study and that melanin requires further scrutiny. Autofluorescence lifetime measurements can help to characterise properties of the arthropod cuticle, especially when coupled with multiphoton excitation to allow for deeper tissue penetration.

M. Balu, G. Lentsch, D.Z. Korta, K. König, K.M. Kelly, B.J. Tromberg, C.B. Zachary.

In vivo multiphoton-microscopy of picosecond-laser-induced optical breakdown in human skin.

Lasers in Surgery and Medicine (2017). Doi:10.1002/lsm.22655.

Abstract

The purpose of this study was to demonstrate the capability of multiphoton microscopy (MPM), a highresolution, label-free imaging technique, to characterize in vivo the skin response to a fractionated non-ablative picosecond-laser treatment.

2016

M. Klemp, M. Meinke, M. Weinigel, HJ. Roewert-Huber, K. König, M. Ulrich, J. Lademann, M. Darvin.

Comparison of morphologic criteria for actinic keratosis and squamous cell carcinoma using in vivo multiphoton tomography.

Exp Dermatol 25 (2016) 218-222. doi: 10.1111/exd.12912.

Abstract

The routine diagnostic procedure of actinic keratosis (AK) and invasive squamous cell carcinoma (SCC) is a histological examination after taking a biopsy. In the past decades, non-invasive optical methods for skin examination have been developed. Patients with clinical diagnosis of AK or SCC were examined. The morphological criteria were determined for healthy, AK and SCC skin and compared for statistically significant differences. In this study, the applicability of multiphoton tomography (MPT) as an in vivo diagnostic tool for AK and SCC was evaluated. Changes in the morphology of the keratinocytes such as broadened epidermis, large intercellular spaces, enlarged nucleus and a large variance in cell shape could easily be recognized. The cell nuclei of AK and SCC were significantly larger compared to healthy skin cells in all cell layers. The nucleus–cytoplasm ratio was also significantly higher for AK and SCC than for the healthy skin cells. It was even higher in SCC compared to spinous and basal cell layer of AK. The cell density in AK and SCC was significantly lower than in the basal and spinous cell layers of healthy skin. In SCC, the cell density was significantly lower than in AK. Concerning the intercellular spaces, significant differences were found for AK and healthy skin in spinous and basal cell layer and for SCC compared to AK and healthy skin. In this study, MPT proved to be a valuable noninvasive imaging method for in vivo detection and discrimination of AK and SCC from healthy skin.

A. Batista, HG. Breunig, A. Uchugonova, AM. Morgado, K. König.

Two-photon spectral fluorescence lifetime and second harmonic generation imaging of the porcine eye with a 12 femtosecond laser microscope.

JBO 21(3), 036002 (2016).

Abstract

Five dimensional microscopy with a 12-fs laser scanning microscope based on spectrally resolved two-photon autofluorescence lifetime and second-harmonic generation (SHG) imaging was used to characterize all layers of the porcine cornea. This setup allowed the simultaneous excitation of both metabolic cofactors, NAD(P)H and flavins, and their discrimination based on their spectral emission properties and fluorescence decay characteristics. Furthermore, the architecture of the stromal collagen fibrils was assessed by SHG imaging in both forward and backward directions. Information on the metabolic state and the tissue architecture of the porcine cornea were obtained with subcellular resolution, and high temporal and spectral resolutions.

S. Kantelhardt, D. Kalasauskas, K. König, E. Kim, M. Weinigel, A. Uchugonova, A. Giese.

In vivo multiphoton tomography and fluorescence lifetime imaging of human brain tumor tissue.

J Neurooncol online 30. Januar 2016. DOI 10.1007/s11060-016-2062-8.

Abstract

High resolution multiphoton tomography and fluorescence lifetime imaging differentiates glioma from adjacent brain in native tissue samples ex vivo. Presently, multiphoton tomography is applied in clinical dermatology and experimentally. We here present the first application of multiphoton and fluorescence lifetime imaging for in vivo imaging on humans during a neurosurgical procedure. We used a MPTflexTM Multiphoton Laser Tomograph (JenLab, Germany). We examined cultured glioma cells in an orthotopic mouse tumor model and native human tissue samples. Finally the multiphoton tomograph was applied to provide optical biopsies during resection of a clinical case of glioblastoma. All tissues imaged by multiphoton tomography were sampled and processed for conventional histopathology. The multiphoton tomograph allowed fluorescence intensity- and fluorescence lifetime imaging with submicron spatial resolution and 200 picosecond temporal resolution. Morphological fluorescence intensity imaging and fluorescence lifetime imaging of tumor-bearing mouse brains and native human tissue samples clearly differentiated tumor and adjacent brain tissue. Intraoperative imaging was found to be technically feasible. Intraoperative image quality was comparable to ex vivo examinations. To our knowledge we here present the first intraoperative application of high resolution multiphoton tomography and fluorescence lifetime imaging of human brain tumors in situ. It allowed in vivo identification and determination of cell density of tumor tissue on a cellular and subcellular level within seconds. The technology shows the potential of rapid intraoperative identification of native glioma tissue without need for tissue processing or staining.

V. Huck, C. Gorzelanny, K. Thomas, V. Getova, V. Niemeyer, K. Zens, TR. Unnerstall, JS. Feger, MA. Fallah, D. Metze, S. Ständer, TA. Luger, K. König, C. Mess, SW. Schneider.

From morphology to biochemical state-intravital multiphoton fluorescence lifetime imaging of inflamed human skin.

Sci. Rep. 6(2016)22789, doi: 10.1038/srep22789 (2016).

Abstract

The application of multiphoton microscopy in the field of biomedical research and advanced diagnostics promises unique insights into the pathophysiology of inflammatory skin diseases. In the present study, we combined multiphoton-based intravital tomography (MPT) and fluorescence lifetime imaging (MPTFLIM) within the scope of a clinical trial of atopic dermatitis with the aim of providing personalised data on the aetiopathology of inflammation in a non-invasive manner at patients’ bedsides. These ‘optical biopsies’ generated via MPT were morphologically analysed and aligned with classical skin histology. Because of its subcellular resolution, MPT provided evidence of a redistribution of mitochondria in keratinocytes, indicating an altered cellular metabolism. Two independent morphometric algorithms reliably showed an even distribution in healthy skin and a perinuclear accumulation in inflamed skin. Moreover, using MPT-FLIM, detection of the onset and progression of inflammatory processes could be achieved. In conclusion, the change in the distribution of mitochondria upon inflammation and the verification of an altered cellular metabolism facilitate a better understanding of inflammatory skin diseases and may permit early diagnosis and therapy.

M. Afshar, M. Leber, W. Poppendieck, K. König, H. Seidel, D. Feili.

On-chip nanostructuring and impedance trimming of transparent and flexible ITO electrodes by laser induced coherent sub-20 nm cuts.

Applied Surface Science 360 (216) 494-501.

Abstract

In this work, the effect of laser-induced nanostructuring of transparent indium tin oxide (ITO) electrodeson flexible glass is investigated. Multi-electrode arrays (MEA) for electrical and optical characterizationof biological cells were fabricated using standard MEMS technologies. Optimal sputter parameters con-cerning oxygen flow, sputter power and ambient pressure for ITO layers with both good optical andelectrical properties were determined. Afterwards, coherent sub-20 nm wide and 150 nm deep nanocutsof many micrometers in length were generated within the ITO electrodes by a sub-15 femtosecond (fs)pulsed laser. The influence of laser processing on the electrical and optical properties of electrodes wasinvestigated. The electrochemical impedance of the manufactured electrodes was measured before andafter laser modification using electrochemical impedance spectroscopy. A small reduction in electrodeimpedance was observed. These nanostructured electrodes show also polarizing effects by the visiblespectrum.

HG Breunig, A. Batista, A. Uchugonova, K. König.

Cell optoporation with a sub-15 fs and a 250-fs laser.

JBO 21(6), 060501 (2016), doi: 10.1117/1.JBO.21.6.060501.

Abstract

We employed two commercially available femtosecond lasers, a Ti:sapphire and a ytterbium-based oscillator, to directly compare from a user’s practical point-of-view in one common experimental setup the efficiencies of transient laser-induced cell membrane permeabilization, i.e., of so-called optoporation. The experimental setup consisted of a modified multiphoton laser-scanning microscope employing high-NA focusing optics. An automatic cell irradiation procedure was realized with custom-made software that identified cell positions and controlled relevant hardware components. The Ti:sapphire and ytterbium-based oscillators generated broadband sub-15-fs pulses around 800 nm and 250-fs pulses at 1044 nm, respectively. A higher optoporation rate and posttreatment viability were observed for the shorter fs pulses, confirming the importance of multiphoton effects for efficient optoporation.

K. König, A. Kasenbacher.

Thermal damage behaviour of human dental pulp stem cells.

ZWP Online 4 (2016).

Abstract

This study was designed to examine the influence of temperatures ranging from 37 to 65 °C on the cell morphology of DPSC stem cells via light and electron microscopy, a synthesis of Heat Shock Proteins (HSP) with fluorescence-marked antibodies and vitality via the Life/Dead Fluorescence Kit.

2015

HG. Breunig, M. Weinigel, K. König.

In vivo imaging of ZnO Nanoparticles from sunscreen on human skin with a mobile multiphoton tomograph.

BioNanoScience 5(2014)42-47. DOI: 10.1007/s12668-014-0155-4.

Abstract

Reports on the toxicity of inorganic nanoparticles used in sunscreen, in particular of zinc oxide and titanium dioxide, have raised concerns about a possible particle penetration through the skin barrier. We used two-photon imaging to visualize the distribution of zinc-oxide nanoparticles after topical application of a commercially available sunscreen on human skin in vivo and to investigate a possible penetration of nanoparticles beyond the stratum corneum. Two-photon imaging is in particular suitable for these investigations since the excitation and the nonlinear signal light from zinc-oxide nanoparticles as well as the endogenous skin autofluorescence are all spectrally well-separated which allows a clear identification of the signal origin by detection in two-spectral channels. Furthermore,microscopic modifications in the cutaneous structure like skin wrinkles which exhibit different thicknesses of the dermal layers and at the same time are regions where nanoparticle accumulation can be specifically investigated. The results indicate no penetration of nanoparticle through the barrier of the stratum corneum of healthy skin even in microscopic wrinkles.

M. Balu, C.B. Zachary, R.M. Harris, T.B. Krasieva, K. König, B.J. Tromberg, K.M. Kelly.

In vivo multiphoton microsocpy of basal cell carcinoma.

JAMA Dermatol. published online April 24, 2015, doi:10.1001/jamadermatol.2015.0453.

Abstract

Basal cell carcinomas (BCCs) are diagnosed by clinical evaluation, which can include dermoscopic evaluation, biopsy, and histopathologic examination. Recent translation of multiphoton microscopy (MPM) to clinical practice raises the possibility of noninvasive, label-free in vivo imaging of BCCs that could reduce the time from consultation to treatment.

K. König, H.G. Breunig.

Two-photon imaging of intact living plants during freezing with a flexible multiphoton tomograph.

Laser Phys. Lett. 12 (2015) 025601.

Abstract

We describe the combination of a flexible multiphoton tomograph (MPTflex) with a heating and cooling stage. The stage allows temperature control in the range of -196°C (77K) to +600°C (873K) with selectable heating/Freezing rates between 0.01 K min-1 and 150 K min-1. To illustrate the imaging capabilities of the combined system, fluorescence intensity and lifetime of intrinsic molecules from aplant leaf were imaged with submicron resolution during freezing in vivo without detaching the leaf from the plant. An increase of fluorescence intensitiy and decay times with decreasing temperature was abserved. The measurements illustrate the usefulness of multiphoton imaging as a non-invasive online tool to investigate temperature-induced effects. The flexible multiphoton tomograph with its adjustable mechano-optical arm and scan head allows imaging at otherwise hardly accessible sample regions.

K. König, T. Liu, P. Anderson, G. Breunig.

Multiphoton imaging with a novel compact diode-pumped Ti:sapphire oscillator.

Microsc Res Technol 78(2015)1154-1158.

Abstract

Multiphoton laser scanning microscopy commonly relies on bulky and expensive femtosecond lasers. We integrated a novel minimal-footprint Ti:sapphire oscillator, pumped by a frequency-doubled distributed Bragg reflector tapered diode laser, into a clinical multiphoton tomograph and evaluated its imaging capability using different biological samples, i.e. cell monolayers, corneal tissue, and human skin. With the novel laser, the realization of very compact Ti:sapphire-based systems for high-quality multiphoton imaging at a significantly size and weight compared to current systems will become possible.

A. Uchugonova, HG. Breunig, A. Batista, K. König.

Optical reprogramming of human somatic cells using ultrashort Bessel-shaped near-infrared femtosecond laser pulses.

JBO 20(2015)11, 115008.

Abstract

We report a virus-free optical approach to human cell reprogramming into induced pluripotent stem cells with low-power nanoporation using ultrashort Bessel-shaped laser pulses. Picojoule near-infrared sub-20 fs laser pulses at a high 85 MHz repetition frequency are employed to generate transient nanopores in the membrane of dermal fibroblasts for the introduction of four transcription factors to induce the reprogramming process. In contrast to conventional approaches which utilize retro- or lentiviruses to deliver genes or transcription factors into the host genome, the laser method is virus-free; hence, the risk of virus-induced cancer generation limiting clinical application is avoided.

A. Uchugonova.

Optical reprogramming of human cells in an ultrashort femtosecond laser microfluidic transfection platform.

J. Biophoton 1-6(2015). DOI 10.1002/jbio.201500240.

Abstract

Induced pluripotent stem cell (iPS cell) technology can be used to produce unlimited numbers of functional cells for both research and therapeutic purposes without ethical controversy. Typically, viruses are applied for efficient intracellular delivery of genes/transcription factors to generate iPS cells. However, the viral genomic integration may cause a risk of mutation as well as tumor formation therefore limits its clinical application. Here we demonstrate that spatially shaped extreme ultrashort laser pulses of sub-20 femtoseconds induce transient membrane permeabilisation which enables contamination-free transfection of cells in a microfluidic tube with multiple genes at the individual cell level in order to achieve optical reprogramming of large cell populations. We found that the ultrashort femtosecond laser-microfluidic cell transfection platform enhanced the efficacy of iPS-like colony-forming following merely a single transfection.

M. Weinigel, HG. Breunig, A. Uchugonova, K. König.

Multipurpose nonlinear optical imaging system for in vivo and ex vivo multimodal histology.

J Medical Imaging 2(2015)0160031-016003110.

Abstract

We report on a flexible multipurpose nonlinear microscopic imaging system based on a femtosecond excitation source and a photonic crystal fiber with multiple miniaturized time-correlated single-photon counting detectors. The system provides the simultaneous acquisition of e.g., two-photon autofluorescence, secondharmonic generation, and coherent anti-Stokes Raman scattering images. Its flexible scan head permits ex vivo biological imaging with subcellular resolution such as rapid biopsy examination during surgery as well as imaging on small as well as large animals. Above all, such an arrangement perfectly matches the needs for the clinical investigation of human skin in vivo where knowledge about the distribution of endogenous fluorophores, second-harmonic generation–active collagen as well as nonfluorescent lipids is of high interest.

M. Afshar, EM. Preiss, T. Sauerwald, M. Rodner, D. Feili, M. Straub, K. König, A. Schütze, H. Seidel.

Indium-tin-oxide single-nanowire gas sensor fabricated via laser writing and subsequent etching.

Sensors and Actuators B215(2015)525-535.

Abstract

We report on the design and nanofabrication of a single nanowire (NW) indium-tin-oxide (ITO) gassensor and on test results obtained with an oxidizing and a reducing gas. As a novel fabrication approach,direct laser writing and a subsequent etching process on sputtered ITO thin-film layers is applied. Forthis technique a near-infrared Ti:sapphire laser with sub-15 fs pulses and a repetition rate of 85 MHz isused. NWs for gas sensors are realized in two versions with a thickness of 125 ± _25 nm; one with 350 nmin width and 90 _m in length the other with 700 nm in width and 200 _m in length. The sensors areexposed to nitrogen dioxide (NO2) in synthetic air with concentrations from 1 ppm to 50 ppm showing asignificant change in resistance (up to 15.8%), whereas the reaction to 2000 ppm carbon monoxide (CO)turns out to be negligible (0.05%). At ambient temperature, the sensor exhibits integrating dosimeter-like behavior with relaxation times of more than 20 h. By self-heating, the NW can be reset to its initialcondition, thus enabling a new dosimeter run at room-temperature. When the sensors are operated inself-heating mode, a conventional behavior is observed, enabling the detection of NO2concentrationsdown to about 1 ppm at a stationary temperature below 200◦C.

HG. Breunig, A. Uchugonova, A. Batista, K. König.

Software-aided automatic laser optoporation and transfection of cells.

Scientific Reports 5(2015)11185. DOI 10.1038/srep11185.

Abstract

Optoporation, the permeabilization of a cell membrane by laser pulses, has emerged as a powerful non-invasive and highly efficient technique to induce transfection of cells. However, the usual tedious manual targeting of individual cells significantly limits the addressable cell number. To overcome this limitation, we present an experimental setup with custom-made software control, for computer-automated cell optoporation. The software evaluates the image contrast of cell contours, automatically designates cell locations for laser illumination, centres those locations in the laser focus, and executes the illumination. By software-controlled meandering of the sample stage, in principle all cells in a typical cell culture dish can be targeted without further user interaction. The automation allows for a significant increase in the number of treatable cells compared to a manual approach. For a laser illumination duration of 100 ms, 7-8 positions on different cells can be targeted every second inside the area of the microscope field of view. The experimental capabilities of the setup are illustrated in experiments with Chinese hamster ovary cells. Furthermore, the influence of laser power is discussed, with mention on post-treatment cell survival and optoporation-efficiency rates.

M. Weinigel, HG. Breunig, ME. Darvin, M. Klemp, J. Röwert-Huber, J. Lademann, K. König.

Impact of refractive index mismatches on coherent anti-Stokes Raman scattering and multiphoton autofluorescence tomography of human skin in vivo.

Physics in Medicine & Biology 60(2015)6881-6899, Doi:10.1088/0031-9155/60/17/6881.

Abstract

Optical non-linear multimodal tomography is a powerful diagnostic imaging tool to analyse human skin based on its autofluorescence and second-harmonic generation signals. Recently, the field of clinical non-linear imaging has been extended by adding coherent anti-Stokes Raman scattering (CARS)— a further optical sectioning method for the detection of non-fluorescent molecules. However, the heterogeneity of refractive indices of different substances in complex tissues like human skin can have a strong influence on CARS image formation and requires careful clinical interpretation of the detected signals. Interestingly, very regular patterns are present in the CARS images, which have no correspondence to the morphology revealed by autofluorescence at the same depth. The purpose of this paper is to clarify this phenomenon and to sensitize users for possible artefacts. A further part of this paper is the detailed comparison of CARS and autofluorescence images of healthy human skin in vivo covering the complete epidermis and part of the upper dermis by employing the flexible medical non-linear tomograph MPTflex CARS.

K. Reinhardt, H.G. Breunig, A. Uchugonova, K. König.

Sperm metabolism is altered during storage by females: evidence from fluorescence lifetime measurements in bedbugs.

J. R. Soc. Interface 12 (2015), doi:10.1098/rsif.2015.0609.

Abstract

We explore the possibility of characterizing sperm cells without the need to stain them using spectral and fluorescence lifetime analyses after multiphoton excitation in an insect model. The autofluorescence emission spectrum of sperm of the common bedbug, Cimex lectularius,was consistent with the presence of flavins andNAD(P)H. The mean fluorescence lifetimes showed smaller variation in sperm extracted from the male (tau m, tm . 1.54–1.84 ns) than in that extracted from the female sperm storage organ (tau m, tm . 1.26–2.00 ns). The fluorescence lifetime histograms revealed four peaks. These peaks (0.18, 0.92, 2.50 and 3.80 ns) suggest the presence ofNAD(P)Hand flavins and show that sperm metabolism can be characterized using fluorescence lifetime imaging. The difference in fluorescence lifetime variation between the sexes is consistent with the notion that female animals alter the metabolism of sperm cells during storage. It is not consistent, however, with the idea that sperm metabolism represents a sexually selected character that provides females with information about the male genotype.

S. Springer, M. Zieger, K. König, M. Kaatz, J. Lademann, ME Darvin.

Optimization of the measurement procedure during multiphoton tomography of human skin in vivo.

Skin Res Tech 0(2015) 1-7, DOI: 10.1111/srt.12273.

Abstract

The in vivo multiphoton tomography has evolved into a useful tool for the non-invasive investigation of morphological and biophysical characteristics of human skin. Until now, changes of skin have been evaluated mainly by clinical and histological techniques. The current study addresses the effects of a changed acquisition time for single scans in a Zstack on the directly related qualitative and quantitative interpretability of the data.

A. Uchugonova, W. Cao, R.M. Hoffman, K. König.

Comparison of label-free and GFP multiphoton imaging of hair-follicle-associated pluripotent (HAP) stem cells in mouse whiskers.

Cell Cycle 14(2015) 3430-3433. doi:10.1080/15384101.2015.1090064.

Abstract

Hair-follicle-associated pluripotent (HAP) stem cells can differentiate into many cell types, including neurons and heart muscle cells, and have been shown to repair peripheral nerves and the spinal cord in mice. HAP stem cells can be obtained from each individual patient for regenerative medicine which overcomes problems with immune rejection. Previously, we have demonstrated that genetically-encoded protein markers such as GFP in transgenic mice can be used to visualize HAP stem cells in vivo by multiphoton tomography. Detection and visualization of stem cells in vivo without exogenous labels such as GFP would be important for human application. In the present report, we demonstrate label-free visualization of hair follicle stem cells in mouse whiskers by multiphoton tomography due to the intrinsic fluorophores such as NAD(P)H/flavins. We compared multiphoton tomography of GFP-labeled HAP stem cells and unlabeled stem cells in isolated mouse whiskers. We show that observation of HAP stem cells by label-free multiphoton tomography is comparable to detection using GFP-labeled stem cells. The results described here have important implications for detection and isolation of human HAP stem cells for regenerative medicine.

2014

K. König, A. Uchugonova, HG Breunig.

High-resolution multiphoton crymicroscopy.

Methods, online July 16,2013, doi: 10.1016.

Abstract

An ultracompact high-resolution multiphoton cryomicroscope with a femtosecond near infrared fiber laser has been utilized to study the cellular autofluorescence during freezing and thawing of cells. Cooling resulted in an increase of the intracellular fluorescence intensity followed by morphological modifications at temperatures below _10 _C, depending on the application of the cryoprotectant DMSO and the cooling rate. Furthermore, fluorescence lifetime imaging revealed an increase of the mean lifetime with a decrease in temperature. Non-destructive, label-free optical biopsies of biomaterial in ice can be obtained with sub-20mW mean powers.

M. Weinigel, H.G.Breunig, J. Lademann, K. König.

In vivo histology: optical biopsies with chemical contrast using multiphoton/CARS tomography.

Laser Phys Lett, 11(2014)055601.

Abstract

The majority of existing coherent anti-Stokes Raman scattering (CARS) imaging systems are still huge and complicated laboratory systems and neither compact nor user-friendly nor mobile medically certified CARS systems. We have developed a new flexible multiphoton/CARS tomograph for imaging in a clinical environment. The system offers exceptional 360° flexibility with a very stable setup and enables label free ‘in vivo histology’ with chemical contrast within seconds. It can be completely operated by briefly trained non-laser experts. The imaging capability and flexibility of the novel in vivo tomograph are shown on optical biopsies with subcellular resolution and chemical contrast of patients suffering from psoriasis and squamous cell carcinoma.

M. Straub, M. Schüle, M. Afshar, D. Feili, H. Seidel, K. König.

Sub-15fs laser-induced nanostructures emerging in Si(100)surfaces immersed in water: analysis of structural phases.

Appl. Phys. A 115(2014)221-228.

Abstract

Nanoscale periodic rifts and subwavelength ripples as well as randomly nanoporous surface structures were generated on Si(100) surfaces immersed in water by tightly focused high-repetition rate sub-15 femtosecond subnanojoule pulsed Ti:sapphire laser light. Subsequent to laser processing, silicon oxide nanoparticles, which originated from a reaction of ablated silicon with water and aggregated on the exposed areas, were etched off by hydrofluoric acid. The structural phases of the three types of silicon nanostructures were investigated by transmission electron microscopy diffraction images recorded on focused ion beam sections. On nanorift patterns, which were produced at radiant exposure extremely close to the ablation threshold, only the ideal Si-I phase at its original bulk orientation was observed. Electron diffraction micrographs of periodic ripples, which were generated at slightly higher radiant exposure, revealed a compression of Si-I in the vertical direction by 6 %, which is attributed to recoil pressure acting during ablation. However, transitions to the high-pressure phase Si-II, which implies compression in the same direction at pressures in excess of 10 GPa, to the metastable phases Si-III or Si-IV that arise from Si-II on pressure relief or to other high-pressure phases (Si-V–Si-XII) were not observed. The nanoporous surfaces featured Si-I material with grains of resolidified silicon occurring at lattice orientations different from the bulk. Characteristic orientational relationships as well as smallangle grain boundaries reflected the rapid crystal growth on the substrate.

M. Schüle, M. Afshar, D. Feili, H. Seidel, K. König, M. Straub.

Incubation and nanostructure formation on n- and p-type Si(100) and Si(111) at various doping levels induced by sub-nanojoule femto- and picosecond near-infrared laser pulses.

Applied Surface Science 314(2014)21-29.

Abstract

N- and p-doped Si(1 0 0) and Si(1 1 1) surfaces with dopant concentrations of 2 × 1014–1 × 1019cm−3were irradiated by tightly focused 85-MHz repetition rate Ti:sapphire laser light (central wavelength 800 nm, bandwidth 120 nm) at pulse durations of 12 fs to 1.6 ps. Dependent on pulse peak intensity and exposure time nanorifts, ripples of period 130 nm as well as sponge-like randomly nanoporous surface structures were generated with water immersion and, thereafter, laid bare by etching off aggregated oxide nanoparticles. The same structure types emerged in air or water with transform-limited 100-fs pulses. At apulse length of 12 fs pronounced incubation occurred with incubation coefficients S = 0.66–0.85, where as incubation was diminished for picosecond pulses (S > 0.95). The ablation threshold strongly rose with dopant concentration. At similar doping level it was higher for n-type than for p-type samples and forSi(1 0 0) compared to Si(1 1 1) surfaces. These observations are attributed to laser-induced defect statesin the bandgap which participate in photoexcitation, deactivation of dopants by complex formation, and different densities of interface states at the boundary with the ultrathin native silicon dioxide surfacelayer. The threshold increase with pulse length revealed predominant single-photon excitation as well as multiphoton absorption.

2013

M. Manfredini, F. Arginelli, C. Dunsby, P. French, C. Talbot, K. König, G. Pellacani, G. Ponti, S. Seidenari.

High-resolution imaging of basal cell carcinoma: a comparison between multiphoton microscopy with fluorescence lifetime imaging and reflectance confocal microscopy

Skin Research and Technology 2013; 19: e433–e443

Abstract

Aims: The aim of this study was to compare morphological aspects of basal cell carcinoma (BCC) as assessed by two different imaging methods: in vivo reflectance confocal microscopy (RCM) and multiphoton tomography with fluorescence lifetime imaging implementation (MPT-FLIM).

Methods: The study comprised 16 BCCs for which a complete set of RCM and MPT-FLIM images were available. The presence of seven MPT-FLIM descriptors was evaluated. The presence of seven RCM equivalent parameters was scored in accordance to their extension. Chi-squared test with Fisher’s exact test and Spearman’s rank correlation coefficient were determined between MPT-FLIM scores and adjusted-RCM scores.

Results: MPT-FLIM and RCM descriptors of BCC were coupled to match the descriptors that define the same pathological structures. The comparison included: Streaming and Aligned elongated cells, Streaming with multiple directions and Double alignment, Palisading (RCM) and Palisading (MPTFLIM), Typical tumor islands, and Cell islands surrounded by fibers, Dark silhouettes and Phantom islands, Plump bright cells and Melanophages, Vessels (RCM), and Vessels (MPTFLIM). The parameters that were significantly correlated were Melanophages/Plump Bright Cells, Aligned elongated cells/ Streaming, Double alignment/Streaming with multiple directions, and Palisading (MPT-FLIM)/Palisading (RCM).

Conclusion: According to our data, both methods are suitable to image BCC’s features. The concordance between MPT-FLIM and RCM is high, with some limitations due to the technical differences between the two devices. The hardest difficulty when comparing the images generated by the two imaging modalities is represented by their different field of view.

A. Alex, J. Weingast, M. Weinigel, M. Kellner-Höfer, R. Nemecek, M. Binder, H. Pehamberger, K. König, W. Drexler.

Three-dimensional multiphoton/optical coherence tomography for diagnostic applications in dermatology

J. Biophotonics 6, No. 4, 352–362 (2013)

Abstract

A preliminary clinical trial using state-of-the-art multiphoton tomography (MPT) and optical coherence tomography (OCT) for three-dimensional (3D) multimodal in vivo imaging of normal skin, nevi, scars and pathologic skin lesions has been conducted. MPT enabled visualization of sub-cellular details with axial and transverse resolutions of <2 mm and <0.5 mm, respectively, from a volume of 0.35 _ 0.35 _ 0.2 mm3 at a frame rate of 0.14 Hz (512 _ 512 pixels). State-of-the-art OCT, operating at a center wavelength of 1300 nm, was capable of acquiring 3D images depicting the layered architecture of skin with axial and transverse resolutions _8 mm and _20 mm, respectively, from a volume of 7 _ 3.5 _ 1.5 mm3 at a frame rate of 46 Hz (1024 _ 1024 pixels). This study demonstrates the clinical diagnostic potential of MPT/OCT for pre-screening relatively large areas of skin using 3D OCT to identify suspicious regions at microscopic level and subsequently using high resolution MPT to obtain zoomed in, sub-cellular level information of the respective regions

H. G. Breunig, M. Weinigel, R. Bückle, M. Kellner-Höfer, J. Lademann, M. E. Darvin, W. STerry, K. König.

Clinical coherent anti-Stokes Raman scattering and multiphoton tomography of human skin with a femtosecond laser and photonic crystal fiber

Laser Phys. Lett. 10 (2013)

Abstract

We report on in vivo coherent anti-Stokes Raman scattering spectroscopy (CARS), two-photon fluorescence and second-harmonic-generation imaging of human skin with a novel multimodal clinical CARS / multiphoton tomograph. CARS imaging is realized by a combination of femtosecond pulses with broadband continuum pulses generated by a photonic crystal fiber. The images reveal the microscopic distribution of non-fluorescent lipids, endogenous fluorophores and the collagen network inside the human skin in vivo with subcellular resolution. Examples of healthy as well as cancer-affected skin are presented.

M. Balu, A. Mazhar, C. K. Hayakawa, R. Mittal, T. B. Krasieva, K. König, V. Venugopalan, B. J. Tromberg.

In Vivo Multiphoton NADH Fluorescence Reveals Depth-Dependent Keratinocyte Metabolism in Human Skin

Abstract

We employ a clinical multiphoton microscope to monitor in vivo and noninvasively the changes in reduced nicotinamide adenine dinucleotide (NADH) fluorescence of human epidermal cells during arterial occlusion. We correlate these results with measurements of tissue oxy- and deoxyhemoglobin concentration during oxygen deprivation using spatial frequency domain imaging. During arterial occlusion, a decrease in oxyhemoglobin corresponds to an increase in NADH fluorescence in the basal epidermal cells, implying a reduction in basal cell oxidative phosphorylation. The ischemia-induced oxygen deprivation is associated with a strong increase in NADH fluorescence of keratinocytes in layers close to the stratum basale, whereas keratinocytes from epidermal layers closer to the skin surface are not affected. Spatial frequency domain imaging optical property measurements, combined with a multilayer Monte Carlo-based radiative transport model of multiphoton microscopy signal collection in skin, establish that localized tissue optical property changes during occlusion do not impact the observed NADH signal increase. This outcome supports the hypothesis that the vascular contribution to the basal layer oxygen supply is significant and these cells engage in oxidative metabolism. Keratinocytes in the more superficial stratum granulosum are either supplied by atmospheric oxygen or are functionally anaerobic. Based on combined hemodynamic and two-photon excited fluorescence data, the oxygen consumption rate in the stratum basale is estimated to be ~0.035 mmoles/106 cells/h.

M. Ulrich, M. Klemp, M. E. Darvin, K. König, J. Lademann, M. C. Meinke.

In vivo detection of basal cell carcinoma: comparison of a reflectance confocal microscope and a multiphoton tomograph

Journal of Biomedical Optics 18(6), 061229 (June 2013)

Abstract

The standard diagnostic procedure for basal cell carcinoma (BCC) is invasive tissue biopsy with timeconsuming histological examination. To reduce the number of biopsies, noninvasive optical methods have been developed providing high-resolution skin examination. We present direct comparison of a reflectance confocal microscope (RLSM) and a multiphoton tomograph (MPT) for BCC diagnosis. Both systems are applied to nine patients prior to surgery, and the results are analyzed, including histological results. Both systems prove suitable for detecting typical characteristics of BCC in various stages. The RLSM allows large horizontal overview images to be obtained, enabling the investigator to find the regions of interest quickly, e.g., BCC nests. Elongated cells and palisading structures are easily recognized using both methods. Due to the higher resolution, changes in nucleus diameter or cytoplasm could be visualized with the MPT. Therefore, the nucleus diameter, nucleus/cytoplasm ratio, and cell density are estimated for normal and BCC cells using the MPT. The nucleus of elongated BCC cells is significantly longer than other measured normal skin cells, whereas the cell density and nucleus/cytoplasm ratio of BCC cannot be significantly distinguished from granular cells.

A. Uchugonova, M. Zhao, M. Weinigel, Y. Zhang, M. Bouvet, R. M. Hoffman, K. König.

Multiphoton Tomography Visualizes Collagen Fibers in the Tumor Microenvironment That Maintain Cancer-Cell Anchorage and Shape

Journal of Cellular Biochemistry 114:99–102 (2013)

Abstract

Second harmonic generation (SHG) multiphoton imaging can visualize fibrillar collagen in tissues. SHG has previously shown that fibrillar collagen is altered in various types of cancer. In the present study, in vivo high resolution SHG multi-photon tomography in living mice was used to study the relationship between cancer cells and intratumor collagen fibrils. Using green fluorescent protein (GFP) to visualize cancer cells and SHG to image collagen, we demonstrated that collagen fibrils provide a scaffold for cancer cells to align themselves and acquire optimal shape. These results suggest a new paradigm for a stromal element of tumors: their role in maintaining anchorage and shape of cancer cells that may enable them to proliferate.

K. König, A. Ostendorf.

Optically generated sub-100 nm structures for biomedical and technical applications

Lasers in Manufacturing Conference 2013

Abstract

Near infrared femtosecond laser - material interactions such as multiphoton ionization and plasma formation provide the possibility to perform 3D nanoprocessing in a variety of organic and non-organic materials. Sub-100 nm structures that are more than one order smaller than the laser wavelength and therefore far below Abbes diffraction limit can be generated on the surface and inside the bulk. Compared to conventional extreme ultraviolet lithography, multiphoton technology provides the chance to produce 3D nanofeatures much simpler, less expensive, more flexible, and even inside transparent bulk material. Rapid prototyping and low power nanosurgery are two examples of applications of this novel nonlinear nanotechnology tool. This paper provides an overview of projects within Germany regarding to multiphoton generation of sub-100 nm structures.

M. Straub, B. Weigand, M. Afshar, D. Feili, H. Seidel, K. König.

Periodic subwavelength ripples on lithium niobate surfaces generated by tightly focused sub-15 femtosecond sub-nanojoule pulsed near-infrared laser light

J. Opt. 15 (2013)

Abstract

On exposure to 85 MHz sub-15 fs pulsed 800 nm Ti:sapphire laser light in the focal spot of a high-numerical aperture oil immersion objective, characteristic periodic nanostructures emerged on the surface of z-cut congruent LiNbO3 crystals. Close to the ablation threshold shallow ripples oriented parallel to the laser polarization appeared on the surface at a periodicity 3k _ 200 nm as well as ripples running perpendicular to the laser polarization at almost the same period (3? _ 190 nm). Nanostructure formation involved melting and resolidification of LiNbO3 as evidenced by scanning electron microscopy images of structural peculiarities. Micro-Raman spectroscopy demonstrated that the resolidified material crystallized in the original structural phase. Ripple formation is attributed to plasma wave interference patterns that arise in the electron–hole plasma generated by multiphoton and avalanche collisional excitation.

K. König, A. Uchugonova, H. G. Breunig.

High-resolution multiphoton cryomicroscopy

Abstract

2012

H. Zhang, M. Afshar, D. Feili, H. Seidel, K. König.

Submicron-dot chains at and beneath surfaces of glasses written by a picojoule 12-fs laser scanning microscope

Abstract

A near-infrared 12-fs laser scanning microscope was employed for structuring glass surfaces. A Ti–sapphire laser operated at 85 MHz and emitted light pulses at wavelengths around 800 nm. A focused laser beam with a mean power of less than 27mW, corresponding to amaximal pulse energy of 318 pJ, was applied for line scanning at and beneath the surfaces of cover slips as well as of a filter glass BG39. Periodic arrangements of dots along the processed lines were produced through digital control of the scanner. Depending on the pulse energy and the scan speed, the diameter of the dots ranged from 550 nm down to 100 nm. For the cover slips, the dots occur as cavities after wet chemical etching. For BG39, which exhibits strong near-infrared absorption, both chains of cavities and bumps can be generated without any etching process. The result shows that structures with a size down to 1/8λ can be generated, probably through nonlinear single-photon processes confined within the focal volume.

M. Straub, M. Afshar, D. Feili, H. Seidel, K. König.

Periodic nanostructures on Si(100) surfaces generated by high-repetition rate sub-15 fs pulsed near-infrared laser light

OPTICS LETTERS / Vol. 37, No. 2 / January 15, 2012

Abstract

Nanoscale rifts and ripples at a periodicity of 130 nm were generated on Si(100) surfaces immersed in water using tightly focused 800 nm 12 fs pulsed 85 MHz laser light at subnanojoule pulse energies. At radiant exposure close to the ablation threshold rifts were typically 20–50 nm in width and 70 nm in depth running perpendicular to the laser

polarization. On increase of the irradiance, the rifts broadened and formed periodic ripples, whereas at highest exposure, a random nanoporous surface topology emerged. Rift and ripple formation is explained by laser-induced standing surface plasma waves, which result in periodic variation of dissipation and ablation.

M. Afshar, M. Straub, H. Voellm, D. Feili, K. Koenig, H. Seidel.

Sub-100 nm structuring of indium-tin-oxide thin films by sub-15 femtosecond pulsed near-infrared laser light

February 15, 2012 / Vol. 37, No. 4 / OPTICS LETTERS

Abstract

In magnetron sputtered indium-tin-oxide thin films of varying oxygen content, nanostructures were formed using tightly focused high-repetition rate near-infrared sub-15 femtosecond pulsed laser light. At radiant exposure well beyond the ablation threshold, cuts of 280–350 nm in width were generated. Illumination close to the ablation threshold resulted in periodic cuts of typically 20 nm in width at periodicities between 50 nm and 180 nm, as well as single sub-20 nm cuts. Subthreshold exposure, in combination with hydrochloric acid etching, yielded nanowires of 50 nm minimum lateral dimensions.

K. König, A. Uchugonova, M. Straub, H. Zhang, M. Licht, M. Afshar, D. Feili, H. Seidel.

Sub-100 nm material processing and imaging with a sub-15 femtosecond laser scanning microscope

JOURNAL OF LASER APPLICATIONS, JULY 2012

Abstract

Low mean powers of 1–10 mW are sufficient for material nanoprocessing when using femtosecond laser microscopes. In particular, near infrared 12 fs laser pulses at peak TW/cm2 intensities, picojoule pulse energies, and 85 MHz repetition rate have been employed. Threedimensional two-photon lithography as well as direct multiphoton ablation have been performed. Subwavelength sub-100 nm cuts have been realized in photoresists, silicon wafers, glass, polymers, metals, and biological targets. When reducing the mean power to the microwatt range, nondestructive two-photon imaging was performed with the same setup taking advantage of the broad laser emission spectrum. Multiphoton microscopes based on low-cost ultracompact sub-20 fs laser sources may become novel nonlinear optical tools for highly precise nanoprocessing and two-photon imaging.

S. Seidenari, F. Arginelli, S. Bassoli, J. Cautela, P.M.W. French, M. Guanti, D. Guardoli, K. König, C. Talbot, C. Dunsby.

Multiphoton LaserMicroscopy and Fluorescence Lifetime Imaging for the Evaluation of the Skin

Dermatology Research and Practice, Volume 2012, Article ID 810749

Abstract

Multiphoton laser microscopy is a new, non-invasive technique providing access to the skin at a cellular and subcellular level, which is based both on autofluorescence and fluorescence lifetime imaging.Whereas the former considers fluorescence intensity emitted by epidermal and dermal fluorophores and by the extra-cellular matrix, fluorescence lifetime imaging (FLIM), is generated by the fluorescence decay rate. This innovative technique can be applied to the study of living skin, cell cultures and ex vivo samples. Although still limited to the clinical research field, the development ofmultiphoton lasermicroscopy is thought to become suitable for a practical application in the next few years: in this paper, we performed an accurate review of the studies published so far, considering the possible fields of application of this imaging method and providing high quality images acquired in the Department of Dermatology of the University of Modena.

H. G. Breunig, R. Bückle, M. Kellner-Höfer, M. Weinigel, J. Lademann , W. Sterry, K. König.

Combined In Vivo Multiphoton and CARS Imaging of Healthy and Disease-Affected Human Skin

Microscopy Research and Technique 75:492–498 (2012)

Abstract

We present combined epi-coherent anti-Stokes Raman scattering (CARS) and multiphoton imaging with both chemical discrimination and subcellular resolution on human skin in vivo. The combination of both image modalities enables label-free imaging of the autofluorescence of endogenous fluorophores by two-photon excited fluorescence, as well as imaging of the distribution of intercellular lipids, topically applied substances and water by CARS. As an example for medical imaging, we investigated healthy and psoriasis-affected human skin with both image modalities in vivo and found indications for different lipid distributions on the cellular level.

M. Licht, A. Uchugonova, K. König, M. Straub.

Sub-15 fs multiphoton lithography of three-dimensional structures for live cell applications

J. Opt. 14 (2012) 065601

Abstract

Development, morphology and intratissue location of cells are influenced by the 3D nano- and microenvironment. In this paper we demonstrate multiphoton photopolymerization to generate three-dimensional structures for cell culture applications with micro- and nanotopographic features using SU-8 photoresist and mr-NIL 6000 nanoimprint resist. Moving the focal spot of high-repetition rate near-infrared sub-15 fs pulsed laser light by a galvanometric beam scanner in combination with a piezoelectric vertical stage, nearly arbitrary trajectories of polymerized photoresist were generated. This technique can be used to generate cage structures with submicron interior features for live cell applications. Preliminary experiments with PC-3 and HT-1080 cells indicate the influence of the structures on cell behavior.

A. Uchugonova, M. Lessel, S. Nietzsche, C. Zeitz, K. Jacobs, C. Lemke, K. König.

Nanosurgery of cells and chromosomes using near-infrared twelve-femtosecond laser pulses

Journal of Biomedical Optics 17(10), 101502 (October 2012)

Abstract

Laser-assisted surgery based on multiphoton absorption of near-infrared laser light has great potential for high precision surgery at various depths within the cells and tissues. Clinical applications include refractive surgery (fs-LASIK). The non-contact laser method also supports contamination-free cell nanosurgery. In this paper we describe usage of an ultrashort femtosecond laser scanning microscope for sub-100 nm surgery of human cells and metaphase chromosomes. A mode-locked 85 MHz Ti:Sapphire laser with an M-shaped ultrabroad band spectrum (maxima∶ 770 nm∕830 nm) and an in situ pulse duration at the target ranging from 12 fs up to 3 ps was employed. The effects of laser nanoprocessing in cells and chromosomes have been quantified by atomic force microscopy. These studies demonstrate the potential of extreme ultrashort femtosecond laser pulses at low mean milliwatt powers for sub-100 nm surgery of cells and cellular organelles.

M.E. Darvin, K. König, M. Kellner-Höfer, H.G. Breunig, W. Werncke, M.C. Meinke, A. Patzelt, W. Sterry, J. Lademann.

Safety Assessment by Multiphoton Fluorescence/ Second Harmonic Generation/Hyper-Rayleigh Scattering Tomography of ZnO Nanoparticles Used in Cosmetic Products

Skin Pharmacol Physiol 2012;25:219–226

Abstract

Zinc oxide nanoparticles (ZnO NPs) are commonly used as UV filters in commercial sunscreen products. Their penetration into the skin is intensively discussed in the literature. In the present in vivo study, penetration of ZnO NPs (30 nm in size) into human skin was investigated by multiphoton tomography. Based on the non-linear effects of a second harmonic generation and hyper-Rayleigh scattering, the distribution of ZnO NPs in the horny layers of the epidermis, as well as the furrows, wrinkles and orifice of the hair follicles was analyzed. This method permitted distinguishing between the particulate and dissolved forms of Zn. A detection limit of 0.08 fg/ _ m 3 was estimated. Taking advantage of this sensitivity, it was clearly shown that ZnO NPs penetrate only into the outermost layers of stratum corneum, furrows and into the orifices of the hair follicles and do not reach the viable epidermis.

M. Straub, M. Afshar, D. Feili, H. Seidel, K. König.

Surface plasmon polariton model of high-spatial frequency laser-induced periodic surface structure generation in silicon

J. Appl. Phys. 111, 124315 (2012)

Abstract

In recent years, high-spatial frequency laser-induced surfaces structures have been generated in a large variety of dielectrics. In silicon subwavelength ripples, some of which featured periodicities below 100 nm, were formed using ultrafast lasers. We demonstrate for Si(100) surfaces that generation of a dense electron-hole plasma in the focal spot of ultrashort-pulsed laser light followed by massive excitation of plasma waves provides an explanation for the formation of such high-spatial frequency surface structures. The applied Drude-like model includes carrier-carrier collisions and is in excellent agreement with the experimentally observed ripple period.

C. Huss, M. Krause, U. Löw, I. Riemann, F. Stracke, P. Mestres, B. Seitz, K. König.

Experimentelle Hornhautbildgebung und Hornhautchirurgie mit nicht verstärkten Femtosekundenlaserpulsen

Ophthalmologe 2012 · 109:995–1000

Abstract

Hintergrund. Ziel war, mit einem unverstärkten Femtosekundenlaser Multiphotoneneffekte zu induzieren und für Hornhautbildgebung und Gewebeabtrag zu nutzen.Material und Methoden. Unverstärkte Titan-Saphir-Laser wurden zur Untersuchung von menschlichen Hornhäuten und Schweinehornhäuten an Laserscanningmikroskope gekoppelt. Bildgebung sowie Erzeugung von Gewebeläsionen erfolgten im gleichen Strahlengang mit Pulsenergien <2 nJ.Ergebnisse. Zelluläre Bestandteile und extrazelluläre Matrix wurden selektiv mittels Autofluoreszenz und Frequenzverdopplung in Submikrometerauflösung dargestellt. Nach intrastromaler linearer Laserabtastung mit höheren Leistungen entstand ein lumineszentes Plasma. Die Läsionsbreite nahm mit zunehmender Gewebetiefe ab und mit zunehmender Laserleistung am Objektiv zu. In der Lichtmikroskopie zeigte sich nach dem Laserabtrag in der Umgebung der Läsion intaktes Stroma.Schlussfolgerung. Mit niederenergetischen Femtosekundenlaserpulsen wurden hochauflösende Bilder sowie präzise Gewebeläsionen in der Kornea erzeugt. Der einfache Wechsel zwischen Bildgebung und Gewebeabtrag lässt diagnostische und therapeutische Anwendungen möglich erscheinen.

M. J. Koehler, A. Preller, P. Elsner, K. König, U. C. Hipler, M. Kaatz.

Non-invasive evaluation of dermal elastosis by in vivo multiphoton tomography with autofluorescence lifetime measurements

Abstract

The non-invasive differentiation of dermal elastic fibres from solar elastosis in vivo is of great interest in dermatologic research, especially for efficacy testing of antiageing products. To date, no studies on multiphoton excited fluorescence lifetime characteristics of human elastic fibres and solar elastosis are reported. The goal of the present work was the identification of differential criteria for elastic fibres and solar elastosis by the analysis of fluorescence decay curves acquired by time-correlated single photon counting in vivo multiphoton tomography. For this purpose, fluorescence lifetime measurements (FLIM) were performed with 47 volunteers of different age groups at sun-protected and sun-exposed localizations. Bi-exponential curve fitting was applied to the FLIM data, and characteristic differences between age groups and localizations were found in both relevant fit parameters describing the decay slope. The FLIM analyses have shown that dermal autofluorescence has different lifetimes depending on age and in part on localization.

R. Patalay, C. Talbot, Y. Alexandrov, M.O. Lenz, S. Kumar, S. Warren, I. Munro, M. A. A. Neil, k. König, P. M. W. French, A. Chu, G. W. H. Stamp, C. Dunsby.

Multiphoton Multispectral Fluorescence Lifetime Tomography for the Evaluation of Basal Cell Carcinomas

Abstract

We present the first detailed study using multispectral multiphoton fluorescence lifetime imaging to differentiate basal cell carcinoma cells (BCCs) from normal keratinocytes. Images were acquired from 19 freshly excised BCCs and 27 samples of normal skin (in & ex vivo). Features from fluorescence lifetime images were used to discriminate BCCs with a sensitivity/ specificity of 79%/93% respectively. A mosaic of BCC fluorescence lifetime images covering .1 mm2 is also presented, demonstrating the potential for tumour margin delineation. Using 10,462 manually segmented cells from the image data, we quantify the cellular morphology and spectroscopic differences between BCCs and normal skin for the first time. Statistically significant increases were found in the fluorescence lifetimes of cells from BCCs in all spectral channels, ranging from 19.9% (425–515 nm spectral emission) to 39.8% (620–655 nm emission). A discriminant analysis based diagnostic algorithm allowed the fraction of cells classified as malignant to be calculated for each patient. This yielded a receiver operator characteristic area under the curve for the detection of BCC of 0.83. We have used both morphological and spectroscopic parameters to discriminate BCC from normal skin, and provide a comprehensive base for how this technique could be used for BCC assessment in clinical practice.

Karsten König.

Hybrid multiphoton multimodal tomography of in vivo human skin

IntraVital 1:1, 11–26; July/August/September 2012

Abstract

Clinical multiphoton tomography based on femtosecond near infrared laser pulses for in vivo high-resolution skin imaging has been employed to thousands of volunteers and patients. Two-photon cellular autofluorescence and second harmonic generation of collagen can be detected with single-photon sensitivity and submicron spatial resolution. Also in vivo clinical CARS has been realized to image intratissue lipids and water. Novel developments focus on multimodal hybrid imaging to generate optical tissue biopsies with subcellular resolution, deep-tissue information, and chemical fingerprints. Widefield imaging tools such as dermoscopes, optical coherence tomographs as well as ultrasound and photoacoustic devices can be integrated. The hybrid tomographs have the potential to trace cosmetics and pharmaceutical components such as sunscreen nanoparticles and anti-aging products in humans. Skin cancer such as malignant melanoma and basal cell carcinoma as well as dermatitis can be detected at an early stage and the efficiency of the treatment can be monitored. These novel hybrid multimodal multiphoton tomographs may become important biopsy-free and label-free imaging tools in personalized medicine, pharmacy, biotechnology as well as cosmetic research.

S. Seidenari, F. Arginelli, C. Dunsby, P. French, K. König, C. Magnoni, M. Manfredini, C. Talbot, G. Ponti.

Multiphoton laser tomography and fluorescence lifetime imaging of basal cell carcinoma: morphologic features for non-invasive diagnostics

Experimental Dermatology, 2012, 21, 831–836

Abstract

Multiphoton laser tomography (MPT) combined with fluorescence lifetime imaging (FLIM) is a non-invasive imaging technique, which gives access to the cellular and extracellular morphology of the skin. The aim of our study was to assess the sensitivity and specificity of MPT/FLIM descriptors for basal cell carcinoma (BCC), to improve BCC diagnosis and the identification of tumor margins. In the preliminary study, FLIM images referring to 35 BCCs and 35 healthy skin samples were evaluated for the identification of morphologic descriptors

characteristic of BCC. In the main study, the selected parameters were blindly evaluated on a test set comprising 63 BCCs, 63 healthy skin samples and 66 skin lesions. Moreover, FLIM values inside a region of interest were calculated on 98 healthy skin and 98 BCC samples. In the preliminary study, three epidermal descriptors and 7 BCC descriptors were identified. The specificity of the diagnostic criteria versus ‘other lesions’ was extremely high, indicating that the presence of at least one BCC descriptor makes the diagnosis of ‘other lesion’ extremely unlikely. FLIM values referring to BCC cells significantly differed from those of healthy skin. In this study, we identified morphological and numerical descriptors enabling the differentiation of BCC from other skin disorders and its distinction from healthy skin in ex vivo samples. In future, MPT/FLIM may be applied to skin lesions to provide direct clinical guidance before biopsy and histological examination and for the identification of tumor margins allowing a complete surgical removal.

2011

K. König, A. Uchugonova, E. Gorjup.

Multiphoton fluorescence lifetime imaging of 3D-stem cell spheroids during differentiation.

Microscopy Research and Technique 74 (2011) 9–17 / DOI 10.1002/jemt.20866

Abstract

Long-term high-resolution multiphoton imaging of nonlabeled human salivary gland stem cell spheroids has been performed with submicron spatial resolution, 10.5-nm spectral resolution, and picosecond temporal resolution. In particular, the two-photon-excited coenzyme NAD(P)H and flavins have been detected by time-correlated single photon counting (TCSPC). Stem cells increased their autofluorescence lifetimes and decreased their total fluorescence intensity during the adipogenic-differentiation process. In addition, the onset of the biosynthesis of lipid
vacuoles was monitored over a period of several weeks in stem-cell spheroids. Time-resolved multiphoton autofluorescence imaging microscopes may become a promising tool for marker-free stemcell characterization and cell sorting.

A. Uchugonova, J. Duong, N. Zhang, K. König, R.M. Hoffman.

The bulge area is the origin of nestin-expressing pluripotent stem cells of the hair follicle.

J Cell Biochem. (2011)

Abstract

Nestin-expressing pluripotent stem cells have been found both in the bulge area (BA) as well as the dermal papilla (DP). Nestin-expressing stem cells of both the BA and DP have been previously shown to be able to form neurons and other non-follicle cell types. The nestinexpressing stem cells from the DP have been termed skin precursor or SKP cells. Both nestinexpressing DP and BA cells have been previously shown to effect repair of the injured spinal cord and peripheral nerve, with the BA being the greater and more constant source of the stem
cells. The BA contains nestin-expressing stem cells throughout the hair cycle, whereas nestinexpressing
dermal papillae stem cells were found in early and mid-anagen only. Our previous studies have shown that the nestin-expressing stem cells in the BA and DP have similar morphological features. The cells from both regions have a small body diameter of approximately 7 μm with long extrusions, as shown by 2-photon confocal microscopy. In the present study, using 2-photon confocal microscopy of whisker follicles from transgenic mice expressing nestin-driven green fluorescent protein (ND-GFP), we demonstrate that the BA is the source of the nestin-expressing stem cells of the hair follicle. The nestin-expressing stem cells migrate from the BA to the DP as well as into the surrounding skin tissues including the epidermis, including during wound healing, suggesting that the BA maybe the source of the stem cells of the skin itself.

F. Liu, A. Uchugonova, H. Kimura, C. Zhang, M. Zhao, L. Zhang, K. König, J. Duong, R. Aki, N. Saito, S. Mii, Y. Amoh, K. Katsuoka, R. M. Hoffman.

The bulge area is the major hair follicle source of nestin-expressing pluripotent stem cells which can repair the spinal cord compared to the dermal papilla

Cell Cycle 5 (2011), 830-9

Abstract

Nestin has been shown to be expressed in the hair follicle, both in the bulge area (BA) as well as the dermal papilla (DP). Nestin-expressing stem cells of both the BA and DP have been previously shown to be pluripotent and be able to form neurons and other non-follicle cell types. The nestin-expressing pluripotent stem cells from the DP have been termed skin precursor or SKP cells. The objective of the present study was to determine the major source of nestin-expressing pluripotent stem cells in the hair follicle and to compare the ability of the nestin-expressing pluripotent stem cells from the BA and DP to repair spinal cord injury. Transgenic mice in which the nestin promoter drives GFP (ND-GFP) were
used in order to observe nestin expression in the BA and DP. Nestin-expressing DP cells were found in early and middle anagen. The BA had nestin expression throughout the hair cycle and to a greater extent than the DP. The cells from both regions had very long processes extending from them as shown by two-photon confocal microscopy. Nestin-expressing stem cells from both areas differentiated into neuronal cells at high frequency in vitro. Both nestin-expressing DP and BA cells differentiated into neuronal and glial cells after transplantation to the injured spinal cord and enhanced injury
repair and locomotor recovery within four weeks. Nestin-expressing pluripotent stem cells from both the BA and DP have potential for spinal cord regeneration, with the BA being the greater and more constant source.

J. Köhler, K. König, M. Speicher, S. Astner, E. Stockfleth, P. Elsner, M. Kaatz.

Clinical application of multiphoton tomography in combination with confocal laser scannig microscopy for evaluation of skin diseases.

Skin Research and Technology.

Abstract

The first-ever application of high-frequency ultrasound combined with multiphoton tomography (MPT) and dermoscopy in a clinical trial is reported. 47 patients with different dermatoses such as benign and malign skin cancers, connective tissue diseases, inflammatory skin diseases, and autoimmune bullous skin diseaseshave been investigated with (i) state-of-the-art and highly sophisticated ultrasound systems for dermatology, (ii) the femtosecond laser multiphoton tomograph and (iii) dermoscopes. Dermoscopy provides two-dimensional color images of the skin surface with a magnification up to 70 . Depending on the ultrasonic frequencies from 7.5 MHz to 100 MHz, the signal depth varies from about 1 mm to 80 mm. Vertical ultrasound wide-field images provide fast information on depth and volume of the lesion. The 100 MHz ultrasound allows imaging with resolutions down to 16 mm (axial) and 32 mm (lateral). Multiphoton tomography provides 0.36 0.36 0.001 mm3 horizontal optical sections of a particular region of interest with submicron resolution down to 200 mm tissue depth. The autofluorescence of mitochondrial coenzymes, keratin, melanin, and elastin as well as the network of collagen structures can be imaged. The combination of ultrasound and MPT opens novel synergistic possibilities in diagnostics of skin diseases with a special focus on the early detection of skin cancer as well as the evaluation of treatments.

M. Koehler, S. Zimmerman, S. Springer, P. Elsner, K. König, M. Kaatz.

Keratinocyte morphology of human skin evaluated by in vivo multiphoton laser tomography.

Skin Research and Technology 17 (2011) / DOI: 10.1111/j.1600-0846.2011.00522.x

Abstract

Multiphoton tomography (MPT) is a novel noninvasive imaging method in dermatology allowing the depiction of the epidermis with sub-cellular resolution. Here, we present a descriptive characterization of unaffected human epidermis, morphometric data on human keratinocytes and some epidermal parameters in vivo and a morphological characterization of keratinocyte changes in actinic keratoses. Methods: In a clinical setting, 57 volunteers of different age groups were examined using MPT. Results: The morphological appearance of keratinocytes showed polygonal cells in the horny layer, a granular cytoplasm in the stratum granulosum, smaller prickle cells in the stratum spinosum and hyperpigmented small round basal cells. Actinic keratoses presented remarkable differences including widened inter-cellular spaces, heterogeneity in cellular fluorescence and shape as well as an increased ratio of nuclear to cellular size. Finally, the thickness of the epidermis was significantly increased in actinic keratoses compared with the control.

M. S. Roberts, Y. Dancik, T. W. Prow, C. A. Thorling, L. L. Lin, J. E. Grice, T. A. Robertson, K. König, W. Becker.

Non-invasive imaging of skin physiology and percutaneous penetration using fluorescence spectral and lifetime imaging with multiphoton and confocal microscopy

European Journal of Pharmaceutics and Biopharmaceutics 77 (2011) 469-488 / DOI: 10.1016/j.ejpb.2010.12.023.

Abstract

New multiphoton and confocal microscope technologies and fluorescence lifetime imaging techniques are now being used to non-invasively image, in space (three dimensions),in time, in spectra, in lifetime and in fluorescence anisotropy (total of 7 dimensions), fluorescent molecules in in situ and in vivo biological tissue, including skin. The process involves scanning a 2D area and measuring fluorescence at a given tissue depth below the surface after excitation by a laser beam with a wavelength within the one-photon or two-photon absorption band of the fluorophores followed by the stacking together of a series of 2D images from different depths to reconstruct the full spatial structure of the sample. Our aim in this work is to describe the principles, opportunities, limitations and applications of this new technology and its application in defining skin morphology, disease and skin penetration in vitro and in vivo by drugs, chemicals and nanoparticles. A key emphasis is in the use of fluorescence lifetime imaging to add additional specificity and quantitation to the detection of the various exogenous chemicals and nanoparticles that may be applied to the skin as well as endogenous fluorescent species in the skin. Examples given include equipment configuration; components in skin autofluorescence in various skin strata; imaging and quantification of coexisting drugs and their metabolites; skin pH; nanoparticle zinc oxide skin penetration; liposome delivery of drugs to deeper tissues; and observations in skin ageing and in various skin diseases.

H. Studier, H.G. Breunig, K. König.

Comparison of broadband and ultrabroadband pulses at MHz and GHz pulse-repetition rates for nonlinear fs-laser scanning microscopy

Journal of Biophotonics 4 (2011) 84–91.

Abstract

Nonlinear optical imaging of human skin and of polychromatic microspheres was carried out to compare and evaluate the imaging properties of three different excitation femtosecond lasers: a spectrally tunable 80 MHz Ti : sapphire oscillator that produced 100 fs pulses (spectral width 10 nm) and two ultrabroadband Ti : sapphire oscillators with repetition rates of 85 MHz and 1 GHz. The latter of these two and the 100 fs laser were combined with a laser scanning microscope (TauMap). The intensities of images of the polychromatic microsphere samples obtained with both lasers are in accordance with the usual dependence of two-photon processes on laser pulse parameters, i.e. the intensity is proportional to the square of the mean laser power and the reciprocal pulse duration. In contrast to that, skin images measured with all three different excitation sources with mean powers of each laser adjusted to the particular pulse length and repetition rate exhibited discrepancies from this relation. For characterization of the ultrabroadband GHz laser, the measurements are supplemented by spectra of second-harmonic-generation signals of urea and collagen.

K. König, H.G. Breunig, R. Bückle, M. Kellner-Höfer, M. Weinigel, E. Büttner, W. Sterry, J. Lademann

Optical skin biopsies by clinical CARS and multiphoton fluorescence/SHG tomography

Laser Physics Letters 8 (2011) 1-4 / DOI: 10.1002/lapl.201110014

Abstract

The ultimate challenge for early diagnostics is labelfree high-resolution intratissue imaging without taking physical biopsies. A novel hybrid femtosecond laser tomograph provides in vivo optical biopsies of human skin based on non-linear excitation of autofluorescence and the detection of lipids and water by CARS. Applications include skin cancer detection, biosafety tests of intradermal nanoparticles, and the testing of anti-aging products.

2010

D. Bruneel, E. Audouard, K. König, R. Le Harzic.

Flexible tool for two-photon laser nanoprocessing and large area mapping with high resolution.

Optics and Lasers in Engineering In press / DOI 10.1016/j.optlaseng.2010.06.003

Abstract

A high accurate, compact and flexible laser nanoprocessing and large area mapping with high-resolution apparatus is presented in this paper. Judicious hardware and software developments of the device are presented, which allow a high range of applications and performances. Coupled to a low average and low cost femtosecond laser system, results on accurate nanoprocessing on biological and biotechnological samples demonstrate the potential of the machine. Nanostructuring of different type of materials with this device is conceivable for a wide spectrum of technological applications in material science, nanobiotechnology and nanomedicine.

D. Bruneel, G. Matras, R. Le Harzic, N. Huot, K. König, E. Audouard.

Micromachining of metals with ultra-short Ti-Sapphire lasers:
Prediction and optimization of the processing time.

Optics and Lasers in Engineering 48 (2010) 268-271 / DOI 10.1016/j.optlaseng.2009.10.010

Abstract

We investigate the processing times of ultrafast laser machining in the case of metals (copper and stainless steel). At a fluence of 2.5 J/cm2, measurements of processing times are in good agreement with the calculations based on the ablation rates. We study the influence of laser repetition rates for 1, 5, 10 and 15 kHz. A linear reduction of the processing time can be expected with an increase of the repetition rate.

H. Studier, H. G. Breunig, K. König.

Comparison of broadband and ultrabroadband pulses at MHz and GHz pulse-repetition rates for nonlinear femtosecond-laser scanning microscopy.

Journal of Biophotonics (2010) DOI 10.1002/jbio.201000010

Abstract

Nonlinear optical imaging of human skin and of polychromatic microspheres was carried out to compare and evaluate the imaging properties of three different excitation femtosecond lasers: a spectrally tunable 80 MHz Ti: sapphire oscillator that produced 100 fs pulses (spectral width 10 nm) and two ultrabroadband Ti: sapphire oscillators with repetition rates of 85 MHz and 1 GHz. The latter of these two and the 100 fs laser were combined with a laser scanning microscope (TauMap). The intensities of images of the polychromatic microsphere samples obtained with both lasers are in accordance with the usual dependence of two-photon processes on laser pulse parameters, i.e. the intensity is proportional to the square of the mean laser power and the reciprocal pulse duration. In contrast to that, skin images measured with all three different excitation sources with mean powers of each laser adjusted to the particular pulse length and repetition rate exhibited discrepancies from this relation. For characterization of the ultrabroadband GHz laser, the measurements are supplemented by spectra of second-harmonic-generation signals of urea and collagen. (© 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim)
Link zum Artikel

M.J. Koehler, T. Vogel, P. Elsner, K. König, R. Bückle, M. Kaatz.

In vivo measurement of the human epidermal thickness in different localizations by multiphoton laser tomography.

Skin Research and Technology 16 (2010) 259-264 / DOI 10.1111/j.1600-0846.2010.00437.x

Abstract

Background: The in vivo measurement of epidermal thickness is still challenging. While ultrasound, optical coherence tomography and confocal laser microscopy are used with moderate success, this issue has not been addressed by multiphoton laser tomography.

Objectives: In the present study, an in vivo measurement of four different morphometric epidermal parameters is performed.

Methods: Thirty healthy volunteers aged 21–82 years were included in the study after informed consent and approval of the local ethics committee. At the dorsal forearm and the dorsum of the hand, the thicknesses of the total epidermis, viable epidermis and stratum corneum and the depth of the papillary dermis were calculated from depth-resolved intensity curves after correlation with multiphoton images.

Results: We have shown consistently that in all age groups, the four morphometric parameters are significantly higher at the hand compared with the forearm, while there were no differences between age groups. This is consistent with most previous findings.

Conclusion: The method presented here provides a novel in vivo investigation tool for the measurement of epidermal morphometric parameters that may be useful for the observation of epidermal changes over time in skin disorders, therapy side effects or in cosmetic science.
Link zum Artikel

R. Bazin, F. Flament, A. Colonna, R. Le Harzic, R. Bückle, B. Piot, F. Laizé, M. Kaatz, K. König, J. W. Fluhr:
Clinical study on the effects of a cosmetic product on dermal extracellular matrix components using a high-resolution multiphoton tomograph. Skin Research and Technology 16 (2010) 305-310 / DOI 10.1111/j.1600-0846.2010.00432.x

Abstract:
Background/purpose:
The aim of this study was to demonstrate the effects of selected plant extracts in a cosmetic cream on the dermal network components after a 3-month treatment using an in vivo multiphoton tomographic device.

Methods:
Twenty-four Caucasian women aged between 45 and 65 applied randomly a cosmetic emulsion B containing active ingredients (soy and jasmine) twice a day on one arm and its vehicle A (without active ingredients) on the other arm during 3 months. Measurements were performed on the internal side of the forearm before starting the treatment (T0), after 4 week (T4) and 12 weeks (T12) treatment. Measurements consisted in a multi-layers acquisitions using a multiphoton tomograph with subcellular resolution. Optical sections (about 6 μm thick) were recorded from 0 to about 200 μm using two different wavelengths: 760 and 820 nm. To compare the series of images and obtain an objective quantification of the signal of second harmonic generation (SHG) and autofluorescence, the method used consisted in taking the integrated brightness of an image (same rectangular area for all images) as a measure of the signal. Following this step a ratio between brightness of images from the area treated with cream A or B and brightness of untreated area was calculated and used as an assessment of treatment efficacy. The parameter used for statistical analysis (variance analysis) is the difference before and after 12 weeks of treatment by either cream A or B of the signal ratios calculated in the upper dermis (118–130 μm) and those from a deeper region of the upper dermis (165–178 μm).

Results:
Signals (autofluorescence+SHG) of extracellular matrix do not change significantly with time (weeks 0, 4 and 12) when cream A (vehicle with no active ingredient) is applied. Treatment with cream B results in an enhancement in the signal level of extracellular matrix at week 12. The comparison of signals, in both areas (118–130 μm and 160–178 μm), show an higher increase in the deeper region than in the more superficial one for product B while we do not notice any change with product A.

Conclusion:
The multiphoton tomograph provided excellent high-resolution images, which describe clearly the different skin layers, single cells and extracellular matrix components in all the 24 volunteers. Statistic analyses reveal a real effect for product B with selected plant extracts, known to increase collagen synthesis. Changes observed are characteristics of modifications in dermal collagen and elastin content. To our knowledge, it is the first time that it was possible to demonstrate in vivo the effect of a cosmetic product on the superficial dermal layer, in a non-invasive and non-destructive process, i.e. without cutting the skin.
Link zum Artikel

M. Kaatz, A. Sturm, P. Elsner, K. König, R. Bückle, M.J. Koehler.

Depth-resolved measurement of the dermal matrix composition by multiphoton laser tomography.

Skin Research and Technology 16 (2010) 131-136 / DOI 10.1111/j.1600-0846.2009.00423.x

Abstract

Background:
In the last years, multiphoton laser tomography (MLT) has emerged as a promising tool for non-invasive diagnostics in dermatology and other medical specialties. The present work is dedicated to the question to what degree the measurement depth and the thickness of the epidermis influence the evaluation of dermal matrix composition and if recommendations for future measurement procedures can be given.

Methods:
In a study group of 30 healthy volunteers aged 21–82 years multiphoton depth-resolved measurements of autofluorescence and second harmonics have been performed in order to evaluate the dermal matrix composition.

Results:
Characteristic intensity curves depending on the penetration depth were derived and differences between age groups were found.

Conclusion:
With the present work we provide evidence for the accuracy of the measurement of dermal matrix composition by MLT and give detailed advice for the measurement procedure. Furthermore, we propose the use of depth-dependent emission intensity curves for monitoring of anti-aging treatment.
Link zum Artikel

M. Kaatz, K. König.

Multiphotonenmikroskopie und In-vivo-Multiphotonentomographie in der dermatologischen Bildgebung.

Der Hautarzt 61(2010)397-409 / DOI 10.1007/s00105-009-1880-4

Abstract

Multiphoton microscopy (MPM) and in vivo multiphoton tomography (MPT) are non-invasive examination techniques that allow for the evaluation of cellular as well as extra-cellular structures by working at a subcellular resolution level. These techniques are thus appropriate not only for clinical diagnostics but also for scientific issues in basic and applied research. MPM and MPT are based on the stimulation of biogenic fluorophores by two or more long-wave, low-energy photons and the evocation of second harmonic generation (SHG). Thus, the evaluation quality of cell clusters and tissues is similar to histological sections. At the same time the dermal fiber network can be assessed. MPT was developed further for the application in non-invasive in vivo diagnostics of skin diseases. This review presents the capabilities of multiphoton-based diagnostics in the evaluation of transcutaneous metabolism. In addition, the multiphoton techniques employed for the evaluation of physiologic and pathologic changes of the dermal fiber network as well as in the diagnosis of dermal and epidermal disorders by visual biopsy. Besides the morphological classification of benign and malignant skin tumors or allergic or inflammatory skin lesions, the techniques also allow for recording metabolic processes.
Link zum Artikel

H. G. Breunig, H. Studier, K. König.

Multiphoton excitation characteristics of cellular fluorophores of human skin in vivo.

Optics Express 18(2010)7857-7871 / DOI 10.1364/OE.18.007857

Abstract

In vivo multiphoton tomography with a wavelength-tunable femtosecond laser has been performed to investigate the autofluorescence intensity of major endogenous fluorophores of human skin in dependence on the excitation wavelength. In high-resolution multiphoton images of different skin layers, clear trends were found for fluorophores like keratin, NAD(P)H, melanin as well as for the elastin and collagen networks. The analysis of the measurements is supplemented by additional measurements of fluorescence lifetime imaging and signal-decay curves by time-correlated single-photon counting.

© 2010 OSA
Link zum Artikel

K. König, A. Uchugonova, E. Gorjup.

Multiphoton fluorescence lifetime imaging of 3D-stem cell spheroids during differentiation.

Microscopy Research and Technique DOI 10.1002/jemt.20866

Abstract

Long-term high-resolution multiphoton imaging of nonlabeled human salivary gland stem cell spheroids has been performed with submicron spatial resolution, 10.5-nm spectral resolution, and picosecond temporal resolution. In particular, the two-photon-excited coenzyme NAD(P)H and flavins have been detected by time-correlated single photon counting (TCSPC). Stem cells increased their autofluorescence lifetimes and decreased their total fluorescence intensity during the adipogenic-differentiation process. In addition, the onset of the biosynthesis of lipid vacuoles was monitored over a period of several weeks in stem-cell spheroids. Time-resolved multiphoton autofluorescence imaging microscopes may become a promising tool for marker-free stem-cell characterization and cell sorting. Microsc. Res. Tech., 2010. © 2010 Wiley-Liss, Inc.
Link zum Artikel

2009

Z. Földes-Papp, K. König, H. Studier, R. Bückle, H.G. Breunig, A. Uchugonova, G.M. Kostner.

Trafficking of mature miRNA-122 into the nucleus of live liver cells.

Current Pharmaceutical Biotechnology 10(2009)569-578 / DOI 10.2174/138920109789069332

Abstract

The binding of superquencher molecular beacon (SQMB) probes to human single-stranded cellular miRNA- 122 targets was detected in various single live cells with femtosecond laser microscopy. For delivery of the SQMBprobes, 3D-nanoprocessing of single cells with sub-15 femtosecond 85 MHz near-infrared laser pulses was applied. Transient nanopores were formed by focusing the laser beam for some milliseconds on the membrane of a single cell in order to import of SQMB-probes into the cells.
In single cells of the human liver cell lines Huh-7D12 and IHH that expressed miRNA-122, we measured target binding in the cytoplasm by two-photon fluorescence imaging. We found increased fluorescence with time in a nonlinear manner up to the point where steady state saturation was reached. We also studied the intracellular distribution of target SQMB and provide for the first time strong experimental evidence that cytoplasmic miRNA travels into the cell nucleus. To interpret nonlinear binding, a number of individual miRNA-122 positive cells (Huh-7D12 and IHH) and negative control cells, human VA13 fibroblasts and Caco-2 cells were analyzed. Our experimental data are consistent with the cytoplasmic assembly of nuclear miRNA and provide further mechanistic insight in the regulatory function of miRNAs in cellular physiology. An open issue in the regulation of gene expression by miRNA is whether miRNA can activate gene expression in addition to the well-known inhibitory effect. A first step for such a regulatory role could be the travelling of miRNA-RISC into the nucleus.
Link zum Artikel

K. König, M. Speicher, R. Bückle, J. Reckfort, G. McKenzie, J. Welzel, M. J. Koehler, P. Elsner, M. Kaatz.

Clinical optical coherence tomography combined with multiphoton tomography of patients with skin diseases.

Journal of Biophotonics 2(2009)389-397 / DOI 10.1002/jbio.200910013

Abstract

We report on the first clinical study based on optical coherence tomography (OCT) in combination with multiphoton tomography (MPT) and dermoscopy. 47 patients with a variety of skin diseases and disorders such as skin cancer, psoriasis, hemangioma, connective tissue diseases, pigmented lesions, and autoimmune bullous skin diseases have been investigated with (i) state-of-the-art OCT systems for dermatology including multibeam swept source OCT, (ii) the femtosecond laser multiphoton tomograph, and (iii) dermoscopes. Dermoscopy provides two-dimensional color images of the skin surface. OCT images reflect modifications of the intratissue refractive index whereas MPT is based on nonlinear excitation of endogenous fluorophores and second harmonic generation. A stack of cross-sectional OCT wide field images with a typical field of view of 5 × 2 mm2 gave fast information on the depth and the volume of the lesion. Multiphoton tomography provided 0.36 × 0.36 mm2 horizontal/diagonal optical sections within seconds of a particular region of interest with superior submicron resolution down to a tissue depth of 200 m. The combination of OCT and MPT provides a unique powerful optical imaging modality for early detection of skin cancer and other skin diseases as well as for the evaluation of the efficiency of treatments. (© 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim)
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M. J. Köhler, A Preller, P. Elsner, N. Kindler, K. König, Karsten, R. Bückle, M. Kaatz.

Intrinsic, solar and sunbed-induced skin aging measured in vivo by multiphoton laser tomography and biophysical methods.

Skin Research and Technology 15(2009)357-363 / DOI 10.1111/j.1600-0846.2009.00372.x

Abstract

Background:
Skin aging is accelerated by extrinsic factors, particularly actinic damage. Over the last decades, both clinical and pathological differences between intrinsic and actinic aging have been characterized. In this work, we aimed at quantifying skin aging by non-invasive in vivo methods.

Methods:
Young healthy volunteers using indoor tanning facilities and aged people were compared with appropriate controls by measurements of skin elasticity with the Cutometer and the Reviscometer and by semi-quantitative evaluation of the dermal matrix composition by the multiphoton laser tomograph DermaInspect.

Results:
We found differences between the sun-protected volar forearm and the dorsal side as well as between young and old test persons with all three methods. No significant differences were found between the skin of indoor-tanned test persons and control. Also, gender had no influence on the severity of skin aging.

Conclusion:
The most consistent results were obtained with the DermaInspect. The considerable inter-individual variation due to the cross-sectional design of the study may have disguised the factual skin damage caused by tanning beds.
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E. Dimitrow, M. Ziemer, M.J. Koehler, J. Norgauer, K. König, P. Elsner, M. Kaatz.

Sensitivity and specificity of multiphoton laser tomography for in vivo and ex vivo diagnosis of malignant melanoma.

Journal of Investigative Dermatology 129(2009)1752-1758 / DOI 10.1038/jid.2008.439

Abstract

The incidence of malignant melanoma has shown a dramatic increase over the past three decades. Patient outcome and curability depend on early diagnosis. In vivo multiphoton laser tomography represents a recently developed diagnostic tool that allows non-invasive tissue imaging. We aim to demonstrate the application of multiphoton laser tomography for the in vivo differentiation and diagnosis of melanoma. Laser radiation in the near infrared spectrum was used to image endogenous fluorophores by multiphoton excitation. Eighty-three melanocytic skin lesions have been investigated. The results showed distinct morphological differences in melanoma compared with melanocytic nevi. In particular, six characteristic features of malignant melanoma were specified and statistically evaluated. Sensitivity values up to 95% (range: 71–95%) and specificity values up to 97% (range: 69–97%) were achieved for diagnostic classification. Logistic regression analysis was performed to identify the most significant diagnostic criteria. We found that architectural disarray of the epidermis, poorly defined keratinocyte cell borders as well as the presence of pleomorphic or dendritic cells were of prime importance. By means of this procedure accuracy values up to 97% were reached. These findings underline the potential applicability of multiphoton laser tomography in melanoma diagnosis of melanocytic skin lesions.
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E. Dimitrow, I. Riemann, A. Ehlers, M.J. Köhler, J. Norgauer, P. Elsner, K. König, M. Kaatz.

Spectral fluorescence lifetime detection and selective melanin imaging by multiphoton laser tomography for melanoma diagnosis.

Experimental Dermatology 18(2009)509 - 515 / DOI 10.1111/j.1600-0625.2008.00815.x

Abstract

Abstract: Multiphoton excited tissue fluorescence summarises the emission of all naturally occurring endogenous fluorescent bio-molecules with their often overlapping fluorescence spectra. Common fluorescence intensity measurements could not be utilised to distinguish between different fluorophores or metabolic states. To overcome this limitation, we investigated new procedures of selective melanin imaging and spectral fluorescence lifetime imaging in combination with high resolution multiphoton laser tomography. Overall 46 melanocytic lesions of human skin were analysed. We suggested that fluorescence light, detected in such a way, may yield additional information for melanoma diagnostics. Remarkable differences in lifetime behaviour of keratinocytes in contrast to melanocytes were observed. Fluorescence lifetime distribution was found in correlation with the intracellular amount of melanin. Spectral analysis of melanoma revealed a main fluorescence peak around 470 nm in combination with an additional peak close to 550 nm throughout all epidermal layers. Excitation at 800 nm shows a selectively observable fluorescence of melanin containing cells and offers the possibility of cell classification. Procedures of selective imaging as well as spectral fluorescence lifetime imaging by means of multiphoton laser tomography support diagnostic decisions and may improve the process of non-invasive early detection of melanoma.
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R. Le Harzic, I. Riemann, M. Weinigel, K. König, B. Messerschmidt.

Rigid and high-numerical-aperture two-photon fluorescence endoscope.

Applied Optics 48(2009)3396-3400 / DOI 10.1364/AO.48.003396

Abstract

We present a rigid miniaturized optical system block fiber-optic two-photon endoscope based on a compact two-axis piezo scanner system and a miniature high (0.65) NA GRIN lens objective. The optical system is scanned as a whole by a piezo scanner allowing always an on-axis beam irradiation of the optical system. A photonic crystal fiber is used for excitation and ultrashort laser pulses can be delivered with typical power up to 100 mW at 800 nm. Two-photon fluorescence signal is collected by the use of a multimode fiber. Lateral resolution values for the system were experimentally measured to be 0.67 μm vertically and 1.08 μm horizontally. Axial resolution was found to be 5.8 μm. The endoscope is highly flexible and controllable in terms of time acquisition, resolution, and magnification. Fluorescence images were acquired over a 420 μm×420 μm field of view. Results presented here demonstrate the ability of the system to resolve subcellular details and the potential of the technology for in vivo applications.

© 2009 Optical Society of America
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R. Le Harzic, K. König, C. Wüllner, K. Vogler, C. Donitzky.

Ultraviolet femtosecond laser creation of corneal flap.

Journal of Refractive Surgery 25(2009)

Abstract

Purpose:
To study parameters for ocular femtosecond laser surgery in terms of process efficiency and safety aspects using ultraviolet (UV) femtosecond laser pulses.

Methods:
Studies on corneal surgery and flap processing on enucleated porcine eyes were performed using a newly developed ytterbium-doped gain media laser source. Ultraviolet femtosecond laser pulses centered at a wavelength of 345 nm and working at a repetition rate of 100 kHz were generated by the third harmonics of the 1035-nm fundamental wavelength.

Results:
Flaps with a diameter of 6 mm and a thickness of 100 µm were created in less than 2 minutes with low energy pulses. Transmissions and spectral measurements were performed during flap processing. Less than 2% UV radiation reaches the retina during corneal flap processing. A detectable transmittance towards the retina of visible light centered on 440 nm was found for UV pulses.

Conclusions:
Ultraviolet corneal refractive surgery is a novel procedure and has the potential to be an alternative to infrared refractive surgery considering safety aspects. [J Refract Surg. 2009;25:383-389.]
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2008

B.G. Wang, P.C. Lohmann, I. Riemann, H. Schubert, K.J. Halbhuber, K. König.

Multiphoton-mediated Corneal Flap Generation Using the 80 MHz Nanojoule Femtosecond Near-Infrared Laser.

Journal of Refractive Surgery 4(2008)

Abstract

Purpose:
To evaluate whether corneal flaps can be generated by the 80 MHz near-infrared, intense nanojoule femtosecond laser based on multiphoton absorption.

Methods:
A solid-state Ti:Sapphire femtosecond laser system was integrated in an inverted JenLab Femt-O-Cut laser scanning microscope. A diffraction-limited 40x objective was used to induce multiphoton ionization and plasma production. New Zealand albino rabbits and porcine eyes were used. Surgical outcomes were determined using frame and line scans with nanojoule pulses at a wavelength of 800 nm.

Results:
Surgical performance was assessed by optical imaging, histology, and electron microscopy. No significant corneal turbidity was observed. Optical imaging and histological examinations detected virtually no perturbation in the surrounding tissue. Corneal flaps and stromal lenticules were generated. Wound repair of the unlifted flaps was observed up to 90 days postoperatively.

Conclusions:
Surgical results and follow-up studies confirm that this femtosecond laser at nanojoule pulse energy is able to generate corneal flaps precisely, without causing visible collateral damage to the surrounding tissue or overlying epithelium.
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M.J. Koehler, S. Hahn, A. Preller, P. Elsner, M. Ziemer, A. Bauer, K König, R. Bückle, J.W. Fluhr, M. Kaatz.

Morphological skin ageing criteria by multiphoton laser scanning tomography : non-invasive in vivo scoring of the dermal fibre network.

Experimental Dermatology 17(2008)519-523 / DOI 10.1111/j.1600-0625.2007.00669.x

Abstract

Background:
Morphological changes in the dermal collagen and elastin fibre network are characteristic for skin ageing and for pathological skin conditions of the dermis.

Objectives:
To characterize pathological and physiological conditions by multiphoton laser scanning tomography (MLT) in vivo, it is necessary to investigate and identify morphological alterations related to ageing.

Methods:
In vivo MLT was used to image two-photon excited autofluorescence (AF) and second harmonics generation (SHG) in human dermis of 18 volunteers of different ages. Criteria for the evaluation of age-dependent morphological changes in MLT images were fibre tension and morphology, network pattern, clot formation and image homogeneity. These criteria were weighted and a score was calculated.

Results:
The resulting MLT-based Dermis Morphology Score is correlated with age (R2 = −0.90) and with the previously published SHG to AF Ageing Index of Dermis (R2 = 0.66). The two groups of young (age 21–38) and old (age 66–84) volunteers showed a significant difference in MLT score values (P < 0.001).

Conclusions:
We could demonstrate an in vivo relationship between morphological characteristics of human dermis assessed by MLT and age. The present score allows the semi-quantitative evaluation of specific morphological changes of the dermal fibre network in ageing skin by in vivo AF and SHG imaging. This method will be useful for diagnostics of pathological conditions and their differentiation from ageing effects.
Link zum Artikel

E. Dimitrow, I. Riemann, A. Ehlers, M.J. Köhler, J. Norgauer, P. Elsner, K. König, M. Kaatz.

Spectral fluorescence lifetime detection and selective melanin imaging by multiphoton laser tomography for melanoma diagnosis.

Experimental Dermatology 18(2008)509-515 / DOI 10.1111/j.1600-0625.2008.00815.x

Abstract

Multiphoton excited tissue fluorescence summarises the emission of all naturally occurring endogenous fluorescent bio-molecules with their often overlapping fluorescence spectra. Common fluorescence intensity measurements could not be utilised to distinguish between different fluorophores or metabolic states. To overcome this limitation, we investigated new procedures of selective melanin imaging and spectral fluorescence lifetime imaging in combination with high resolution multiphoton laser tomography. Overall 46 melanocytic lesions of human skin were analysed. We suggested that fluorescence light, detected in such a way, may yield additional information for melanoma diagnostics. Remarkable differences in lifetime behaviour of keratinocytes in contrast to melanocytes were observed. Fluorescence lifetime distribution was found in correlation with the intracellular amount of melanin. Spectral analysis of melanoma revealed a main fluorescence peak around 470 nm in combination with an additional peak close to 550 nm throughout all epidermal layers. Excitation at 800 nm shows a selectively observable fluorescence of melanin containing cells and offers the possibility of cell classification. Procedures of selective imaging as well as spectral fluorescence lifetime imaging by means of multiphoton laser tomography support diagnostic decisions and may improve the process of non-invasive early detection of melanoma.
Link zum Artikel

A. Uchugonova, A. Isemann, E. Gorjup, G. Tempea, R. Bückle, W. Watanabe, K. König.

Optical knock out of stem cells with extremely ultrashort femtosecond laser pulses.

Journal of Biophotonics 1(2008)463-469 / DOI 10.1002/jbio.200810047

Abstract

Novel ultracompact multiphoton sub-20 femtosecond near infrared 85 MHz laser scanning microscopes and conventional 250 fs laser microscopes have been used to perform high spatial resolution two-photon imaging of stem cell clusters as well as selective intracellular nanoprocessing and knock out of living single stem cells within an 3D microenvironment without any collateral damage. Also lethal cell exposure of large parts of cell clusters was successfully probed while maintaining single cells of interest alive. The mean power could be kept in the milliwatt range for 3D nanoprocessing and even in the microwatt range for two-photon imaging. Ultracompact low power sub-20 fs laser systems may become interesting tools for optical nanobiotechnology such as optical cleaning of stem cell clusters as well as optical transfection. (© 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim)
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K. König, M. Weinigel, D. Hoppert, R. Bückle, H. Schubert, M.J. Köhler, M. Kaatz, P. Elsner.

Multiphoton tissue imaging using high-NA microendoscopes and flexible scan heads for clinical studies and small animal research.

Journal of Biophotonics 1(2008)506-513 / DOI 10.1002/jbio.200810049

Abstract

Multiphoton tomographs based on femtosecond laser and GRIN lens technology in combination with flexible scan heads have been developed for clinical high-resolution tissue imaging and small animal research. The novel tissue tomograph possesses a 0.5 m long flexible mirror arm in combination with piezodriven focusing optics and multiple single photon counting PMT detectors. The photodetectors are in particular useful to obtain information on the extracellular matrix by the simultaneous measurement of the two-photon autofluorescence of elastin as well as the second harmonic generation of collagen. A major application is the in vivo determination of the skin age index. The rigid two-photon microendoscope with a high numerical aperture of 0.8 is based on a combination of silver-doped 1.0 mm rod-shaped GRIN lens with a hemispheric front optics. It was used in combination with the multiphoton tomograph for clinical studies as well as for inner organ and eye imaging of small animals. (© 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim)
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K. König.

Clinical multiphoton tomography.

Journal of Biophotonics 1(2008)13-23 / DOI 10.1002/jbio.200710022

Abstract

Clinical multiphoton tomography and two-photon microendoscopy provide clinicians and researchers with high-resolution in vivo optical biopsies based on two-photon autofluorescence, second harmonic generation, and fluorescence lifetime imaging. This review reflects state of the art technology and reports on applications in the fields of early stage melanoma detection, skin aging, nanoparticle imaging, tissue engineering, and in situ screening of pharmaceutical and cosmetical products. So far, more than 500 patients and volunteers in Europe, Asia, and Australia have been investigated with these novel molecular imaging tools. (© 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim)
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R. Le Harzic, M. Weinigel, I. Riemann, K. König, B. Messerschmidt.

Nonlinear optical endoscope based on a compact two axes piezo scanner and a miniature objective lens.

Optics Express 16(2008)20588-20596 / DOI 10.1364/OE.16.020588

Abstract

We report on a nonlinear optical endoscope that adopts a hollow core photonic crystal fiber for single-mode illumination delivery and a multimode one for signal collection. Femtosecond laser pulses up to 100 mW can be delivered at a centered wavelength of 800 nm. The two-photon fluorescence response of our system is shown to have axial and lateral resolutions of 5.8um and 0.6um respectively. Fluorescence detection was obtained at different wavelengths between 790 and 840 nm which could allow SHG detection for example. The maximal field-of-view of the acquired images is 420 µm x 420 µm. Detection efficiency is greater by using an avalanche photodiode in comparison to a photo multiplier tube. Results presented here demonstrate the ability of the system to resolve cellular details and the potential of the device for future in vivo imaging diagnosis

© 2008 Optical Society of America
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S.G. Toropygin, M. Krause, I. Riemann, B. Seitz, P. Mestres-Ventura, K.W. Ruprecht, K. König.

In vitro femtosecond laser-assisted nanosurgery of porcine posterior capsule.

Journal of Cataract & Refractive Surgery 34(2008)2128-2132 DOI 10.1016/j.jcrs.2008.08.038

Abstract

Purpose:
To investigate femtosecond laser–assisted nanosurgery of the posterior capsule in a prospective in vitro animal study.

Setting:
Department of Ophthalmology and Eye Hospital, University of Saarland, Homburg, and Fraunhofer Institute for Biomedical Engineering, St. Ingbert, Germany.

Methods:
The posterior capsules of 12 porcine eyes were irradiated with a nonamplified 90 MHz near-infrared 750 nm titanium:sapphire femtosecond laser. Intratissue and superficial laser cuts of laser-ablated (5 capsules) and control (1 capsule) specimens were examined by femtosecond multiphoton laser scanning microscopy (MLSM) and transmission electron microscopy (TEM).

Results:
Laser exposure time and pulse power determined the width of the lesions, which ranged from 0.69 mm G 0.19 (SD) to 2.81 G 0.5 mm. Both MLSM and TEM revealed minimal collateral alterations in the tissue surrounding the laser cuts.

Conclusions:
Nonamplified near-infrared femtosecond laser pulses at low pulse energies may be a promising strategy for precise lamellar noncontact nanosurgery of the posterior capsule, with minimal structural collateral damage to surrounding tissue. High-resolution MLSM offers 3-dimensional, noninvasive, nondestructive imaging at submicrometer resolution within seconds before and after ablation.
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S.G. Toropygin, M. Krause, I. Riemann, M. Hild, P. Mestres-Ventura, B. Seitz, E. Khurieva, K.W. Ruprecht, U. Löw, Z. Gatzioufas, K. König.

In Vitro Noncontact Intravascular Femtosecond Laser Surgery in Models of Branch Retinal Vein.

Current Eye Research 33(2008)277-283 / DOI 10.1080/02713680701875299

Abstract

Purpose:
To investigate intravenous femtosecond laser surgery in models of branch retinal vein occlusion.

Materials and Methods:
Non-amplified near infrared femtosecond laser was used to ablate polyamide sutures and human hairs inserted into the vascular lumina of porcine retinal veins in vitro. Specimens were subjected to multiphoton laser scanning microscopy and electron microscopy.

Results:
Regular laser cuts within sutures and hairs were detected with laser microscopy and electron microscopy. Neither laser microscopy nor histology revealed collateral damage of the vascular wall.

Conclusions:
Non-amplified femtosecond lasers may allow precise atraumatic non-contact intravenous retinal surgery controlled by high-resolution imaging of the target.
Link zum Artikel

M. Hild, M. Krause, I. Riemann, P. Mestres-Ventura, S.G. Toropygin, U. Löw, K. Brückner, B. Seitz, C. Jonescu-Cuypers, K. König.

Femtosecond laser-assisted retinal imaging and ablation : experimental pilot study.

Current Eye Research 33(2008)351-363 / DOI 10.1080/02713680801956452

Abstract

Purpose:
To investigate retinal imaging and ablation using femtosecond laser pulses.

Materials and Methods:
Two non-amplified near-infrared femtosecond lasers were used to irradiate porcine retinal specimens in vitro. The lasers were used for tissue removal as well as multiphoton laser scanning microscopy.

Results:
Ablation of the nerve fiber layer was performed at pulse energies of 1.0 nJ to 3.9 nJ. Control laser scanning images were acquired within seconds after irradiation. Specimens were additionally investigated with electron microscopy.

Conclusions:
Non-amplified femtosecond lasers may allow precise surgery controlled by fast high-resolution imaging of the target.
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R. Le Harzic, M. Stark, P. Becker, E. Lai, D. Bruneel, F. Bauerfeld, D. Sauer, T. Velten, K. König.

Nanostructuring with nanojoule femtosecond laser pulses.

Journal of Laser Micro/Nanoengineering 3(2008)106-113

Abstract

Sub-wavelength multiphoton nanoprocessing of silicon wafers, direct nanowriting of metals, boro-silicate glass or polymers, laser processing for nanofluidics applications as well as 3D maskless li-thography by two-photon polymerization have been performed using several compact near infrared MHz femtosecond lasers as tuneable turn-key, one-box Chameleon (coherent) and Mai-Tai (spectra physics) oscillators as well as a special femtoTrain system (High Q laser production GmbH). The lasers were coupled with the scanning microscopes FemtOcut (JenLab GmbH) and a modified ZEISS LSM510-NLO system. MHz femtosecond laser pulses with nanojoule photon energies can be considered as novel tools for nanoprocessing in material science, nanobiotechnology and nanomedicine.
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A. Uchugonova, K. König.

Two-photon autofluorescence and second-harmonic imaging of adult stem cells.

Journal of Biomedical Optics 13(2008)054068 / DOI 10.1117/1.3002370

Abstract

Human and animal stem cells (rat and human adult pancreatic stem cells, salivary gland stem cells, and human dental pulp stem cells) are investigated by femtosecond laser 5-D two-photon microscopy. Autofluorescence and second-harmonic generation (SHG) are imaged with submicron spatial resolution, 270 ps temporal resolution, and 10 nm spectral resolution. In particular, the reduced coenzyme nicotinamide adenine (phosphorylated) dinucleotide [NAD(P)H] and flavoprotein fluorescence is detected in stem cell monolayers and stem cell spheroids. Major emission peaks at 460 and 530 nm with typical long fluorescence lifetimes (2) of 1.8 and 2.0 ns, respectively, are measured using spectral imaging and time-correlated single photon counting. Differentiated stem cells produce the extra cellular matrix (ECM) protein collagen, detected by SHG signals at 435 nm. Multiphoton microscopes may become novel noninvasive tools for marker-free optical stem cell characterization and for on-line monitoring of differentiation within a 3-D microenvironment.

©2008 Society of Photo-Optical Instrumentation Engineers
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A. Uchugonova, K. König, R. Bückle, A. Isemann, G. Tempea.

Targeted transfection of stem cells with sub-20 femtosecond laser pulses.

Optics Express 16(2008)9357-9364 / DOI 10.1364/OE.16.009357

Abstract

F. Garbe, U. Bauerschäfer, A. Csaki, A. Steinbrück, K. Ritter, A. Bochmann, J. Bergmann, A. Weise, D. Akimov, G. Maubach, K. König, G. Hüttmann, W. Paa, J. Popp, W. Fritzsche.

Optically controlled thermal management on the nanometer length scale.

Nanotechnology 19(2008) 055207 / DOI 10.1088/0957-4484/19/05/055207

Abstract

eine Liste aller Publikationen finden sie hier.

Proceedings
1. K. König, H. Schneckenburger, A. Rück, S. Auchter: Photoproduct formation of endogeneous protoporphyrin and its photodynamic activity. SPIE-Proceedings vol. 1525(1991)412-419 2. K. König, A. Rück, S. Auchter, W. Strauss, H. Schneckenburger: Photoinduced reactions of porphyrin photosensitizers. (A) Hematoporphyrin Derivative (HpD). In: Laser in Medicine, eds. W. Waidelich, R. Waidelich, A. Hofstetter, Springer Verlag 1992, 117-121 3. W. Strauss, A. Rück, T. Köllner, K. König, H. Schneckenburger: Photoinduced reactions of porphyrin photosensitizers. (B) Hydrophilic meso-tetraphenylporphyrins. In: Laser in Medicine, eds. W. Waidelich, R. Waidelich, A. Hofstetter, Springer Verlag 1992, 122-127 4. K. König, J. Hemmer, H. Schnecknburger: Laser-induced autofluorescence of squamous cell carcinoma. In: P. Spinelli, M. Dal Fante, R. Marchesini: Photodynamic Therapy and Biomedical Lasers. Elsevier Science Publishers (1992)903-906. 5. K. König et al.: Photodynamic therapy with texaphyrins. In: P. Spinelli, M. Dal Fante, R. Marchesini: Photodynamic Therapy and Biomedical Lasers. Elsevier Science Publishers (1992) 6. K. König, H. Schneckenburger, A. Rück, H. Meyer: Fluorescence Diagnosis and Photodynamic Therapy of Acne vulgaris. In: P. Spinelli, M. Dal Fante, R. Marchesini: Photodynamic Therapy and Biomedical Lasers. Elsevier Science Publishers (1992) 7. K. König, F. Nowak, F. Genze, H. Schneckenburger: In-vivo autofluorescence measurements during photodynamic damage of cells and tumor tissue. In: Laser in Medicine 1993, W. Waidelich, R. Waidelich, A. Hofstetter (eds.), Springer Verlag, 1994, 95-99. 8. K. König, K. Kunzi-Rapp: On-line measurement of photodynamically induced lysis of erythrocytes with and without nucleus by small angle light scattering and video-intensified microscopy. In: Laser in Medicine 1993, eds. W. Waidelich, R. Waidelich, A. Hofstetter, Springer Verlag, 1994, 91-94. 9. R. Sailer, W. Strauss, K. König, A. Rück, R. Steiner: Untersuchungen zu Porphyrinstoffwechsel and photodynamische Inaktivierung am Gram-negativen Bakterium Pseudomonas aeruginosa. In: Laser in Medicine 1993, W. Waidelich, R. Waidelich, A. Hofstetter (ed.), Springer Verlag, 109-112 10. H. Schneckenburger, K. König, T. Dienersberger, R. Hahn: Time-gated video microscopy and spectroscopy. In: Laser in Medicine 1993, eds. W. Waidelich, R. Waidelich, A. Hofstetter, Springer Verlag,1994,497-501 11. G. Beck, K. König, R. Steiner: Optical detection of topically applied photosensitizers by in vivo remission spectroscopy. In: Laser in Medicine 1993, eds. W. Waidelich, R. Waidelich, A. Hofstetter, Springer Verlag, 1994, 469-472 12. W. Strauss, W. Mohr, K. König, K. Miller, R. Sailer, M. Gschwend, A. Rück, H. Schneckenburger, R. Steiner: MesoTetra(4-Carboxyphenyl)Porphyrin-Organverteilung und photodynamische Therapie. In: Laser in Medicine 1993, eds. W. Waidelich, R. Waidelich, A. Hofstetter, Springer Verlag,1994, 104-108 13. K. König, R. Hibst, H. Schneckenburger, G. Flemming: Laserinduced autofluorescence of caries. SPIE -Proceedings, vol. 1880(1993)125-131 14. K. König, H. Schneckenburger, A. Rück, R. Steiner, H. Walt:Autofluorescence of cells and tissue. SPIE-Proceedings, vol. 1887: "Physiological Imaging, Spectroscopy, and Early-Detection Diagnostic Methods", 1993, 213-221 15. K. König, R. Hibst, H. Meyer, G. Flemming, H. Schneckenburger: Laser-induced autofluorescence of carious regions of human teeth and caries-involved bacteria. SPIE-Proceedings, vol. 2080. In Druck 16. K. König, A. Kienle, W-H. Boehncke, R. Kaufmann, A. Rück, T. Meier, R. Steiner: Photodynamic tumour therapy and on-line fluorescence spectroscopy after ALA administration using 633 nm-light as therapeutic and fluorescence excitation radiation. SPIE-Proceedings, vol. 2078. In Druck 17. K. König, G. Beck, W-H Boehncke, R. Kaufmann, R. Hibst: In vivo remission spectroscopy on tattoos and topically applied photosensitizers in man, SPIE-Proceedings, vol. 2086. In Druck 18. K. König, W-H. Boehncke, A. Rück, R. Kaufmann, R. Steiner, W. Sterry: Photodynamic effects on T-cells and skin lesions of a patient with mycosis fungoides using porphyrin photosensitizers. SPIE-Proceedings, vol. 2086. In Druck 19. H. Schneckenburger, M. Gschwend, K. König, A. Rück, R. Sailer, W. Strauss: Subcellular distribution of photodynamic photosensitizers. SPIE-Proceedings, vol. 2086, in press 20. K. König, H. Schneckenburger, H. Walt, T. Leemann, M.T. Wyss-Desserich, A. Rück,B. Tromberg: Microscopic Studies on ALA-incubated tumor cells and tumor spheroids.SPIE-Proceedings, vol. 2133: Optical Methods for Tumor Treatment and Detection, 238-248 21. K. König, H. Schneckenburger, J. Hemmer, B. Tromberg, R. Steiner: In-vivo fluorescence detection and imaging of porphyrin-producing bacteria in the human skin and in the oral cavity for diagnosis of acne vulgaris, caries, and squamous cell carcinoma. SPIE -Proceedings, vol. 2135: Advances in Laser and Light Spectroscopy to Diagnose Cancer and other Diseases, 129-138 22. K. König, H. Schneckenburger: Laser-induced dental caries and plaque diagnosis on patients by sensitive autofluorescence spectroscopy and time-gated video imaging. Preliminary studies. SPIE-Proceedings, vol. 2128: Lasers in Surgery. In Druck 23. K. König, H. Schneckenburger, A. Rück, R. König: Studies on Porphyrin Photoproducts in Solution, Cells, and Tumor Tissue. SPIE-Proceedings 2133: Optical Methods for Tumor Treatment and Detection, 226-237 24. K. König, H. Schneckenburger, H. Boehncke, R. Hibst: In vivo fluorescence spectroscopy and imaging of ALA-induced endogenous porphyrins in skin after Er:YAG ablation of stratum corneum. SPIE-Proceedings, vol. 2128: Lasers in Surgery. In Druck. 25. K. König: NAD(P)H and porphyrin attributed laser induced autofluorescence. Proceedings of the International Conference: Lasers & Applications. Advances in science, medicine and technology, NILES 1994, Cairo, March 26-30, 1994. In Druck. 26. K. König, Y. Liu, GJ. Sonek, MW. Berns, BJ. Tromberg: Photoinduced autofluorescence modifications of cells in an optical trap. SPIE-Proceedings, vol. 2329: "Optical and Imaging Techniques in Biomedicine". 193-203.. 27. S. Kimel, K. König, MW. Berns: Photodynamic effects on human and chicken erythrocytes. SPIE-Proceedings, vol. 2329: "Optical and Imaging Techniques in Biomedicine". 269-279. 28. K. König, Y. Liu, T. Krasieva, P. Patrizio, Y. Tadir, GJ. Sonek, MW. Berns, BJ. Tromberg. Invited Paper: Fluorescence imaging and spectroscopy of motile cells and CHO cells in an optical trap ("optical tweezers"). SPIE-Proceed. "Laser-Tissue Interaction VI", vol. 2391, 238-249 29. K. König, P. So, WW. Mantulin, E. Gratton, T. Krasieva, MW. Berns, BJ Tromberg. Two-photon excited cellular autofluorescence induced by cw- and femtosecond NIR microradiation. SPIE-Proceed. vol. 2628: 12-19. 30. K. König, T.Krasieva, E. Bauer, U. Fiedler, MW. Berns, BJ. Tromberg, KO. Greulich.UVA-Induced Oxidative Stress in Single Cells Probed by Autofluorescence Modifications, Cloning Assay, and Comet Assay. SPIE-Procced. vol. 2628: 43-49. 31. KO. Greulich, E. Bauer, U. Fiedler, C. Hoyer, K. König, S. Monajembashi. Single cell and single cell biotechnology. SPIE-Proceed. vol. 2629. In Druck. 32. L. Norwang, K. König, L. Svaasand. Comparison between reflectance spectra obtained with an integrating sphere and a fiber-optic collection system. SPIE-Procced. vol. 2624. In Druck. 33. K. König, P. Fergin, MW. Berns, BJ. Tromberg. Rapid spectrally-resolved fluorescence imaging of skin after topical ALA-administration. In: Laser in Medicine 1995, eds. W. Waidelich, G. Staehler, R. Waidelich, Springer Verlag. 587-590. 34. K. König, BJ. Tromberg, MW. Berns. One- and Two Photon Excited Fluorescence of Motile Cells in the Optical Trap. In: Laser in Medicine 1995, eds. W. Waidelich, H. Hügel, H. Opower, H. Tiziani, R. Wallenstein, W. Zinth, Springer Verlag. 164-167. 35. H. Schneckenburger, M. Gschwend, K. König, R. Sailer, W. Strauss. Fluorescence Lifetime Imaging and Spectroscopy in Photobiology and Photomedicine. In: "Fluorescence Microscopy and Fluorescent Probes", ed. J. Slavik, Plenum Press, New York - London. In Druck. 36. H. Schneckenburger, M. Gschwend, K. König, K. Kunzi-Rapp, R. Sailer, W. Strauss. Laser in der Diagnostik am Beispiel der Fluoreszenz-Diagnostik und Laser-Mikroskopie. In: "Lasertechnik und Lasermedizin", ed. H.D. Reichenbach, Ecomed-Verlag, Lundsberg, In Druck. 37. K. König, T. Krasieva, Y. Liu, M.W. Berns, B.J. Tromberg. Invited Paper: Two-photon excitation in living cells induced by low-power CW laser beams. SPIE Proceed.: Optical Diagnostics of Living Cells and Biofluids. Vol. 2678 (1996) 30-37. 38. K. König. Invited Paper: Porphyrin- and NAD(P)H attributed autofluorescence for medical diagnosis. Proceedings "LASER 95" of the Society for Optical and Quantum Electronics.In Druck. 39. K. König, P. So, W.W. Mantulin, E. Gratton: Cell damage in Two-Photon Microscopes. SPIE-Proceed.1996: In Druck. 40. K. König, L. Svaasand, Y. Tadir, B. Tromberg, M.W. Berns: Optical determination of motility forces in human spermatozoa with laser tweezers. SPIE-Proceed.1996: In Druck. 41. K. König, S. Kimel, L.O. Svaasand, B. Tromberg, T. Krasieva, M.W. Berns, P. So, W.W. Mantulin, E. Gratton, K.J. Halbhuber: Cell damage in UVA and cw/femtosecond NIR microscopes. SPIE-Proceed.1997, vol. 2984: 37-44. 42. K. König, K.-J. Halbhuber: Zwei-Photonen-Femtosekundenmikroskopie vitaler Zellen. In: W. Waidelich, R. Waidelich, A. Hofstetter (eds.). Laser in Medicine. Proceedings of the 13th International Congress LASER 97. Springer-Verlag. In Druck. 43. K. König, H. Oehring, K.-J. Halbhuber, U. Fiedler, E. Bauer, K.O. Greulich. Comet assay, cloning assay, histochemistry, light- and electron microscopy on one preselected cell. SPIE-Proceed.1997, vol. 3199: 148-155. 44. K. König. Laser tweezers as novel nonlinear tools in cell and biomolecule diagnostics. SPIE-Proceed.1997, vol. 3199: 178-182. 45. K. König. How safe is the gamete micromanipulation by laser tweezers? SPIE-Proceed.1998, vol. 3260: In Druck. 46. K. König, M. Teschke, W. Pfister, H. Meyer: Photodynamically inactivation of propionibacterium acnes. SPIE-Proceed.1998, vol. 3247: In Druck. 47. K. König. Invited paper: Cloning assay thresholds on cells exposed to ultrafast laser pulses. SPIE-Proceed.1999, vol. 3616: In Druck. 48. K. König, P. Fischer. I. Riemann, K.-J. Halbhuber. PDT by non-resonant two-photon excitation.SPIE-Proceed.1999, vol. 3592: In Druck. 49. K. König, P. Fischer. I. Riemann, K.-J. Halbhuber. Intracellular nanosurgery with compactfemtosecond laser. In: 18^th Congress of the International Commission for Optics: Optics for the next Millenium, SPIE-Proceed.1999, vol. 3749, Seite 390. 50. K. König. Applikationen der optischen Mikromanipulation und Zweiphotonenanregung vitaler Zellen mittels Naher Infrarot Mikroskopie. Shaker-Verlag. Aachen.1999. ISBN: 3-8265-6788-9. 51. K. König. Lasertechnik. Chapter in: Laser in der Dermatologie. 2000. In Press. 52. K. König, I. Riemann, A. Göhlert, P. Fischer, T. Liehr, I.F. Loncarevic, U. Claussen, K.J. Halbhuber. Multiphoton Multicolor FISH. SPIE-Proceed.2000, vol. 4164: In Druck. 53. K. König. Chapter: Cellular response to laser radiation in fluorescence microscopes. In: Methods in Cellular Imaging. Editor: A. Perisamy. Oxford University Press. 2001, 236-250. 54. C.Y. Dong, E.A. Bevan, L. Hsu, K. König, P.T.C. So. Invited paper: Characterization of two-photon point spread function in turbid medium by direct measurements, multicolocr imaging, and blind deconvolution. SPIE-Proceed. 4262(2001)73-81. 55. W. Becker, A. Bergmann, K. König, U. Tirlapur. Picosecond Fluorescence Lifetime Microscopy by TCSPC Imaging. SPIE-Proceed. 4262(2001)414-419. 56. K. König, U.K. Tirlapur: Chapter: Cellular and subcelluar pertubations during multiphoton microscopy. In: Confocal and two-photon microscopy. Editor: A. Diasporo. John Wiley $ Sons, Inc. 2002, 191-206 + cover page for the book. 57. U.K. Tirlapur, K. König: Two-photon near infrared femtosecond laser scanning microscopy in plant biology. In: Confocal and two-photon microscopy. Editor: A. Diasporo. John Wiley & Sons, Inc. 2002, 449-468. 58. K. König, I. Riemann, O. Krauss, W. Fritzsche. Invited paper: Nanodissection of human chromosomes and ultraprecise eye surgery with anojoule near infrared femtoseond laser pulses. SPIE-Proceed. 4633(2002)11-22. 59. K. König, U. Wollina, I. Riemann, C. Peuckert, K.J. Halbhuber, V. Fünfstück, T.W. Fischer, P. Elsner. Optical tomography of human skin with subcellular spatial and picosecond time resolution. SPIE-Proceed. 4620(2002)191-201. 60. K. König, I. Riemann, U.K. Tirlapur. Optical gene transfer by femtosecond laser pulses. SPIE-Proceed. 4963(2003)81-88. 61. K. König. High-resolution multiphoton imaging and nanosurgery of the cornea using femtosecond laser pulses. In: Lasers in ophthalmology. Basics, diagnostic and surgical aspects. Eds. F. Fankhauser & S. Kwasniewska. Kugler Publications pp. 79-89 (2003) ISBN: 9062991890. 62. K. König, B. Wang, O. Krauss, I. Riemann, H. Schubert, S. Kirtse, P. Fischer. First in vivo animal studies on intraocular nanosurgery and multiphoton tomography with low-energy 80 MHz near infrared femtosecond laser pulses. SPIE-Proceed. 5314 (2004) 63. K. König, I. Riemann, G. Ehrlich, V. Ulrich, P. Fischer. Multiphoton FLIM and spectral imaging of cells and tissues. SPIE-Proceed. (2004) 64. F. Fischer, K. König, S. Puschmann, R. Wepf, I. Riemann, V. Ulrich, P. Fischer. Characterization of multiphoton laser scanning device optical parameters for image restoration. SPIE-Proceed. (2004) in press 65. I. Riemann, P. Fischer, K. König. Photodynamic therapy and knocking out of single tumor cells by multiphoton excitation processes. SPIE-Proceed. (2004) in press 66. I. Riemann, E. Dimitrow, P. Fischer, A. Reif, M. Kaatz, P. Elsner, K. König. High resolution multiphoton tomography of human skin in vivo and in vitro. SPIE-Proceeding 5312 (2004) 67. W. Becker, A. Bergmann, G. Biscotti, K. König. High-Speed FLIM Data Acquisition by Time-Correlated Single Photon Counting. SPIE-Proceeding 5323 (2004). 68. K.Schenke-Layland, I. Riemann, V. Ulrich, F. Opitz, U. A. Stock, K. König. Multiphoton imaging of collagen and elastin of native and tissue-engineered heart valves. SPIE-Proceeding 5323 (2004). 69. K. Schenke-Layland, I. Riemann, U. A. Stock, K. König. Non-invasive multiphoton imaging of cardiovascular structures using NIR femtosecond laser scanning microscopy. SPIE-Proceeding 5312 (2004)300-308. 70. I. Riemann, P. Fischer, K. König. In vivo multiphoton tomography of melanoma. SPIE-Procced (2004). 71. K. König. Multiphoton tomography, transfection and nanosurgery with <2nJ, 80 MHz Femtosecond Laser Pulses. SPIE-Proceed. 5340(2004)37-46. 72. K. König: Femtosecond laser application in biotechnology and medicine. LPM2004 in Nara, Japan. SPIE-Proceed. 5662(2004)255-267. 73. T. Velten, H. Schuck, I. Riemann, F. Bauerfeld, D. Sauer, K. König. Time-Resolved and Spectrally-Resolved 5D Multiphoton Microscopy for Analysis and Nanoprocessing of Biological and Non-Biological Materials, LANE 2004. 74. K. König, F. Garwe, A. Czaki, G. Maubach, I. Riemann, W. Fritzsche. Nanoprocessing of DNA with femtosecond laser. SPIE-Proceed. 5462(2004)27- 75. I. Riemann, P. Fischer, M. Kaatz, T.W. Fischer, P. Elsner, E. Dimitrov, A. Reif, K. König. Optical Tomography of pigmented human skin biopsies. SPIE-Proceed. 5312(2004)24-34. 76. A. Czaki, G. Maubach, F. Garwe, A. Steinbrück, K. König, W. Fritzsche. A novel DNA restriction technology based on laser pulse energy conversion on sequence-specific bound metal nanoparticles. SPIE-Proceed. 2005. 77. W. Fritzsche, A. Czaki, A. Steinbrück, F. Garwe, K. König, M. Raschke. Metal nanoparticles as passive and active tools for bioanalytics. SPIE-Proceed. 2005. 78. T. Anhut, K. Hassler, T. Lasser, K. König, R. Rigler. Fluorescence Correlation Spectroscopy on dielectric surfaces in total internal reflection geometries. SPIE-Proceed. 2005. 79. B. Wang, I. Riemann, H. Schubert, S. Kirste, K. König. In vivo animal follow-up studies on intrastromal surgery with near infrared nanojoule femtosecond laser pulses. SPIE-Proceed. 2005. 80. K. König, I. Riemann, H. Schuck, D. Sauer, T. Velten, R. LeHarzic. Time-resolved and spectrally resolved 5D multiphoton microscopy for analysis and nanoprocessing of materials. SPIE-Proceed. 2005. 81. I. Riemann, A. Ehlers, A. Reif, J. Kobow, K. König. In vivo multiphoton tomography of skin as a tool to study the effects of topically applied probes and UV exposure. SPIE-Proceed. 2005. 82. I. Riemann, T. Anhut, F. Stracke, R. LeHarzic, K. König. Multiphoton nanosurgery in cells and tissues. SPIE-Proceed. 2005. 83. I. Riemann, E. Dimitrow, M. Kaatz, J. Fluhr, P. Elsner, J. Kobow, K. König. In vivo multiphoton tomography of inflammatory tissue and melanoma. SPIE-Proceed. 2005. 84. K. König, B. Wang, I. Riemann, J. Kobow. Cornea surgery with nanojoule femtosecond laser pulses. SPIE-Proceed. 2005. 85. W. Becker, K. König. Fluorescence lifetime images and correlation spectra obtained by multi-dimensional TCSPC. SPIE-Proceed. 2005. 86. K. König, H. Schuck, D. Sauer, F. Bauerfeld, F. Stracke, T. Velten, A. Tchernook, S. Martin, R. LeHarzic. Invited paper: Femtosecond laser nanoprocessing using near infrared nanojoule pulses at MHZ repetition frequency. SPIE-Proceedings, vol. 6400. 87. K. König, I. Riemann, A. Ehles, R. Bückle, E. Dimitrow. In vivo multiphoton tomography of skin cancer. SPIE-Proceedings, vol. 6089. 88. B.G. Wang, K. König, I. Riemann, H. Schubert, K.-J. Halbhuber. Multiphoton imaging of corneal tissue with near-infrared femtosecond laser pulses: corneal optical tomography and ist use in refractive surgery. SPIE-Proceedings, vol. 6089. 89. R. LeHarzic, S. Martin, R. Bückle, C. Wullner, C. Donitzky, I. Riemann, K. König. New developments in corneal refractive surgery with femtosecond laser pulses. SPIE-Proceed. 2006, vol. 6138. 90. I. Riemann, K. Schenke-Layland, A. Ehlers, E. Dimitrov, M. Kaatz, P. Elsner, S. Martin, K. König. High-resolution multiphoton optical tomography of tissues – an in vitro and in vivo study. SPIE-Proceedings, vol. 6142. 91. I. Riemann, F. Stracke, D. Sauer, S. Martin, K. König. Multiphoton nanosurgery in cells and tissues. SPIE-Proceedings, vol. 6089. 92. A. Ehlers, I. Riemann, T. Anhut, M. Kaatz, P. Elsner, K. König. Fluorescence lifetime imaging of human skin and hair. SPIE-Proceedings, vol. 6089. 93. A. Csaki, F. Garwe, A. Steinbrück, A. Weise, K. König, W. Fritzsche. Localization of laser energy conversion by metal nanoparticles: basic effects and applications. SPIE- Proceedings, vol. 6191. 94. B. Messerschmidt, A. Kraeplin, S. Schenkl, I. Riemann, M. Stark, A. Ehlers, A. Tchernook, R. LeHarzic, K. König. Novel concept of GRIN optical systems for high resolution microendoscopy: Part 1. Physical aspects. SPIE-Proceedings, vol. 6432. 95. A. Uchugonova, I. Riemann, F. Stracke, E. Gorjup, R. LeHarzic, K. König. The influence of NIR femtosecond alser radiation on the viability of 3D stem cell clusters and tumor spheroids. SPIE-Proceedings, vol. 6442. 96. A. Ehlers, S. Schenkl, I. Riemann, B. Messerschmidt, M. Kaatz, R. Bückle, K. König. In vivo multiphoton endoscopy of endogenous skin fluorophores. SPIE-Proceedings, vol. 6442. 97. F. Stracke, M. Schneider, B. Weiss, C.-M. Lehr, U. F. Schäfer, K. König. Multiphoton microscopy for the investigation of trans-cutaneous drug delivery. SPIE-Proceedings, vol. 6630. 98. M. Stark, B. Manz, I. Riemann, F. Volke, W. Weschke, K. König. Multiphoton and magnetic resonance imaging of barley embryos: comparing micro-imaging techniques across scale and parameter barriers. SPIE-Proceedings, vol. 6442. 99. M. Stark, D. Dörr,A. Ehlers, D. Sauer, R. Bückle, S. Martin, F. Ehrhart, J. Baunach, A. Katsen-Globa, H. Zimmermann, K. König. Multiphoton imaging and fluorescence lifetime studies on unstained cells and tissue at cryogenic conditions. SPIE-Proceedings, vol. 6628. 100. I. Riemann, A. Ehlers, R. LeHarzic, S. Martin, A. Reif, K. König. In vivo multiphoton tomography of skin during wound healing and scar formation. SPIE-Proceedings, vol. 6442. 101. I. Riemann, A. Ehlers, D. Dill-Müller, S. Martin, K. König. Multiphoton tomography of skin tumors after ALA application. SPIE-Proceedings, vol. 6424. 102. I. Riemann, F. Stracke, A. Uchugonova, S. Martin, R. Bückle, K. König. Optical nanoinjection into cells and 3D stem cell-clusters via a NIR femtosecond laser. SPIE-Proceedings, vol. 6442. 103. H. Schuck, F. Bauerfeld, D. Sauer, R. LeHarzic, T. Velten, I. Riemann, K. König. Rapid prototyping of 3D micro- nanostructures to explore cell behaviour. Vortrag anlässlich der 3rd International Conference on Multi-Material Micro Manufacture (4M) in Borovets (Bulgarien), 03.-05.10.2007 Proceedings (2007). 104. S. Schenkl, A. Ehlers, I. Riemann, B. Messerschmidt, K. König. Rigid and high NA fluorescence GRIN-endoscopes. SPIE-Proceedings, vol. 6631. 105. S. Schenkl, E. Weiss, M. Stark, F. Stracke, I. Riemann, R. Lemor, K. König. Imaging living cells with a combined high-resolution multi-photon-acoustic microscope. SPIE-Proceedings, vol. 6437. 106. S. Schenkl, A. Ehlers. I. Riemann, B. Messerschmidt, R. Bückle, K. König. Applications of rigid and flexible GRIN-endoscopes. SPIE-Proceedings, vol. 6433. 107. K. König, A. Ehlers, I. Riemann, S. Schenkl, B. Messerschmidt, R. Bückle, R. Le Harzic, P. Elsner, M. Kaatz. SPIE-Proceedings, 6442. 108. R. Le Harzic, C. Wüllner, C. Donitzky, K. König. New developments in femtosecond laser corneal refractive surgery. SPIE-Proceedings, vol. 6460.. 109. R. Le Harzic, C. Wüllner, D. Bruneel, C. Donitzky, K. König. Femtosecond refractive eye surgery: study of laser parameters for even more efficiency and safety. SPIE-Proceedings, vol. 6633. 110. R. Le Harzic, A. Colonna, R. Bückle, A. Ehlers, C. Hadjur, F. Leroy, F. Flament, R. Bazin, B. Piot, I. Riemann, K. König. In vivo multiphoton tomography: a non invasive powerful tool for biochemical investigation of human skin. SPIE-Prooceedings, vol. 6630. 111. F. Ehrhart, D. Dörr, M. Stark, K. König, H. Zimmermann. Laser assisted processing of cross-linked alginate hydrogel. WLT Conference, München 2007. 112. M Stark, D Dörr, F Ehrhart, J Schulz, J Baunach, A Katsen-Globa, A Ehlers, K König, H Zimmermann. Multiphoton Fluorescence Imaging at Cryogenic Conditions. Microscopy and Microanalysis / Volume13 / SupplementS03 / September 2007, pp 110-111 113. A. Uchugonova, J. Müller, R. Bückle G. Tempea, A. Isemann, A. Stingl, K. König. Negatively chirped laser enables nonlinear excitation and nanoprocessing with sub-20-fs pulses. Proc. SPIE 6860. 114. A. Uchugonova, K. König. Two-photon imaging of stem cells. Proc. SPIE 6860. 115. P. Becker, D. Sauer, F. Bauerfeld, K. König, R. LeHarzic. Surface and bulk micro- and nano-structering with nanojoule femtosecond laser pulses at high repetition rate. Proc. SPIE 6879. 116. K. König. Multiphoton tomography for tissue engineering. Proc. SPIE 6858. 117. K. König, J. Müller, M. Höfer, C. Müller, M. Weinigel, R. Bückle, P. Elsner, M. Kaatz, B. Messerschmidt. Invited review: Two-photon scanning systems for clinical high-resolution in vivo tisuse imaging. Proc. SPIE 6860. 118. I. Riemann, S. Schenkl, R. LeHarzic, D. Sauer, A. Ehlers, B. Messerschmidt, R. Bückle, K. König. Two-photon imaging using a flixible endoscope. Proc. SPIE 6851. 119. I. Riemann, A. Ehlers, R. LeHarzic, E. Dimitrow, M. Kaatz, P. Elsner, R. Bückle, K. König. Non-invasive analysis/diagnosis of human normal and melanoma skin tissues with two-photon FLIM in vivo Proc. SPIE 6842. 120. V.K. Pustovalov, K. König, L.G. Astafyeva, W. Fritzsche. Optical properties of core-shell gold-silver and silver-gold nanoparticles for some laser wavelengths. Proc. SPIE 6879. 121. V.K. Pustovalov, K. König, L.G. Astafyeva. Distributions of laser radiation intensity inside gold nanoparticles during laser radiation. Proc. SPIE 6879. 122. K. König, J. Müller, M. Höfer, C. Müller, M. Weinigel, R. Bückle, P. Elsner, M. Kaatz, B. Messerschmidt: Two-photon scanning systems for clinical high resolution in vivo tissue imaging. Proc. SPIE, Vol. 6860. 123. K. König, R. Bückle, M. Weinigel, P. Elsner, M. Kaatz: Clinical multiphoton tomography and clinical two-photon microendoscopy. Proc. SPIE 7183. 124. K. König, R. Bückle, M. Weinigel, J. Köhler, P. Elsner, M. Kaatz: In vivo multiphoton tomography in skin aging studies. Proc. SPIE 7161. 125. M. Schwarz, I. Riemann, M. Weinigel, K. König, B. Messerschmidt, R. Le Harzic: New developments in two photon endoscopy. Proc. SPIE 7172. 126. A. Uchugonova, A. Isemann, R. Bückle, W. Watanabe, K. König: Two-photon imaging and nanoprocessing of stem cells with sub-20 fs laser pulses. Proc. SPIE 7183. 127. D. Bruneel, M. Schwarz, E. Audouard, K. König, R. Le Harzic: Development of a powerful tool for nanostructuring and multiphoton imaging with nanojoule femtosecond laser pulses. Proc. SPIE 7201. 128. I. Riemann, M. Schwarz, F. Stracke, A. Ehlers, E. Dimitrow, M. Kaatz, K. König, R. Le Harzic: New developments in two-photon analysis of human skin in vivo. Proc. SPIE 7203. 129. K. König, M. Weinigel, H. G. Breunig, A. Gregory, P. Fischer, M. Kellner-Höfer, R. Bückle, M. Schwarz, I. Riemann, F. Stracke, V. Huck, C. Gorzelanny, S. W. Schneider: 5D-intravital tomography as a novel tool for non-invasive in-vivo analysis of human skin. Proc. SPIE 7555. 130. K. König, A. Uchugonova, M. Schug, H. Zhang, S. Saremi, D. Feili, H. Seidel: Two-photon lithography and nanoprocessing with picojoule extreme ultrashort 12 femtosecond laser pulses. Proc. SPIE 7584. 131. A. Uchugonova, Z. Földes-Papp, G. M. Kostner, K. König: Long-term marker-free multiphoton imaging, targeted transfection, optical cleaning of stem cell clusters, and optical transport of microRNA with extreme ultrashort laser pulses. Proc. SPIE 7569. 132. K. König, M. Weinigel, H. G. Breunig, A. Gregory, P. Fischer, M. Kellner-Höfer, R. Bückle: Current developments in clinical multiphoton tomography. Proc. SPIE 7569. 133. M. Schwarz, I. Riemann, F. Stracke, V, Huck, C. Gorzelanny, S. W. Schneider, K. König, S. Puschmann, V. Lutz, N. Sommer, C. Rahn, S. Gallinat, H. Wenck, K.-P. Wittern, F. Fischer: A comparative study of different instrumental concepts for spectrally and lifetime-resolved multiphoton intravital tomography (5D-IVT) in dermatological applications. Proc. SPIE 7568. 134. K. König, M. Speicher, M. J. Koehler, R. Scharenberg, P. Elsner, M. Kaatz: Clinical combination of multiphoton tomography and high frequency ultrasound imaging for evaluation of skin diseases. Proc. SPIE 7564. 135. V. Huck, C. Gorzelanny, K. Thomas, V. Niemeyer, T. A. Luger, K. König, S. W. Schneider: Intravital multiphoton tomography as a novel tool for non-invasive in vivo analysis of human skin affected with atopic dermatitis. Proc. SPIE 7548. 136. K. König, M. Speicher, R. Bückle, J. Reckfort, G. McKenzie, J. Welzel, M. J. Koehler, P. Elsner, M. Kaatz: Clinical optical coherence tomography combined with multiphoton tomography for evaluation of several skin disorders. Proc. SPIE 7554. 137. H. G. Breunig, H. Studier, K. König: Excitation-wavelength dependence of multiphoton excitation of fluorophores of human skin in vivo. Proc. SPIE 7548. 138. H. Studier, H. G. Breunig, K. König: Two-photon imaging with 80 MHz and 1-GHz repetition rate Ti:sapphire oscillators. Proc. SPIE 7569. 139. M. Weinigel, H. G. Breunig, A. Gregory, P. Fischer, M. Kellner-Höfer, R. Bückle, K. König: A novel flexible clinical multiphoton tomograph for early melanoma detection, skin analysis, testing of anti-age products, and in situ nanoparticle tracking. 140. K. König, A. Uchugonova, M. Straub, H. Zhang, M. Afshar, D. Feili, H. Seidel: Sub-100 nm material processing with sub-15 femtosecond picojoule near infrared laser pulses. Proc. SPIE 7903. 141. M. Straub, K. König: Nanostructure formation on silicon surfaces by high repetition rate sub-15 femtosecond near infrared laser pulses. Proc. SPIE 7920. 142. H. Zhang, M. Straub, K. König, M. Afshar, D. Feili, H. Seidel: Nanoprocessing of glass and PMMA by means of near-infrared sub-15 femtosecond laser pulses. Proc. SPIE 7921. 143. M. Licht, M. Straub, K. König, M. Afshar, D. Feili, H. Seidel: Three-dimensional nanostructures for applications in cell biology generated by high-repetition rate sub-15 fs near-infrared laser pulses. Proc. SPIE 7908. 144. A. Uchugonova, H. Zhang, C. Lemke, K. König: Nanosurgery with near-infrared 12-femtosecond and picosecond laser pulses. Proc. SPIE 7903. 145. M. Afshar, S. Saremi, H. Völlm, D. Feili, H. Seidel, M. Straub, H. Zhang, K. König: Multiphoton lithography and ITO structuring by high-repetition-rate sub-15 femtosecond laser pulses. Proc. SPIE 7920. 146. C.B. Talbot, R. Patalay, I. Munro, H.G. Breunig, K. König, Y. Alexandrov, S. Warren, A. Chu, G.W. Stamp, M.A.A. Neil, P.M.W. French, C. Dunsby: A multispectral FLIM tomograph for in-vivo imaging of skin cancer. Proc. SPIE 7903. 147. K. König: High-resolution multimodal clinical multiphoton tomography of skin. Proc. SPIE 7883. 148. K. König: New developments in multimodal clinical multiphoton tomography. Proc. SPIE 7903. 149. V. Huck, C. Gorzelanny, K. Thomas, C. Mess, V. Dimitrova, M. Schwarz, I. Riemann, V. Niemeyer, T.A. Luger, K. König, S.W. Schneider: Intravital multiphoton tomography as an appropriate tool for non-invasive in vivo analysis of human skin affected with atopic dermatitis. Proc. SPIE 7883. 150. R. Patalay, C. Talbot, I. Munro, H.G. Breunig, K. König, Y. Alexandrov, S. Warren, M.A.A. Neil, P.M.W. French, A. Chu, G.W. Stamp and C. Dunsby: Fluorescence lifetime imaging of skin cancer. Proc. SPIE 7883. 151. H.G. Breunig, K. König: Spectral characteristics of two-photon autofluorescence and second harmonic generation from human skin in vivo. Proc. SPIE 7883. 152. H.G. Breunig, M. Weinigel, J. Lademann, W. Sterry, I. Latka, B. Dietzek, J. Popp, K. König: Combining multiphoton and CARS microscopy for skin imaging. Proc. SPIE 7903. 153. Patalay, C. Talbot, Y. Alexandrov, I. Munro, H. G. Breunig, K. König, S. Warren, M. A. A. Neil, P. M. W. French, A. Chu, G. W. Stamp, C. Dunsby: Non-invasive imaging of skin cancer with fluorescence lifetime imaging using two photon tomography. Proc. SPIE 8087. 154. K. König. Multiphoton Tomography if Intratissue Tattoo Nanoparticles. Proc. SPIE 8207. 155. M. Weinigel, H.G. Breunig, P. Fischer, M. Kellner-Höfer, R. Bückle, K. König. Compact clinical high-NA multiphoton endoscopy. Proc. SPIE 8217. 156. H. G. Breunig, M. Weinigel, K. König. Multiphoton Spectroscopy of human skin in vivo. Proc. SPIE 8225. 157. H.G. Breunig, C. Köhler, K. König. Two-photon cryomicroscope. Proc. SPIE 8225. 158. K. König. Clinical multiphoton FLIM tomography. SPIE Proceed. 8226. 159. A. Uchugonova, R. Hoffmann, M. Weinigel, K. König. Watching stem cells at work with a flexible multiphoton tomograph. Proc. SPIE 8226. 160. H.G. Breunig, M. Weinigel, M.E. Darvin, J. Lademann, K. König. Clinical multiphoton and CARS microscopy. Proc. SPIE 8226 161. M. Weinigel, H.G. Breunig, P. Fischer, M. Kellner-Höfer, R. Bückle, K. König. Studies on wide-field-of-view multiphoton imaging using the flexible clinical multiphoton tomograph MPTflex. Proc. SPIE 8226. 162. M. Straub, A. Uchugonova, K. König. Silicon cell culture templates with nanotopography: Periodic nanostructures and random nanoporous topologies generated by high-repetition rate sub-15 fs pulsed near-infrared laser light. Proc. SPIE 8231. 163. K. König, M. Licht, M. Straub, A. Uchugonova. Material Processing with 12 Femtosecond Picojoule Laser Pulses. Proc. SPIE 8249. 164. M. Straub, B. Weigand, K. König. Nanostructure formation on lithium niobate surfaces by high-repetition rate sub-15 fs near-infrared laser pulses. 165. K. König. In vivo CARS tomography combined with two-photon autofluorescence and SHG imaging in patients with dermatological disorders. Proceedings of the 30th International Congress on High Speed Imagng & Photonics, Pretoria, South Africa, September 2012. 166. A. Uchugonova, R. Hofmann, K. König. Multiphoton Tomography with Submicron Spatial Resolution of Living Tumor-Bearing Mice. Proceedings of the 30th International Congress on High Speed Imagng & Photonics, Pretoria, South Africa, September 2012. 167. M. Afshar, D. Feili, H. Voellm, M. Straub, K. Koenig, H. Seidel. Nanoscale Laser Writing of Indium-Tin-Oxide Nanowires. NEMS 2012, Kyoto, Japan, März 2012. 168. H.G. Breunig, M. Weinigel, M. Kellner-Höfer, R. Bückle, M.E. Darvin, J. Lademann, K. König. Combining multiphoton and CARS microscopy for skin imaging. SPIE-Proceed. 85880N1. 169. M. Weinigel, H.G. Breunig, M. Kellner-Höfer, R. Bückle, M. Darvin, J. Lademann, K. König. New developments in clinical CARS. SPIE-Proceed. 85881E-1. 170. G.H. Breunig, A. Uchugonova, K. König. Multiphoton cryo microscope with sample temperature control. SPIE-Proceed. 858819-1. 171. M. Weinigel, H.G. Breunig, P. Fischer, M. KellnerHöfer, R. Bückle, K. König. Clinical multiphoton endoscopy with FLIM capability. Spie-Proceed. 85882E-1. Best Poster Award. 172. M. Balu, K.M. Kelly, C.B. Zachary, R.M. Harris, T.B. Krasieva, K. König, B.J. Tromberg. Invited paper: Clinical studies of pigmented lesions in human skin by using a multiphoton tomograph. SPIE-Proceed. 858812-1. 173. M.C. Meinke, M. Klemp, M.E. Darvin, K. König, J. Lademann, M. Ulrich. Comparison of a reflectance confocal microscope and a multiphtoon tomograph to detect different skin cancers. 174. H.G. Breunig, A. Uchugonova, K. König. Multiphoton imaging of biological samples during freezing and heating. SPIE-Proceed. 8948-43. 175. H.G. Breunig, A. Uchugonova, K. König. Towards optical cell transfection inside a micro-flow cell. SPIE-Proceed. 8947-55. 176. K. König, M. Weinigel, H.G. Breunig, A. Uchugonova. Quantitative Multiphoton Imaging. Plenar Lecture. SPIE-Proceed. 8948-3. 177. M. Weinigel, H.G. Breunig, K. König. A novel clinical multimodal multiphoton tomograph for AF, SHG, CARS imaging, and FLIM. SPIE-Proceed. 8948-61. 178. A. Uchugonova, A. Batista, K. König. Fluorescence Lifetime Imaging of Pluripotent Stem Cells. SPIE-Proceed. 8948-52. 179. A. Batista, A.M. Morgado, K. König. Detection of back-reflected SHG from corneal histological sections. SPIE-Proc. 8948-83. 180. A. Batista, H.G. Breunig, A. Uchugonova, B. Seitz, A.M. Morgado, K. König. Label-free SHG imaging and spectral FLIM of corneas using a sub-15 fs laser microscope. SPIE-Proc. 8930-28. 181. K. König, A. Ostendorf. Femtosecond laser generated sub-100nm-structures for biomedical and technical applications. SLPC2014. 182. K. König, M. Weinigel, R. Bückle, M. Kaatz, C. Hipler, K. Zens, S. Schneider, V. Huck. Monitoring wound healing by multiphoton tomography / endoscopy. SPIE-Proc. 9303. 183. A. Batista, HG Breunig, A. Uchugonova, B. Seitz, AM Morgado, K. König. Two-Photon autofluorescence lifetime and SHG imaging of healthy and diseased human corneas. SPIE-Proc. 9307. 184. HG Breunig, A. Batista, A. Uchugonova, K. König. Software-aided automatic cell optoporation system. SPIE-Proc. 9328. 185. A. Batista, HG Breunig, A. Uchugonova, AM Morgado, K. König. Characterization of porcine eyes based on autofluorescence lifetime imaging. SPIE-Proc. 9329. 186. A. Uchugonova, HG Breunig, A. Batista, K. König. Optical Reprogramming with ultrashort femtosecond laser pulses. SPIE-Proc. 9329. 187. M. Weinigel, HG Breunig, K. König. Histology in vivo: chemical contrast combined with clinical multimodal multiphoton tomography. SPIE-Proc. 9329. 188. HG Breunig, A. Batista, A. Uchugonova, K. König. Motionless polarization-resolved second harmonic generation imaging of corneal collagen. SPIE-Proc. 9329. 189. A. Uchugonova, HG Breunig, A. Batista, K. König. Optical cell cleaning with NIR femtosecond laser pulses. SPIE-Proc. 9328. 190. K. König, M. Weinigel, A. Pietruszka, R. Bückle, N. Gerlach, U. Heinrich. Multiphoton Tomography of Astronauts. SPIE-Proc. 9329. 191.F. Leinenbach, HG. Breunig, K. König. Mobile laser lithography station for microscopic two-photon polymerization. SPIE-Proc. 9350. 192. M. Klötzer, M. Afshar, D. Feili, H. Seidel, K. König, M. Straub. Generation of laser-induced periodic surfaces structures in indium-tin-oxide thin films and two-photon lithography of ma-N photoresist by sub-15 femtosecond laser microscopy for liquid crystal cell application. SPIE-Proc. 9351. 193. K. König. Multiphoton Tomography to detect Chemo- and Biohazards. SPIE-Proc. 932933. 194. K. König, S. Kantelhardt, D. Kalasauskas, E. Kim, A. Giese. First Multiphoton Tomography of Brain in Man. SPIE-Proc. 9690. 195. A. Uchugonova, H.G. Breunig, T. Le, K. König. Multiphoton Tomography with tunable Ti:sapphire laser. SPIE-Proc. 9712. 196. A. Batista, H.G. Breunig, A. Uchugonova, K. König. In vivo multiphoton imaging of the eyelid skin. SPIE-Proc. 197. A. Batista, A. Uchugonova, HG. Breunig, K. König. Towards in vivo breast skin characterization using multiphoton tomography. SPIE-Proc. 10069. 198. HG. Breunig, A. Uchugonova, A. Batista, K. König. Femtosecond-laser assisted cell reprogramming. SPIE-Proc. 10068. 199. K. König. A. Batista, T. Hager, B. Seitz. Multiphoton tomography of the human eye. SPIE-Proc. 10069. 200. C. Mess, K. Zens, C. Gorzelanny, D. Metze, TA Luger, K. König, SW Schneider, V. Huck. From morphology to clinical pathophysiology: multiphoton fluorescence lifetime imaging at patients`bedside. SPIE-Proc. 10069.

Proceedings

1. K. König, H. Schneckenburger, A. Rück, S. Auchter: Photoproduct formation of endogeneous protoporphyrin and its photodynamic activity. SPIE-Proceedings vol. 1525(1991)412-419

2. K. König, A. Rück, S. Auchter, W. Strauss, H. Schneckenburger: Photoinduced reactions of porphyrin photosensitizers. (A) Hematoporphyrin Derivative (HpD). In: Laser in Medicine, eds. W. Waidelich, R. Waidelich, A. Hofstetter, Springer Verlag 1992, 117-121

3. W. Strauss, A. Rück, T. Köllner, K. König, H. Schneckenburger: Photoinduced reactions of porphyrin photosensitizers. (B) Hydrophilic meso-tetraphenylporphyrins. In: Laser in Medicine, eds. W. Waidelich, R. Waidelich, A. Hofstetter, Springer Verlag 1992, 122-127

4. K. König, J. Hemmer, H. Schnecknburger: Laser-induced autofluorescence of squamous cell carcinoma. In: P. Spinelli, M. Dal Fante, R. Marchesini: Photodynamic Therapy and Biomedical Lasers. Elsevier Science Publishers (1992)903-906.

5. K. König et al.: Photodynamic therapy with texaphyrins. In: P. Spinelli, M. Dal Fante, R. Marchesini: Photodynamic Therapy and Biomedical Lasers. Elsevier Science Publishers (1992)

6. K. König, H. Schneckenburger, A. Rück, H. Meyer: Fluorescence Diagnosis and Photodynamic Therapy of Acne vulgaris. In: P. Spinelli, M. Dal Fante, R. Marchesini: Photodynamic Therapy and Biomedical Lasers. Elsevier Science Publishers (1992)

7. K. König, F. Nowak, F. Genze, H. Schneckenburger: In-vivo autofluorescence measurements during photodynamic damage of cells and tumor tissue. In: Laser in Medicine 1993, W. Waidelich, R. Waidelich, A. Hofstetter (eds.), Springer Verlag, 1994, 95-99.

8. K. König, K. Kunzi-Rapp: On-line measurement of photodynamically induced lysis of erythrocytes with and without nucleus by small angle light scattering and video-intensified microscopy. In: Laser in Medicine 1993, eds. W. Waidelich, R. Waidelich, A. Hofstetter, Springer Verlag, 1994, 91-94.

9. R. Sailer, W. Strauss, K. König, A. Rück, R. Steiner: Untersuchungen zu Porphyrinstoffwechsel and photodynamische Inaktivierung am Gram-negativen Bakterium Pseudomonas aeruginosa. In: Laser in Medicine 1993, W. Waidelich, R. Waidelich, A. Hofstetter (ed.), Springer Verlag, 109-112

10. H. Schneckenburger, K. König, T. Dienersberger, R. Hahn: Time-gated video microscopy and spectroscopy. In: Laser in Medicine 1993, eds. W. Waidelich, R. Waidelich, A. Hofstetter, Springer Verlag,1994,497-501

11. G. Beck, K. König, R. Steiner: Optical detection of topically applied photosensitizers by in vivo remission spectroscopy. In: Laser in Medicine 1993, eds. W. Waidelich, R. Waidelich, A. Hofstetter, Springer Verlag, 1994, 469-472

12. W. Strauss, W. Mohr, K. König, K. Miller, R. Sailer, M. Gschwend, A. Rück, H. Schneckenburger, R. Steiner: MesoTetra(4-Carboxyphenyl)Porphyrin-Organverteilung und photodynamische Therapie. In: Laser in Medicine 1993, eds. W. Waidelich, R. Waidelich, A. Hofstetter, Springer Verlag,1994, 104-108

13. K. König, R. Hibst, H. Schneckenburger, G. Flemming: Laserinduced autofluorescence of caries. SPIE -Proceedings, vol. 1880(1993)125-131

14. K. König, H. Schneckenburger, A. Rück, R. Steiner, H. Walt:Autofluorescence of cells and tissue. SPIE-Proceedings, vol. 1887: "Physiological Imaging, Spectroscopy, and Early-Detection Diagnostic Methods", 1993, 213-221

15. K. König, R. Hibst, H. Meyer, G. Flemming, H. Schneckenburger: Laser-induced autofluorescence of carious regions of human teeth and caries-involved bacteria. SPIE-Proceedings, vol. 2080. In Druck

16. K. König, A. Kienle, W-H. Boehncke, R. Kaufmann, A. Rück, T. Meier, R. Steiner: Photodynamic tumour therapy and on-line fluorescence spectroscopy after ALA administration using 633 nm-light as therapeutic and fluorescence excitation radiation. SPIE-Proceedings, vol. 2078. In Druck

17. K. König, G. Beck, W-H Boehncke, R. Kaufmann, R. Hibst: In vivo remission spectroscopy on tattoos and topically applied photosensitizers in man, SPIE-Proceedings, vol. 2086. In Druck

18. K. König, W-H. Boehncke, A. Rück, R. Kaufmann, R. Steiner, W. Sterry: Photodynamic effects on T-cells and skin lesions of a patient with mycosis fungoides using porphyrin photosensitizers. SPIE-Proceedings, vol. 2086. In Druck

19. H. Schneckenburger, M. Gschwend, K. König, A. Rück, R. Sailer, W. Strauss: Subcellular distribution of photodynamic photosensitizers. SPIE-Proceedings, vol. 2086, in press

20. K. König, H. Schneckenburger, H. Walt, T. Leemann, M.T. Wyss-Desserich, A. Rück,B. Tromberg: Microscopic Studies on ALA-incubated tumor cells and tumor spheroids.SPIE-Proceedings, vol. 2133: Optical Methods for Tumor Treatment and Detection, 238-248

21. K. König, H. Schneckenburger, J. Hemmer, B. Tromberg, R. Steiner: In-vivo fluorescence detection and imaging of porphyrin-producing bacteria in the human skin and in the oral cavity for diagnosis of acne vulgaris, caries, and squamous cell carcinoma.

SPIE -Proceedings, vol. 2135: Advances in Laser and Light Spectroscopy to Diagnose Cancer and other Diseases, 129-138

22. K. König, H. Schneckenburger: Laser-induced dental caries and plaque diagnosis on patients by sensitive autofluorescence spectroscopy and time-gated video imaging. Preliminary studies. SPIE-Proceedings, vol. 2128: Lasers in Surgery. In Druck

23. K. König, H. Schneckenburger, A. Rück, R. König: Studies on Porphyrin Photoproducts in Solution, Cells, and Tumor Tissue. SPIE-Proceedings 2133: Optical Methods for Tumor Treatment and Detection, 226-237

24. K. König, H. Schneckenburger, H. Boehncke, R. Hibst: In vivo fluorescence spectroscopy and imaging of ALA-induced endogenous porphyrins in skin after Er:YAG ablation of stratum corneum. SPIE-Proceedings, vol. 2128: Lasers in Surgery. In Druck.

25. K. König: NAD(P)H and porphyrin attributed laser induced autofluorescence. Proceedings of the International Conference: Lasers & Applications. Advances in science, medicine and technology, NILES 1994, Cairo, March 26-30, 1994. In Druck.

26. K. König, Y. Liu, GJ. Sonek, MW. Berns, BJ. Tromberg: Photoinduced autofluorescence modifications of cells in an optical trap. SPIE-Proceedings, vol. 2329: "Optical and Imaging Techniques in Biomedicine". 193-203..

27. S. Kimel, K. König, MW. Berns: Photodynamic effects on human and chicken erythrocytes. SPIE-Proceedings, vol. 2329: "Optical and Imaging Techniques in Biomedicine". 269-279.

28. K. König, Y. Liu, T. Krasieva, P. Patrizio, Y. Tadir, GJ. Sonek, MW. Berns, BJ. Tromberg. Invited Paper: Fluorescence imaging and spectroscopy of motile cells and CHO cells in an optical trap ("optical tweezers"). SPIE-Proceed. "Laser-Tissue Interaction VI", vol. 2391, 238-249

29. K. König, P. So, WW. Mantulin, E. Gratton, T. Krasieva, MW. Berns, BJ Tromberg. Two-photon excited cellular autofluorescence induced by cw- and femtosecond NIR microradiation. SPIE-Proceed. vol. 2628: 12-19.

30. K. König, T.Krasieva, E. Bauer, U. Fiedler, MW. Berns, BJ. Tromberg, KO. Greulich.UVA-Induced Oxidative Stress in Single Cells Probed by Autofluorescence Modifications, Cloning Assay, and Comet Assay. SPIE-Procced. vol. 2628: 43-49.

31. KO. Greulich, E. Bauer, U. Fiedler, C. Hoyer, K. König, S. Monajembashi. Single cell and single cell biotechnology. SPIE-Proceed. vol. 2629. In Druck.

32. L. Norwang, K. König, L. Svaasand. Comparison between reflectance spectra obtained with an integrating sphere and a fiber-optic collection system. SPIE-Procced. vol. 2624. In Druck.

33. K. König, P. Fergin, MW. Berns, BJ. Tromberg. Rapid spectrally-resolved fluorescence imaging of skin after topical ALA-administration. In: Laser in Medicine 1995, eds. W. Waidelich, G. Staehler, R. Waidelich, Springer Verlag. 587-590.

34. K. König, BJ. Tromberg, MW. Berns. One- and Two Photon Excited Fluorescence of Motile Cells in the Optical Trap. In: Laser in Medicine 1995, eds. W. Waidelich, H. Hügel, H. Opower, H. Tiziani, R. Wallenstein, W. Zinth, Springer Verlag. 164-167.

35. H. Schneckenburger, M. Gschwend, K. König, R. Sailer, W. Strauss. Fluorescence Lifetime Imaging and Spectroscopy in Photobiology and Photomedicine. In: "Fluorescence Microscopy and Fluorescent Probes", ed. J. Slavik, Plenum Press, New York - London. In Druck.

36. H. Schneckenburger, M. Gschwend, K. König, K. Kunzi-Rapp, R. Sailer, W. Strauss. Laser in der Diagnostik am Beispiel der Fluoreszenz-Diagnostik und Laser-Mikroskopie. In: "Lasertechnik und Lasermedizin", ed. H.D. Reichenbach, Ecomed-Verlag, Lundsberg, In Druck.

37. K. König, T. Krasieva, Y. Liu, M.W. Berns, B.J. Tromberg. Invited Paper: Two-photon excitation in living cells induced by low-power CW laser beams. SPIE Proceed.: Optical Diagnostics of Living Cells and Biofluids. Vol. 2678 (1996) 30-37.

38. K. König. Invited Paper: Porphyrin- and NAD(P)H attributed autofluorescence for medical diagnosis. Proceedings "LASER 95" of the Society for Optical and Quantum Electronics.In Druck.

39. K. König, P. So, W.W. Mantulin, E. Gratton: Cell damage in Two-Photon Microscopes. SPIE-Proceed.1996: In Druck.

40. K. König, L. Svaasand, Y. Tadir, B. Tromberg, M.W. Berns: Optical determination of motility forces in human spermatozoa with laser tweezers. SPIE-Proceed.1996: In Druck.

41. K. König, S. Kimel, L.O. Svaasand, B. Tromberg, T. Krasieva, M.W. Berns, P. So, W.W. Mantulin, E. Gratton, K.J. Halbhuber: Cell damage in UVA and cw/femtosecond NIR microscopes. SPIE-Proceed.1997, vol. 2984: 37-44.

42. K. König, K.-J. Halbhuber: Zwei-Photonen-Femtosekundenmikroskopie vitaler Zellen. In: W. Waidelich, R. Waidelich, A. Hofstetter (eds.). Laser in Medicine. Proceedings of the 13th International Congress LASER 97. Springer-Verlag. In Druck.

43. K. König, H. Oehring, K.-J. Halbhuber, U. Fiedler, E. Bauer, K.O. Greulich. Comet assay, cloning assay, histochemistry, light- and electron microscopy on one preselected cell. SPIE-Proceed.1997, vol. 3199: 148-155.

44. K. König. Laser tweezers as novel nonlinear tools in cell and biomolecule diagnostics. SPIE-Proceed.1997, vol. 3199: 178-182.

45. K. König. How safe is the gamete micromanipulation by laser tweezers? SPIE-Proceed.1998, vol. 3260: In Druck.

46. K. König, M. Teschke, W. Pfister, H. Meyer: Photodynamically inactivation of propionibacterium acnes. SPIE-Proceed.1998, vol. 3247: In Druck.

47. K. König. Invited paper: Cloning assay thresholds on cells exposed to ultrafast laser pulses. SPIE-Proceed.1999, vol. 3616: In Druck.

48. K. König, P. Fischer. I. Riemann, K.-J. Halbhuber. PDT by non-resonant two-photon excitation.SPIE-Proceed.1999, vol. 3592: In Druck.

49. K. König, P. Fischer. I. Riemann, K.-J. Halbhuber. Intracellular nanosurgery with compactfemtosecond laser. In: 18^th Congress of the International Commission for Optics: Optics for the next Millenium, SPIE-Proceed.1999, vol. 3749, Seite 390.

50. K. König. Applikationen der optischen Mikromanipulation und Zweiphotonenanregung vitaler Zellen mittels Naher Infrarot Mikroskopie. Shaker-Verlag. Aachen.1999. ISBN: 3-8265-6788-9.

51. K. König. Lasertechnik. Chapter in: Laser in der Dermatologie. 2000. In Press.

52. K. König, I. Riemann, A. Göhlert, P. Fischer, T. Liehr, I.F. Loncarevic, U. Claussen, K.J. Halbhuber. Multiphoton Multicolor FISH. SPIE-Proceed.2000, vol. 4164: In Druck.

53. K. König. Chapter: Cellular response to laser radiation in fluorescence microscopes. In: Methods in Cellular Imaging. Editor: A. Perisamy. Oxford University Press. 2001, 236-250.

54. C.Y. Dong, E.A. Bevan, L. Hsu, K. König, P.T.C. So. Invited paper: Characterization of two-photon point spread function in turbid medium by direct measurements, multicolocr imaging, and blind deconvolution. SPIE-Proceed. 4262(2001)73-81.

55. W. Becker, A. Bergmann, K. König, U. Tirlapur. Picosecond Fluorescence Lifetime Microscopy by TCSPC Imaging. SPIE-Proceed. 4262(2001)414-419.

56. K. König, U.K. Tirlapur: Chapter: Cellular and subcelluar pertubations during multiphoton microscopy. In: Confocal and two-photon microscopy. Editor: A. Diasporo. John Wiley $ Sons, Inc. 2002, 191-206 + cover page for the book.

57. U.K. Tirlapur, K. König: Two-photon near infrared femtosecond laser scanning microscopy in plant biology. In: Confocal and two-photon microscopy. Editor: A. Diasporo. John Wiley & Sons, Inc. 2002, 449-468.

58. K. König, I. Riemann, O. Krauss, W. Fritzsche. Invited paper: Nanodissection of human chromosomes and ultraprecise eye surgery with anojoule near infrared femtoseond laser pulses. SPIE-Proceed. 4633(2002)11-22.

59. K. König, U. Wollina, I. Riemann, C. Peuckert, K.J. Halbhuber, V. Fünfstück, T.W. Fischer, P. Elsner. Optical tomography of human skin with subcellular spatial and picosecond time resolution. SPIE-Proceed. 4620(2002)191-201.

60. K. König, I. Riemann, U.K. Tirlapur. Optical gene transfer by femtosecond laser pulses. SPIE-Proceed. 4963(2003)81-88.

61. K. König. High-resolution multiphoton imaging and nanosurgery of the cornea using femtosecond laser pulses. In: Lasers in ophthalmology. Basics, diagnostic and surgical aspects. Eds. F. Fankhauser & S. Kwasniewska. Kugler Publications pp. 79-89 (2003) ISBN: 9062991890.

62. K. König, B. Wang, O. Krauss, I. Riemann, H. Schubert, S. Kirtse, P. Fischer. First in vivo animal studies on intraocular nanosurgery and multiphoton tomography with low-energy 80 MHz near infrared femtosecond laser pulses. SPIE-Proceed. 5314 (2004)

63. K. König, I. Riemann, G. Ehrlich, V. Ulrich, P. Fischer. Multiphoton FLIM and spectral imaging of cells and tissues. SPIE-Proceed. (2004)

64. F. Fischer, K. König, S. Puschmann, R. Wepf, I. Riemann, V. Ulrich, P. Fischer. Characterization of multiphoton laser scanning device optical parameters for image restoration. SPIE-Proceed. (2004) in press

65. I. Riemann, P. Fischer, K. König. Photodynamic therapy and knocking out of single tumor cells by multiphoton excitation processes. SPIE-Proceed. (2004) in press

66. I. Riemann, E. Dimitrow, P. Fischer, A. Reif, M. Kaatz, P. Elsner, K. König. High resolution multiphoton tomography of human skin in vivo and in vitro. SPIE-Proceeding 5312 (2004)

67. W. Becker, A. Bergmann, G. Biscotti, K. König. High-Speed FLIM Data Acquisition by Time-Correlated Single Photon Counting. SPIE-Proceeding 5323 (2004).

68. K.Schenke-Layland, I. Riemann, V. Ulrich, F. Opitz, U. A. Stock, K. König. Multiphoton imaging of collagen and elastin of native and tissue-engineered heart valves. SPIE-Proceeding 5323 (2004).

69. K. Schenke-Layland, I. Riemann, U. A. Stock, K. König. Non-invasive multiphoton imaging of cardiovascular structures using NIR femtosecond laser scanning microscopy. SPIE-Proceeding 5312 (2004)300-308.

70. I. Riemann, P. Fischer, K. König. In vivo multiphoton tomography of melanoma. SPIE-Procced (2004).

71. K. König. Multiphoton tomography, transfection and nanosurgery with <2nJ, 80 MHz Femtosecond Laser Pulses. SPIE-Proceed. 5340(2004)37-46.

72. K. König: Femtosecond laser application in biotechnology and medicine. LPM2004 in Nara, Japan. SPIE-Proceed. 5662(2004)255-267.

73. T. Velten, H. Schuck, I. Riemann, F. Bauerfeld, D. Sauer, K. König. Time-Resolved and Spectrally-Resolved 5D Multiphoton Microscopy for Analysis and Nanoprocessing of Biological and Non-Biological Materials, LANE 2004.

74. K. König, F. Garwe, A. Czaki, G. Maubach, I. Riemann, W. Fritzsche. Nanoprocessing of DNA with femtosecond laser. SPIE-Proceed. 5462(2004)27-

75. I. Riemann, P. Fischer, M. Kaatz, T.W. Fischer, P. Elsner, E. Dimitrov, A. Reif, K. König. Optical Tomography of pigmented human skin biopsies. SPIE-Proceed. 5312(2004)24-34.

76. A. Czaki, G. Maubach, F. Garwe, A. Steinbrück, K. König, W. Fritzsche. A novel DNA restriction technology based on laser pulse energy conversion on sequence-specific bound metal nanoparticles. SPIE-Proceed. 2005.

77. W. Fritzsche, A. Czaki, A. Steinbrück, F. Garwe, K. König, M. Raschke. Metal nanoparticles as passive and active tools for bioanalytics. SPIE-Proceed. 2005.

78. T. Anhut, K. Hassler, T. Lasser, K. König, R. Rigler. Fluorescence Correlation Spectroscopy on dielectric surfaces in total internal reflection geometries. SPIE-Proceed. 2005.

79. B. Wang, I. Riemann, H. Schubert, S. Kirste, K. König. In vivo animal follow-up studies on intrastromal surgery with near infrared nanojoule femtosecond laser pulses. SPIE-Proceed. 2005.

80. K. König, I. Riemann, H. Schuck, D. Sauer, T. Velten, R. LeHarzic. Time-resolved and spectrally resolved 5D multiphoton microscopy for analysis and nanoprocessing of materials. SPIE-Proceed. 2005.

81. I. Riemann, A. Ehlers, A. Reif, J. Kobow, K. König. In vivo multiphoton tomography of skin as a tool to study the effects of topically applied probes and UV exposure. SPIE-Proceed. 2005.

82. I. Riemann, T. Anhut, F. Stracke, R. LeHarzic, K. König. Multiphoton nanosurgery in cells and tissues. SPIE-Proceed. 2005.

83. I. Riemann, E. Dimitrow, M. Kaatz, J. Fluhr, P. Elsner, J. Kobow, K. König. In vivo multiphoton tomography of inflammatory tissue and melanoma. SPIE-Proceed. 2005.

84. K. König, B. Wang, I. Riemann, J. Kobow. Cornea surgery with nanojoule femtosecond laser pulses. SPIE-Proceed. 2005.

85. W. Becker, K. König. Fluorescence lifetime images and correlation spectra obtained by multi-dimensional TCSPC. SPIE-Proceed. 2005.

86. K. König, H. Schuck, D. Sauer, F. Bauerfeld, F. Stracke, T. Velten, A. Tchernook, S. Martin, R. LeHarzic. Invited paper: Femtosecond laser nanoprocessing using near infrared nanojoule pulses at MHZ repetition frequency. SPIE-Proceedings, vol. 6400.

87. K. König, I. Riemann, A. Ehles, R. Bückle, E. Dimitrow. In vivo multiphoton tomography of skin cancer. SPIE-Proceedings, vol. 6089.

88. B.G. Wang, K. König, I. Riemann, H. Schubert, K.-J. Halbhuber. Multiphoton imaging of corneal tissue with near-infrared femtosecond laser pulses: corneal optical tomography and ist use in refractive surgery. SPIE-Proceedings, vol. 6089.

89. R. LeHarzic, S. Martin, R. Bückle, C. Wullner, C. Donitzky, I. Riemann, K. König. New developments in corneal refractive surgery with femtosecond laser pulses. SPIE-Proceed. 2006, vol. 6138.

90. I. Riemann, K. Schenke-Layland, A. Ehlers, E. Dimitrov, M. Kaatz, P. Elsner, S. Martin, K. König. High-resolution multiphoton optical tomography of tissues – an in vitro and in vivo study. SPIE-Proceedings, vol. 6142.

91. I. Riemann, F. Stracke, D. Sauer, S. Martin, K. König. Multiphoton nanosurgery in cells and tissues. SPIE-Proceedings, vol. 6089.

92. A. Ehlers, I. Riemann, T. Anhut, M. Kaatz, P. Elsner, K. König. Fluorescence lifetime imaging of human skin and hair. SPIE-Proceedings, vol. 6089.

93. A. Csaki, F. Garwe, A. Steinbrück, A. Weise, K. König, W. Fritzsche. Localization of laser energy conversion by metal nanoparticles: basic effects and applications. SPIE- Proceedings, vol. 6191.

94. B. Messerschmidt, A. Kraeplin, S. Schenkl, I. Riemann, M. Stark, A. Ehlers, A. Tchernook, R. LeHarzic, K. König. Novel concept of GRIN optical systems for high resolution microendoscopy: Part 1. Physical aspects. SPIE-Proceedings, vol. 6432.

95. A. Uchugonova, I. Riemann, F. Stracke, E. Gorjup, R. LeHarzic, K. König. The influence of NIR femtosecond alser radiation on the viability of 3D stem cell clusters and tumor spheroids. SPIE-Proceedings, vol. 6442.

96. A. Ehlers, S. Schenkl, I. Riemann, B. Messerschmidt, M. Kaatz, R. Bückle, K. König. In vivo multiphoton endoscopy of endogenous skin fluorophores. SPIE-Proceedings, vol. 6442.

97. F. Stracke, M. Schneider, B. Weiss, C.-M. Lehr, U. F. Schäfer, K. König. Multiphoton microscopy for the investigation of trans-cutaneous drug delivery. SPIE-Proceedings, vol. 6630.

98. M. Stark, B. Manz, I. Riemann, F. Volke, W. Weschke, K. König. Multiphoton and magnetic resonance imaging of barley embryos: comparing micro-imaging techniques across scale and parameter barriers. SPIE-Proceedings, vol. 6442.

99. M. Stark, D. Dörr,A. Ehlers, D. Sauer, R. Bückle, S. Martin, F. Ehrhart, J. Baunach, A. Katsen-Globa, H. Zimmermann, K. König. Multiphoton imaging and fluorescence lifetime studies on unstained cells and tissue at cryogenic conditions. SPIE-Proceedings, vol. 6628.

100. I. Riemann, A. Ehlers, R. LeHarzic, S. Martin, A. Reif, K. König. In vivo multiphoton tomography of skin during wound healing and scar formation. SPIE-Proceedings, vol. 6442.

101. I. Riemann, A. Ehlers, D. Dill-Müller, S. Martin, K. König. Multiphoton tomography of skin tumors after ALA application. SPIE-Proceedings, vol. 6424.

102. I. Riemann, F. Stracke, A. Uchugonova, S. Martin, R. Bückle, K. König. Optical nanoinjection into cells and 3D stem cell-clusters via a NIR femtosecond laser. SPIE-Proceedings, vol. 6442.

103. H. Schuck, F. Bauerfeld, D. Sauer, R. LeHarzic, T. Velten, I. Riemann, K. König. Rapid prototyping of 3D micro- nanostructures to explore cell behaviour. Vortrag anlässlich der 3rd International Conference on Multi-Material Micro Manufacture (4M) in Borovets (Bulgarien), 03.-05.10.2007 Proceedings (2007).

104. S. Schenkl, A. Ehlers, I. Riemann, B. Messerschmidt, K. König. Rigid and high NA fluorescence GRIN-endoscopes. SPIE-Proceedings, vol. 6631.

105. S. Schenkl, E. Weiss, M. Stark, F. Stracke, I. Riemann, R. Lemor, K. König. Imaging living cells with a combined high-resolution multi-photon-acoustic microscope. SPIE-Proceedings, vol. 6437.

106. S. Schenkl, A. Ehlers. I. Riemann, B. Messerschmidt, R. Bückle, K. König. Applications of rigid and flexible GRIN-endoscopes. SPIE-Proceedings, vol. 6433.

107. K. König, A. Ehlers, I. Riemann, S. Schenkl, B. Messerschmidt, R. Bückle, R. Le Harzic, P. Elsner, M. Kaatz. SPIE-Proceedings, 6442.

108. R. Le Harzic, C. Wüllner, C. Donitzky, K. König. New developments in femtosecond laser corneal refractive surgery. SPIE-Proceedings, vol. 6460..

109. R. Le Harzic, C. Wüllner, D. Bruneel, C. Donitzky, K. König. Femtosecond refractive eye surgery: study of laser parameters for even more efficiency and safety. SPIE-Proceedings, vol. 6633.

110. R. Le Harzic, A. Colonna, R. Bückle, A. Ehlers, C. Hadjur, F. Leroy, F. Flament, R. Bazin, B. Piot, I. Riemann, K. König. In vivo multiphoton tomography: a non invasive powerful tool for biochemical investigation of human skin. SPIE-Prooceedings, vol. 6630.

111. F. Ehrhart, D. Dörr, M. Stark, K. König, H. Zimmermann. Laser assisted processing of cross-linked alginate hydrogel. WLT Conference, München 2007.

112. M Stark, D Dörr, F Ehrhart, J Schulz, J Baunach, A Katsen-Globa, A Ehlers, K König, H Zimmermann. Multiphoton Fluorescence Imaging at Cryogenic Conditions. Microscopy and Microanalysis / Volume13 / SupplementS03 / September 2007, pp 110-111

113. A. Uchugonova, J. Müller, R. Bückle G. Tempea, A. Isemann, A. Stingl, K. König. Negatively chirped laser enables nonlinear excitation and nanoprocessing with sub-20-fs pulses. Proc. SPIE 6860.

114. A. Uchugonova, K. König. Two-photon imaging of stem cells. Proc. SPIE 6860.

115. P. Becker, D. Sauer, F. Bauerfeld, K. König, R. LeHarzic. Surface and bulk micro- and nano-structering with nanojoule femtosecond laser pulses at high repetition rate. Proc. SPIE 6879.

116. K. König. Multiphoton tomography for tissue engineering. Proc. SPIE 6858.

117. K. König, J. Müller, M. Höfer, C. Müller, M. Weinigel, R. Bückle, P. Elsner, M. Kaatz, B. Messerschmidt. Invited review: Two-photon scanning systems for clinical high-resolution in vivo tisuse imaging. Proc. SPIE 6860.

118. I. Riemann, S. Schenkl, R. LeHarzic, D. Sauer, A. Ehlers, B. Messerschmidt, R. Bückle, K. König. Two-photon imaging using a flixible endoscope. Proc. SPIE 6851.

119. I. Riemann, A. Ehlers, R. LeHarzic, E. Dimitrow, M. Kaatz, P. Elsner, R. Bückle, K. König. Non-invasive analysis/diagnosis of human normal and melanoma skin tissues with two-photon FLIM in vivo Proc. SPIE 6842.

120. V.K. Pustovalov, K. König, L.G. Astafyeva, W. Fritzsche. Optical properties of core-shell gold-silver and silver-gold nanoparticles for some laser wavelengths. Proc. SPIE 6879.

121. V.K. Pustovalov, K. König, L.G. Astafyeva. Distributions of laser radiation intensity inside gold nanoparticles during laser radiation. Proc. SPIE 6879.

122. K. König, J. Müller, M. Höfer, C. Müller, M. Weinigel, R. Bückle, P. Elsner, M. Kaatz, B. Messerschmidt: Two-photon scanning systems for clinical high resolution in vivo tissue imaging. Proc. SPIE, Vol. 6860.

123. K. König, R. Bückle, M. Weinigel, P. Elsner, M. Kaatz: Clinical multiphoton tomography and clinical two-photon microendoscopy. Proc. SPIE 7183.

124. K. König, R. Bückle, M. Weinigel, J. Köhler, P. Elsner, M. Kaatz: In vivo multiphoton tomography in skin aging studies. Proc. SPIE 7161.

125. M. Schwarz, I. Riemann, M. Weinigel, K. König, B. Messerschmidt, R. Le Harzic: New developments in two photon endoscopy. Proc. SPIE 7172.

126. A. Uchugonova, A. Isemann, R. Bückle, W. Watanabe, K. König: Two-photon imaging and nanoprocessing of stem cells with sub-20 fs laser pulses. Proc. SPIE 7183.

127. D. Bruneel, M. Schwarz, E. Audouard, K. König, R. Le Harzic: Development of a powerful tool for nanostructuring and multiphoton imaging with nanojoule femtosecond laser pulses. Proc. SPIE 7201.

128. I. Riemann, M. Schwarz, F. Stracke, A. Ehlers, E. Dimitrow, M. Kaatz, K. König, R. Le Harzic: New developments in two-photon analysis of human skin in vivo. Proc. SPIE 7203.

129. K. König, M. Weinigel, H. G. Breunig, A. Gregory, P. Fischer, M. Kellner-Höfer, R. Bückle, M. Schwarz, I. Riemann, F. Stracke, V. Huck, C. Gorzelanny, S. W. Schneider: 5D-intravital tomography as a novel tool for non-invasive in-vivo analysis of human skin. Proc. SPIE 7555.

130. K. König, A. Uchugonova, M. Schug, H. Zhang, S. Saremi, D. Feili, H. Seidel: Two-photon lithography and nanoprocessing with picojoule extreme ultrashort 12 femtosecond laser pulses. Proc. SPIE 7584.

131. A. Uchugonova, Z. Földes-Papp, G. M. Kostner, K. König: Long-term marker-free multiphoton imaging, targeted transfection, optical cleaning of stem cell clusters, and optical transport of microRNA with extreme ultrashort laser pulses. Proc. SPIE 7569.

132. K. König, M. Weinigel, H. G. Breunig, A. Gregory, P. Fischer, M. Kellner-Höfer, R. Bückle: Current developments in clinical multiphoton tomography. Proc. SPIE 7569.

133. M. Schwarz, I. Riemann, F. Stracke, V, Huck, C. Gorzelanny, S. W. Schneider, K. König, S. Puschmann, V. Lutz, N. Sommer, C. Rahn, S. Gallinat, H. Wenck, K.-P. Wittern, F. Fischer: A comparative study of different instrumental concepts for spectrally and lifetime-resolved multiphoton intravital tomography (5D-IVT) in dermatological applications. Proc. SPIE 7568.

134. K. König, M. Speicher, M. J. Koehler, R. Scharenberg, P. Elsner, M. Kaatz: Clinical combination of multiphoton tomography and high frequency ultrasound imaging for evaluation of skin diseases. Proc. SPIE 7564.

135. V. Huck, C. Gorzelanny, K. Thomas, V. Niemeyer, T. A. Luger, K. König, S. W. Schneider: Intravital multiphoton tomography as a novel tool for non-invasive in vivo analysis of human skin affected with atopic dermatitis. Proc. SPIE 7548.

136. K. König, M. Speicher, R. Bückle, J. Reckfort, G. McKenzie, J. Welzel, M. J. Koehler, P. Elsner, M. Kaatz: Clinical optical coherence tomography combined with multiphoton tomography for evaluation of several skin disorders. Proc. SPIE 7554.

137. H. G. Breunig, H. Studier, K. König: Excitation-wavelength dependence of multiphoton excitation of fluorophores of human skin in vivo. Proc. SPIE 7548.

138. H. Studier, H. G. Breunig, K. König: Two-photon imaging with 80 MHz and 1-GHz repetition rate Ti:sapphire oscillators. Proc. SPIE 7569.

139. M. Weinigel, H. G. Breunig, A. Gregory, P. Fischer, M. Kellner-Höfer, R. Bückle, K. König: A novel flexible clinical multiphoton tomograph for early melanoma detection, skin analysis, testing of anti-age products, and in situ nanoparticle tracking.

140. K. König, A. Uchugonova, M. Straub, H. Zhang, M. Afshar, D. Feili, H. Seidel: Sub-100 nm material processing with sub-15 femtosecond picojoule near infrared laser pulses. Proc. SPIE 7903.

141. M. Straub, K. König: Nanostructure formation on silicon surfaces by high repetition rate sub-15 femtosecond near infrared laser pulses. Proc. SPIE 7920.

142. H. Zhang, M. Straub, K. König, M. Afshar, D. Feili, H. Seidel: Nanoprocessing of glass and PMMA by means of near-infrared sub-15 femtosecond laser pulses. Proc. SPIE 7921.

143. M. Licht, M. Straub, K. König, M. Afshar, D. Feili, H. Seidel: Three-dimensional nanostructures for applications in cell biology generated by high-repetition rate sub-15 fs near-infrared laser pulses. Proc. SPIE 7908.

144. A. Uchugonova, H. Zhang, C. Lemke, K. König: Nanosurgery with near-infrared 12-femtosecond and picosecond laser pulses. Proc. SPIE 7903.

145. M. Afshar, S. Saremi, H. Völlm, D. Feili, H. Seidel, M. Straub, H. Zhang, K. König: Multiphoton lithography and ITO structuring by high-repetition-rate sub-15 femtosecond laser pulses. Proc. SPIE 7920.

146. C.B. Talbot, R. Patalay, I. Munro, H.G. Breunig, K. König, Y. Alexandrov, S. Warren, A. Chu, G.W. Stamp, M.A.A. Neil, P.M.W. French, C. Dunsby: A multispectral FLIM tomograph for in-vivo imaging of skin cancer. Proc. SPIE 7903.

147. K. König: High-resolution multimodal clinical multiphoton tomography of skin. Proc. SPIE 7883.

148. K. König: New developments in multimodal clinical multiphoton tomography. Proc. SPIE 7903.

149. V. Huck, C. Gorzelanny, K. Thomas, C. Mess, V. Dimitrova, M. Schwarz, I. Riemann, V. Niemeyer, T.A. Luger, K. König, S.W. Schneider: Intravital multiphoton tomography as an appropriate tool for non-invasive in vivo analysis of human skin affected with atopic dermatitis. Proc. SPIE 7883.

150. R. Patalay, C. Talbot, I. Munro, H.G. Breunig, K. König, Y. Alexandrov, S. Warren, M.A.A. Neil, P.M.W. French, A. Chu, G.W. Stamp and C. Dunsby: Fluorescence lifetime imaging of skin cancer. Proc. SPIE 7883.

151. H.G. Breunig, K. König: Spectral characteristics of two-photon autofluorescence and second harmonic generation from human skin in vivo. Proc. SPIE 7883.

152. H.G. Breunig, M. Weinigel, J. Lademann, W. Sterry, I. Latka, B. Dietzek, J. Popp, K. König: Combining multiphoton and CARS microscopy for skin imaging. Proc. SPIE 7903.

153. Patalay, C. Talbot, Y. Alexandrov, I. Munro, H. G. Breunig, K. König, S. Warren, M. A. A. Neil, P. M. W. French, A. Chu, G. W. Stamp, C. Dunsby: Non-invasive imaging of skin cancer with fluorescence lifetime imaging using two photon tomography. Proc. SPIE 8087.

154. K. König. Multiphoton Tomography if Intratissue Tattoo Nanoparticles. Proc. SPIE 8207.

155. M. Weinigel, H.G. Breunig, P. Fischer, M. Kellner-Höfer, R. Bückle, K. König. Compact clinical high-NA multiphoton endoscopy. Proc. SPIE 8217.

156. H. G. Breunig, M. Weinigel, K. König. Multiphoton Spectroscopy of human skin in vivo. Proc. SPIE 8225.

157. H.G. Breunig, C. Köhler, K. König. Two-photon cryomicroscope. Proc. SPIE 8225.

158. K. König. Clinical multiphoton FLIM tomography. SPIE Proceed. 8226.

159. A. Uchugonova, R. Hoffmann, M. Weinigel, K. König. Watching stem cells at work with a flexible multiphoton tomograph. Proc. SPIE 8226.

160. H.G. Breunig, M. Weinigel, M.E. Darvin, J. Lademann, K. König. Clinical multiphoton and CARS microscopy. Proc. SPIE 8226

161. M. Weinigel, H.G. Breunig, P. Fischer, M. Kellner-Höfer, R. Bückle, K. König. Studies on wide-field-of-view multiphoton imaging using the flexible clinical multiphoton tomograph MPTflex. Proc. SPIE 8226.

162. M. Straub, A. Uchugonova, K. König. Silicon cell culture templates with nanotopography: Periodic nanostructures and random nanoporous topologies generated by high-repetition rate sub-15 fs pulsed near-infrared laser light. Proc. SPIE 8231.

163. K. König, M. Licht, M. Straub, A. Uchugonova. Material Processing with 12 Femtosecond Picojoule Laser Pulses. Proc. SPIE 8249.

164. M. Straub, B. Weigand, K. König. Nanostructure formation on lithium niobate surfaces by high-repetition rate sub-15 fs near-infrared laser pulses.

165. K. König. In vivo CARS tomography combined with two-photon autofluorescence and SHG imaging in patients with dermatological disorders. Proceedings of the 30th International Congress on High Speed Imagng & Photonics, Pretoria, South Africa, September 2012.

166. A. Uchugonova, R. Hofmann, K. König. Multiphoton Tomography with Submicron Spatial Resolution of Living Tumor-Bearing Mice. Proceedings of the 30th International Congress on High Speed Imagng & Photonics, Pretoria, South Africa, September 2012.

167. M. Afshar, D. Feili, H. Voellm, M. Straub, K. Koenig, H. Seidel. Nanoscale Laser Writing of Indium-Tin-Oxide Nanowires. NEMS 2012, Kyoto, Japan, März 2012.

168. H.G. Breunig, M. Weinigel, M. Kellner-Höfer, R. Bückle, M.E. Darvin, J. Lademann, K. König. Combining multiphoton and CARS microscopy for skin imaging. SPIE-Proceed. 85880N1.

169. M. Weinigel, H.G. Breunig, M. Kellner-Höfer, R. Bückle, M. Darvin, J. Lademann, K. König. New developments in clinical CARS. SPIE-Proceed. 85881E-1.

170. G.H. Breunig, A. Uchugonova, K. König. Multiphoton cryo microscope with sample temperature control. SPIE-Proceed. 858819-1.

171. M. Weinigel, H.G. Breunig, P. Fischer, M. KellnerHöfer, R. Bückle, K. König. Clinical multiphoton endoscopy with FLIM capability. Spie-Proceed. 85882E-1. Best Poster Award.

172. M. Balu, K.M. Kelly, C.B. Zachary, R.M. Harris, T.B. Krasieva, K. König, B.J. Tromberg. Invited paper: Clinical studies of pigmented lesions in human skin by using a multiphoton tomograph. SPIE-Proceed. 858812-1.

173. M.C. Meinke, M. Klemp, M.E. Darvin, K. König, J. Lademann, M. Ulrich. Comparison of a reflectance confocal microscope and a multiphtoon tomograph to detect different skin cancers.

174. H.G. Breunig, A. Uchugonova, K. König. Multiphoton imaging of biological samples during freezing and heating. SPIE-Proceed. 8948-43.

175. H.G. Breunig, A. Uchugonova, K. König. Towards optical cell transfection inside a micro-flow cell. SPIE-Proceed. 8947-55.

176. K. König, M. Weinigel, H.G. Breunig, A. Uchugonova. Quantitative Multiphoton Imaging. Plenar Lecture. SPIE-Proceed. 8948-3.

177. M. Weinigel, H.G. Breunig, K. König. A novel clinical multimodal multiphoton tomograph for AF, SHG, CARS imaging, and FLIM. SPIE-Proceed. 8948-61.

178. A. Uchugonova, A. Batista, K. König. Fluorescence Lifetime Imaging of Pluripotent Stem Cells. SPIE-Proceed. 8948-52.

179. A. Batista, A.M. Morgado, K. König. Detection of back-reflected SHG from corneal histological sections. SPIE-Proc. 8948-83.

180. A. Batista, H.G. Breunig, A. Uchugonova, B. Seitz, A.M. Morgado, K. König. Label-free SHG imaging and spectral FLIM of corneas using a sub-15 fs laser microscope. SPIE-Proc. 8930-28.

181. K. König, A. Ostendorf. Femtosecond laser generated sub-100nm-structures for biomedical and technical applications. SLPC2014.

182. K. König, M. Weinigel, R. Bückle, M. Kaatz, C. Hipler, K. Zens, S. Schneider, V. Huck. Monitoring wound healing by multiphoton tomography / endoscopy. SPIE-Proc. 9303.

183. A. Batista, HG Breunig, A. Uchugonova, B. Seitz, AM Morgado, K. König. Two-Photon autofluorescence lifetime and SHG imaging of healthy and diseased human corneas. SPIE-Proc. 9307.

184. HG Breunig, A. Batista, A. Uchugonova, K. König. Software-aided automatic cell optoporation system. SPIE-Proc. 9328.

185. A. Batista, HG Breunig, A. Uchugonova, AM Morgado, K. König. Characterization of porcine eyes based on autofluorescence lifetime imaging. SPIE-Proc. 9329.

186. A. Uchugonova, HG Breunig, A. Batista, K. König. Optical Reprogramming with ultrashort femtosecond laser pulses. SPIE-Proc. 9329.

187. M. Weinigel, HG Breunig, K. König. Histology in vivo: chemical contrast combined with clinical multimodal multiphoton tomography. SPIE-Proc. 9329.

188. HG Breunig, A. Batista, A. Uchugonova, K. König. Motionless polarization-resolved second harmonic generation imaging of corneal collagen. SPIE-Proc. 9329.

189. A. Uchugonova, HG Breunig, A. Batista, K. König. Optical cell cleaning with NIR femtosecond laser pulses. SPIE-Proc. 9328.

190. K. König, M. Weinigel, A. Pietruszka, R. Bückle, N. Gerlach, U. Heinrich. Multiphoton Tomography of Astronauts. SPIE-Proc. 9329.

191.F. Leinenbach, HG. Breunig, K. König. Mobile laser lithography station for microscopic two-photon polymerization. SPIE-Proc. 9350.

192. M. Klötzer, M. Afshar, D. Feili, H. Seidel, K. König, M. Straub. Generation of laser-induced periodic surfaces structures in indium-tin-oxide thin films and two-photon lithography of ma-N photoresist by sub-15 femtosecond laser microscopy for liquid crystal cell application. SPIE-Proc. 9351.

193. K. König. Multiphoton Tomography to detect Chemo- and Biohazards. SPIE-Proc. 932933.

194. K. König, S. Kantelhardt, D. Kalasauskas, E. Kim, A. Giese. First Multiphoton Tomography of Brain in Man. SPIE-Proc. 9690.

195. A. Uchugonova, H.G. Breunig, T. Le, K. König. Multiphoton Tomography with tunable Ti:sapphire laser. SPIE-Proc. 9712.

196. A. Batista, H.G. Breunig, A. Uchugonova, K. König. In vivo multiphoton imaging of the eyelid skin. SPIE-Proc.

197. A. Batista, A. Uchugonova, HG. Breunig, K. König. Towards in vivo breast skin characterization using multiphoton tomography. SPIE-Proc. 10069.

198. HG. Breunig, A. Uchugonova, A. Batista, K. König. Femtosecond-laser assisted cell reprogramming. SPIE-Proc. 10068.

199. K. König. A. Batista, T. Hager, B. Seitz. Multiphoton tomography of the human eye. SPIE-Proc. 10069.

200. C. Mess, K. Zens, C. Gorzelanny, D. Metze, TA Luger, K. König, SW Schneider, V. Huck. From morphology to clinical pathophysiology: multiphoton fluorescence lifetime imaging at patients`bedside. SPIE-Proc. 10069.

Buchbeiträge
1. K. König, F. Genze, E. Reich, R. Miller, A. Rück, D. Repassy. PDT of tumor-bearing mice using liposome delivered texaphyrins. In: Spinelli, DalFante, Marchesini (eds.) Photodynamic therapyand biomedical lasers. Exerpta Medica. 1992. ISBN 0444814302 2. K. König. Biomedizinische Applikationen der optischen Mikromanipulation und Zweiphotonen-Anregung vitaler Zellen mittels Naher-Infrarot Laser-Mikroskopie. Shaker Verlag. 1999. ISBN 3-8265-6788-9 3. K. König. Photoproduct formation during porphyrin photodynamic therapy. In: Wyss, Tadir, Tromberg, Haller (eds.): Photomedicine in Gynecology and Reproduction, pp. 86-95. Karger. 2000. ISBN 3-8055-6905-X 4. K. König. Cellular response to laser radiation in fluorescence microscopes. In: Periasamy (ed.). Methods in Cellular Imaging, pp 236-251. Oxford 2001. 5. K. König. Lasertechnik. Biophysikalische Grundlagen. In: Raulin, Greve (eds.): Laser und IPL-Technologie in der Dermatologie und Ästhetischen Medizin, pp. 4-14. Schattauer 2003. ISBN 3-7945-2236-2 6. K. König. Cellular and Subcellular Perturbations During Multiphogon Microscopy. Two-Photon Near-Infrared Femtosecond Laser Scanning Microscopy in Plant Biology. In: Diaspro (ed.) Confocal and Two-Photon Microscopy, Foundations, Applications and Advances. ISBN 0-471-40920-0. 7. K. König. Multiphoton Multicolor FISH and Chromosome nanoprocessing with near infrared femtosecond laser pulses. In: Hemmerich, Dieckmann (eds.). Visions of the Nucleus-Eukaryotic DNA. American Scientific Publishers. In press.

Buchbeiträge

1. K. König, F. Genze, E. Reich, R. Miller, A. Rück, D. Repassy. PDT of tumor-bearing mice using liposome delivered texaphyrins. In: Spinelli, DalFante, Marchesini (eds.) Photodynamic therapyand biomedical lasers. Exerpta Medica. 1992. ISBN 0444814302

2. K. König. Biomedizinische Applikationen der optischen Mikromanipulation und Zweiphotonen-Anregung vitaler Zellen mittels Naher-Infrarot Laser-Mikroskopie. Shaker Verlag. 1999. ISBN 3-8265-6788-9

3. K. König. Photoproduct formation during porphyrin photodynamic therapy. In: Wyss, Tadir, Tromberg, Haller (eds.): Photomedicine in Gynecology and Reproduction, pp. 86-95. Karger. 2000. ISBN 3-8055-6905-X

4. K. König. Cellular response to laser radiation in fluorescence microscopes. In: Periasamy (ed.). Methods in Cellular Imaging, pp 236-251. Oxford 2001.

5. K. König. Lasertechnik. Biophysikalische Grundlagen. In: Raulin, Greve (eds.): Laser und IPL-Technologie in der Dermatologie und Ästhetischen Medizin, pp. 4-14. Schattauer 2003. ISBN 3-7945-2236-2

6. K. König. Cellular and Subcellular Perturbations During Multiphogon Microscopy. Two-Photon Near-Infrared Femtosecond Laser Scanning Microscopy in Plant Biology. In: Diaspro (ed.) Confocal and Two-Photon Microscopy, Foundations, Applications and Advances. ISBN 0-471-40920-0.

7. K. König. Multiphoton Multicolor FISH and Chromosome nanoprocessing with near infrared femtosecond laser pulses. In: Hemmerich, Dieckmann (eds.). Visions of the Nucleus-Eukaryotic DNA. American Scientific Publishers. In press.

Tagungsvorträge
1. E. Knoth, H. Kneipp, M. Rentsch, K. König: Kupfer- und Golddampflaser zur bronchiolog. Verwendung. Symposium "Neue Diagnostik- und Therapieverfahren in der Bronchol. (1986) Bad Berka 2. V. Bockhorn, W. Dietel, K. König, U. Krause: Erste Erfahrungen im Forschungsprojekt: Laserinduzierte Fluoreszenz- und Photochemotherapie des Harnblasencarcinoms. III. Symposium: Methoden und Ergebnisse tierexperimenteller Arbeiten (1986) Jena 3. V. Bockhorn, W. Dietel, K. König: Fluoreszenzdiagnostik und Photochemotherapie maligner Tumoren. Workshop mit internationaler Beteiligung: Laseranwendung in der Medizin (1987) Dresden 4. L.P. Löbe, U. Krause, P. Lotz, W. Dietel, K. König, H. Schubert: Photodynamische Tumortherapie im HNO-Gebiet. Prinzip und erste experimentelle Ergebnisse. 34. Gemeinschaftstagung der med.-wissensch. Gesellschaft für HNO-Heilkunde (1987) Rostock 5. V. Bockhorn, W. Dietel, K. König, A. Werth, H. Schubert: Laserinduzierte Fluoreszenz und Photochemotherapie von Tumoren. (Poster) VII. Onkologie-Symposium (1987) Jena 6. K. König, W. Dietel: Fluorescence Investigations on Animal Tumors. (Poster) 5. Symposium Optical Spectroscopy (1988) Eisenach 7. K. König, E. Welsch, H.G. Walther: Photoacoustic absorption measurements on marked tissue.(Poster) Fifth Symposium Optical Spectroscopy (1988) Eisenach 8. I. Bugiel, K. König, H. Wabnitz: Fluoreszenzmikroskopische Untersuchungen an Zellen mit sub-ns Zeitauflösung. PDT-Schule (1988) Berlin 9. W. Dietel, K. König: Laserinduzierte in-vivo Fluoreszenz tierexperimenteller Tumoren. PDT-Schule (1988) Berlin 10. K. König, V. Bockhorn, W. Dietel, H. Schubert: Photochemotherapie tierexperimenteller Tumoren. PDT-Schule (1988) Berlin 11. E. Knoth, H. Kneipp, M. Rentsch, K. König: Experimentelle PDT mittels Kupfer- und Golddampflaser. PDT-Schule (1988) Berlin 12. E. Knoth, K. König, W. Grassme, W. Dietel: HpD-induzierte Tumorfluoreszenz. PDT-Schule (1988) Berlin 13. U. Krause, P. Lotz, L.P. Löbe, K. König, H. Schubert: Versuche zur Darstellung von HpD und seines Einsatzes zur PDT an Mäuse-Ascites-Tumoren. PDT-Schule (1988) Berlin 14. U. Krause, L.P. Löbe, P. Lotz, J. Barth, H. Schubert, K. König: Die Wirkung klassischer Strahlungsquellen in der photodynamischen HpD-Therapie von malignen Geschwülsten. 35. Gemeinschaftstagung der med.-wissensch. Gesellschaft für HNO-Heilkunde (1988) Rostock 15. L.P. Löbe, U. Krause, P. Lotz, K. König, W. Dietel, J. Barth: Photodynamic therapy with the HpD-Halle II. Results of animal experiments. International Conference: Photodynamic Therapy and Medical Laser Application (1988) London Proceedings: Vortrag 141 16. U. Krause, L.P. Löbe, P. Lotz, K. König, H. Schubert, J. Barth: Tierexperimentelle Untersuchungen zur PDT mit dem HpD-Typ Halle. 4. Photodynamisches Kolloquium mit intern. Beteiligung der AG Photodermatologie der Gesellsch. für Dermatologie der DDR (1988) Wilthen 17. L.P. Löbe, U. Krause, P. Lotz, K. König, H. Schubert, J. Barth: Photodynamische Tumortherapie im HNO-Gebiet mit einem Hämatoporphyrinderivat. III. Symposium über Tumorforschung in der HNO-Heilkunde (1988) Mannheim 18. W. Dietel, K. König: Laser stimulated in vivo Fluorescence of animal tumors. Second Intern. Conference on Laser Scattering Spectroscopy of Biological Objects. (1988) Pecs, Hungaria 19. V. Bockhorn, W. Dietel, K. König, H. Schubert: Fluoreszenzdiagnostik und PCT oberflächlicher Tumoren. XIX. Urologenkongress der DDR (1988) Erfurt 20. W. Dietel, K. König: Photobleaching of the HpD fluorescence and photoproduct formation in-vivo and in solution. Third Congress of the European Society for Photobiology. (Poster) (1989) Budapest 21. K. König, H. Schneckenburger, A. Rück, S. Auchter: Photoproduct formation of endogeneous protoporphyrin and its photodynamic activity. SPIE: Future Trends in Biomedical Applications of Lasers (Poster) (1991) Berlin, Proceedings vol. 1525, 412-419 22. K. König, A. Rück, S. Auchter, W. Strauss, H. Schneckenburger: Photoinduced reactions of porphyrin photosensitizers. (A) HpD. Laser in Medicine (Laser 91) (1991) München 23. W. Strauss, A. Rück, T. Köllner, K. König, H. Schneckenbur-ger: Photoinduced reactions of porphyrin photosensitizers. (B) Hydrophile Tetraphenylporphyrines. Laser in Medicine (Laser 91) (1991) München 24. C. Westphal, K. König, J. Krauter, G. Heil: Morphological changes of human leukaemic cells during photodynamic therapy. Jahrestagung der Deutschen und Österreichischen Gesellsch. für Hämatologie und Onkologie (1991) Innsbruck. 25. K. König, E. Welsch, H.G. Walther: Photoacoustic absorption measurements on tumor tissue stained with the photosensitizer Methylene Blue. Fourth Congress of the European Society for Photobiology (Poster) (1991) Amsterdam 26. K. König, H. Schneckenburger, H. Meier, A. Rück, R. Kaufmann: Laserinduced in-vivo autofluorescence of the human skin caused on the porphyrin production of Propionibacterium acnes. Fourth Congress of the European Society for Photobiology (Poster) (1991) Amsterdam 27. K. König, R. Fischer, Puhl, A. Rück, R. Steiner: Selective Excimer ablation of intervertebral discs using fluorescence spectroscopy. 2nd Conference on Methods and Applications of Fluorescence Spectroscopy (Poster) (1991) Graz 28. K. König, H. Schneckenburger, H. Meier, A. Rück, R. Kaufmann: Laserinduced in-vivo autofluorescence of the human skin caused on the porphyrin production of Propionibacterium acnes. 2nd Conference on Methods and Applications of Fluorescence Spectroscopy (Poster) (1991) Graz 29. R. Fischer, K. König, Puhl, A. Rück, R. Steiner: Selective Excimer ablation of intervertebral discs using fluorescence spectroscopy. 1st Conference of the European Orthopaedic Research Society (EORS) (11.-12.11.1991) Paris 30. R. Fischer, K. König, W. Puhl: Selektive Ablation von Bandscheibengewebe mit dem Excimer-Laser mittels Fluoreszenz-spektroskopie - eine in vitro Untersuchung. 40. Jahrestagung der Vereinigung Süddeutscher Orthopäden e.V. (30.4.-3.5.1992) Baden-Baden 31. A. Rück, H. Schneckenburger, K. König, C. Westphal-Frösch, K. Kunzi-Rapp, W. Strauss, H. Wald: Observation of light-induced intracellular reactions and photodynamic organelle destruction of cationic and anionic mesotetraphenylporphyrins. Gordon Conference, New Hampshire, 1992 32. K. König, H. Meyer: Photodynamische Aktivität von Methylenblau. Photodermatologie-Kongreß (1992) Düsseldorf 33. K. König, H. Meyer, H. Schneckenburger, A. Rück: Fluoreszenzverhalten und photodynamische Wirksamkeit von Propionibacterium acnes. Photodermatologie-Kongreß (1992) Düsseldorf 34. A. Rück, H. Schneckenburger, W. Strauß, C. Westphal-Frösch, K. König, K. Kunzi-Rapp: Detektion von intrazellulären photodyna-mischen Reaktionen mit Hilfe hochauflösender mikroskopischer Techniken. Jahreskongreß der Deutschen Gesellschaft für Lasermedizin (1992) Münster, Abstract: Laser in Medicine and Surgery 8(1992)127 35. W.H. Boehncke, K. König, A. Rück, R. Kaufmann, W. Sterry: Proliferationsinhibition einer menschlichen T-Zell-Linie durch Photodynamische- und PUVA-Therapie. Jahreskongreß der Deutschen Gesellschaft für Lasermedizin (1992) Münster, Abstract: Laser in Medicine and Surgery 8(1992)93 36. K. König, A. Rück, W. Strauß, H. Schneckenburger, H. Wabnitz: Photobleaching and Photoproduct Formation of HPD. International Conference Photodynamic Therapy and Medical Laser Applications (1992) Mailand, Abstract: Lasers in Medical Science 7(1992)251 37. K. König, F. Genze, E. Reich, R. Miller, A. Rück, d. Repassy: PDT of tumor-bearing mice using liposome-delivered texaphyrins. International Conference Photodynamic Therapy and Medical Laser Applications (1992) Mailand, Abstract: Lasers in Medical Science 7(1992)268 38. K. König, J. Hemmer, H. Schneckenburger: Laser-induced autofluorescence of squamous cell carcinoma. International Conference Photodynamic Therapy and Medical Laser Applications (1992) Mailand, Abstract: Lasers in Medical Science 7(1992)233 39. K. König, H. Schneckenburger, A. Rück, H. Meyer: Fluorescence diagnosis and photodynamic therapy of acne vulgaris. International Conference Photodynamic Therapy and Medical Laser Applications (1992) Mailand, Abstract: Lasers in Medical Science 7(1992)291 40. A. Rück, K. König, W. Strauss, H. Schneckenburger: Light-induced reactions of cationic and anionic meso-tetraphenylporphyrins in-vitro and in-vivo probed by different spectroscopic techniques. International Conference Photodynamic Therapy and Medical Laser Applications (1992) Mailand, Abstract: Lasers in Medical Science 7(1992)280 41. R. Fischer, K. König, W. Puhl, A. Rück, R. Steiner: Selective Ablation of Intervertebral Discs using Fluorescence Spectroscopy - an in-vitro Examination. 3rd World Congress of the International Society for Low Power Laser Application in Medicine. Bologna, 9.-12.9.1992 42. A. Rück, H. Schneckenburger, C. Westphal-Frösch, K. Kunzi-Rapp, K. König, W. Strauss: Direct observation of photodynamically induced organelle destruction probed by video-enhanced contrast microscopy and fiber optical spectroscopy. 11th International Congress on Photobiology (1992) Kyoto (Abstract V 09-13) 43. E. Reich, K. Miller, D. Repassy, K. König, A. Rück, R. Bachor: Liposome-administered tetrametyhlhematoporphyrin (TMHP) as a photodynamic agent for bladder tumor cells. 11th International Congress on Photobiology (1992) Kyoto (Abstract P201) 44. R. Bachor, E.Reich, K. Miller, A. Rück, K. König, R. Hautmann: Phototoxicity of aminolevulinic acid (ALA) in vitro. 11th International Congress on Photobiology (1992) Kyoto (Abstract V 17-02) 45. K. König, E. Reich, F. Genze, R. Miller: In-vivo fluorescence behaviour and photodynamic activity of liposomal Tetramethyl-Hematoporphyrin (TMHP). 11th International Congress on Photobiology (1992) Kyoto (Abstract P 144) 46. K. König, A. Rück, F. Genze, E. Reich, K. Miller, R. Bachor Distribution and photodynamic activity of ALA-induced endogeneous protoporphyrin. 11th International Congress on Photobiology (1992) Kyoto (Abstract P 145) 47. K. König, H. Schneckenburger, A. Rück, E. Reich, K.Miller, R. Bachor: On-line fluorescence spectroscopy during photodynamic therapy with ALA-induced endogeneous porphyrin. 11th International Congress on Photobiology (1992) Kyoto (Abstract V 09-12) 48. H. Schneckenburger, K. König, P. Gessler, I. Pavenstädt-Grupp: Laser-induzierte Autofluoreszenz für die Diagnostik. Jahreskongreß der Deutsch. Gesellsch. für Lasermedizin 1992, Münster, Abstract: Laser in Medicine and Surgery 8(1992)128 49. W. Strauss, K. König, K. Miller, W. Mohr, A. Rück, H. Schneckenburger, R. Steiner: Meso-Tetra(4-carboxyphenyl)porphyrin-Fluoreszenzverhalten und photodynamische Therapie an xenotransplantierten Blasentumoren. Jahreskongreß der Deutsch. Gesellsch. für Lasermedizin 1992, Münster, Abstract: Laser in Medicine and Surgery 8(1992)135 50. K. König, H. Schneckenburger, A. Rück, R. Steiner, E. Reich, K. Miller, R. Bachor: On-line Fluoreszenzspektroskopie während der PDT nach ALA-Applikation. Jahreskongreß der Deutsch. Gesellsch. für Lasermedizin 1992, Münster, Abstract: Laser 92. Reihe Medizin. Eds.: G.H. Willital, M. Maragakis, R.R. Lehmann. Verlag Shaker, Aachen 1992, Seite 22 52 K. König, A. Rück, F. Genze, E. Reich, K. Miller, R. Bachor: Pharmakokinetik und photodynamische Aktivität von ALA-induziertem Protoporphyrin IX. Jahreskongreß der Deutsch. Gesellsch. für Lasermedizin 1992, Münster, Abstract: Laser 92, Seite 241 (s.o.) 53. K. König, E. Reich, F. Genze, K. Miller: In-vivo Fluoreszenzverhalten und photodynamische Wirkung von Tetramethyl-Hämatoporphyrin. Jahreskongreß der Deutsch. Gesellsch. für Lasermedizin 1992, Münster, Abstract: Laser 92, Seite 242 (s.o.) 54. R. Fischer, K. König, W. Puhl: Möglichkeiten und Grenzen der percutanen Nukleotomie mit dem Excimer Laser - Ergebnisse von in-vitro Untersuchungen. Kongreß der Deutsch. Gesellsch. für Orthopädie und Traumatologie. Mannheim, 16.-20.9.1992 55. W.H. Boehncke, K. König, A. Rück, K. Kaltoft, R. Kaufmann, W. Sterry: Proliferationsinhibition einer menschlichen T-Zell Linie durch photodynamische und PUVA Therapie. Lasermedizin 8(1992)93 56. K. König, R. Hibst, H. Schneckenburger, G. Flemming: Laserinduced autofluorescence of caries. SPIE (Januar 1993) Los Angeles 57. K. König, H. Schneckenburger, A. Rück, H. Wald, R. Steiner: Autofluorescence of cells and tissue. SPIE (Januar 1993) Los Angeles 58. R. Kaufmann, W.H. Boehncke, K. König, R. Hibst: Comparitive study of Q-switched Nd:YAG- and Alexandrite laser treatment of tattoos. 13th annual meeting of the American Society for Laser Medicine and Surgery (April 1993) New Orleans 59. K. König (invited), H. Schneckenburger, A. Rück: The Nature and Role of Protoporphyrin Photoproducts: In Vitro and In Vivo. 21st Annual Meeting of the American Society for Photobiology (26.-30.6.93) Chicago, abstract: Photochem. Photobiol. 57(1993)76S-77S 60. W.H. Boehncke, K. König, A. Rück, K. Kaltoft, R. Kaufmann, W. Sterry: Inhibitory effects of photodynamic therapy on malignant transformed T cells suggest its potential in the treatment of cutaneous T cell lymphomas. Kongress der Arbeitsgemeinschaft Dermatol. Forschung, ADF. Mainz, 1992. Abstract: Arch Dermatol Res 285(1993)64-65. 61. R. Kaufmann, W-H Boehncke, K. König, R. Hibst: Comparative study of Q-switched Nd:YAG- and alexandrite laser treatment of tattoos. Congress of the American Society of Lasermedicine, New Orleans, 1993. Abstract: Laser Surg Med 1993, Suppl 5:54 62. W.H. Boehncke, K. König, W. Scheffold, W. Sterry: Application of photodynamic therapy in psoriasis. Tricontinental Meeting, Kyoto, 1993. Abstract: J Invest Dermatol 101(1993)465. 63. W. Sterry, K. König, A. Rück, R. Kaufmann, W-H Boehncke: Application of photodynamic therapy in mycosis fungoides. Tricontinental Meeting, Kyoto, 1993. Abstract: J Invest Dermatol 101(1993)464 64. W-H Boehncke, K. König, W. Scheffold, W. Sterry. Photodynamic therapy in psoriasis. Meeting of the Skin Pharmacology Society, Hongkong, 1993. Abstract: Skin Pharmacol, in press 65. W-H Boehncke, K. König, A. Rück, K. Kaltoft, R. Kaufmann, W. Sterry: Proliferationsinhibition einer menschlichen T-Zell Linie durch photodynamische und PUVA Therapie. Kongress der Deutsch. Gesellschaft für Lasermedizin, Münster. Abstract: Lasermedizin 8(1992)93 66. K. König, R. Hibst, H. Meyer, G. Flemming, H. Schneckenburger: Laser-induced autofluorescence of carious regions of human teeth and caries-involved bacteria. International Symposium on Biomedical Optics Europe '93, 1993, Sept 1-5, Budapest, abstract: 67. K. König, A. Kienle, W-H. Boehncke, R. Kaufmann, A. Rück, T. Meier, R. Steiner: Photodynamic tumour therapy and on-line fluorescence spectroscopy after ALA administration using 633 nm-light as therapeutic and fluorescence excitation radiation. International Symposium on Biomedical Optics Europe '93, 1993, Sept 1-5, Budapest, abstract: 68. K. König, G. Beck, R. Steiner: International Symposium on Biomedical Optics Europe '93, 1993, Sept 1-5, Budapest, abstract: 69. K. König, W-H. Boehncke, A. Rück, R. Kaufmann, R. Steiner, W. Sterry: Photodynamic effects on T-cells and skin lesions of a patient with mycosis fungoides using porphyrin photosensitizers. International Symposium on Biomedical Optics Europe '93, 1993, Sept 1-5, Budapest, abstract: 70. H. Schneckenburger, M. Gschwend, K. König, A. Rück, R. Sailer, W. Strauss: Subcellular distribution of photodynamic photosensitizers. International Symposium on Biomedical Optics Europe '93, 1993, Sept 1-5, Budapest, abstract: 71. K. König, K. Kunzi-Rapp, H. Schneckenburger, A. Rück: Natural and ALA stimulated autofluorescence and PDT of xenotransplanted tumors in the CAM of chicken embryos.(Poster), International Congress on Photobiology, Marburg, 1994, Sept., abstract: V-10/P 10 72. R. Sailer, W. Strauss, K. König, A. Rück, R. Steiner: Fluorescence properties and photodynamic inactivation of the gram-negative bacterium Pseudomonas aeruginosa in comparison to the amount of endogenous porphyrins. International Congress on Photobiology, Marburg, 1994, Sept., abstract: V-10/p15 73. H. Schneckenburger, K. König, T. Dienersberger, M. Gschwend: Time-gated microscopic imaging and spectroscopy in photobiology and medical diagnosis. International Congress on Photobiology, Marburg, 1994, Sept., abstract: VI-6/01 74. A. Rück, M. Gschwend, K. König, H. Schneckenburger, R. Sailer, W. Strauss: Photoproducts and formation of molecular species during PDT, International Congress on Photobiology, Marburg, 1994, Sept., abstract: V-2/03 75. H. Schneckenburger, K. König, T. Dienersberger, M. Gschwend: Time-gated microscopic imaging and spectroscopy in photobiology and diagnostics. UPS'93, 1993, Sept 26-30, Vilnius 76. A. Rück, W.H. Boehncke, K. König, K. Kunzi-Rapp, T. Meier, R. Kaufmann: Polychromatic vs. monochromatic PDT-comparative effects in vitro and in vivo. 10th Congress, International Society for Laser Surgery and Medicine, 1993, Nov 12-15, Bangkok, Thailand 77. K. König (invited), H. Schneckenburger: Laser-Induced Autofluorescence for Medical Diagnosis. Congress on Fluorescence. Prag, Febr. 1994 78. K. König, H. Schneckenburger, H. Walt, T. Leemann, M.T. Wyss-Desserich, A. Rück, B. Tromberg: Microscopic Studies on ALA-incubated tumor cells and tumor spheroids. 1994, Los Angeles, Jan 22-29, abstract: 2133-40 79. K. König, H. Schneckenburger, J. Hemmer, B. Tromberg, R. Steiner: In-vivo fluorescence detection and imaging of porphyrin-producing bacteria in the human skin and in the oral cavity for diagnosis of acne vulgaris, caries, and squamous cell carcinoma. SPIE 1994, Los Angeles, Jan 22-29, abstract: 2135-16 80. K. König, H. Schneckenburger: Laser-induced dental caries and plaque diagnosis on patients by sensitive autofluorescence spectroscopy and time-gated video imaging. Preliminary studies. SPIE 1994, Los Angeles, Jan 22-29, abstract: 2128-64 81. K. König, H. Schneckenburger, A. Rück, R. König: Studies on Porphyrin Photoproducts in Solution, Cells, and Tumor Tissue. SPIE 1994, Los Angeles, Jan 22-29, abstract: 2133-39 82. K. König, H. Schneckenburger, H. Boehncke, R. Hibst: In vivo fluorescence spectroscopy and imaging of ALA-induced endogenous porphyrins in skin after Er:YAG ablation of stratum corneum. SPIE 1994, Los Angeles, Jan 22-29, abstract: 2128-38 83. A. Rück, M. Gschwend, W. Strauss, K. König, R. Sailer, H. Schneckenburger: Photoproducts and new spectral bands during PDT probed by cw and time-gated microscopy and spectroscopy. SPIE 1994, Los Angeles, Jan 22-29, abstract: 84. H. Schneckenburger, K. König, K. Kunzi-Rapp, M. Gschwend: Zeitaufgelöste Untersuchungen zur Intrazellulären Verteilung und Licht-induzierten Reaktion von ALA-induziertem Protoporphyrin. 1. Münchner ALA-Workshop, 17./18.3.94, München. 85. K. König (invited): Laser-induced autofluorescence in medicine. International Conference: Lasers & Applications. Advances in science, medicine and technology, NILES 1994, Cairo, March 26-30, 1994 86. BJ. Tromberg, TB. Krasieva, SJ. Madsen, K. König: Principles of Laser Based Diagnostics. 14th Annual meeting of the American Society for Laser Medicine and Surgery, Ontario, April 8-10, 1994, abstract 305, Lasers Surg Med, Suppl 6, 1994, 58 87. K. König, T. Krasieva, B. Tromberg: Spectrally-resolved fluorescence imaging of porphyrin-producing bacteria in human skin. CLEO, May 8-13, 1994, Anaheim, abstract: CTuA3 in "Biomedical Optical Imaging" 88. BJ. Tromberg, C. Chapman, K. König, MW. Berns, Y. Liu, G. Sonek: Microirradiation effects on cells. Gordon Conference, New Hampshire, July, 1994 89. K. König , Y. Liu, GJ. Sonek, MW. Berns, BJ. Tromberg: Photoinduced modifications of cells in an optical trap. BIOS Europe '94, Lille, Sept 6-10, 1994, abstract: 2329-40 90. S. Kimel, K. König, MW. Berns: Photodynamic effects on human and chicken erythrocytes. SPIE-Proceedings, BIOS Europe '94, Lille, Sept 6-10, 1994, abstract: 2329-46 91. K. König (invited): Fluorescence imaging and spectroscopy of motile sperm cells and CHO cells in an optical trap, SPIE '95, San Jose, Febr 4-10, 1995, abstract: 92. K. König, BJ Tromberg, MW Berns: One- and Two Photon Excited Fluorescence of Motile Cells in the Optical Trap, LASER 95, München 93. K. König, P. Fergin, MW. Berns, BJ. Tromberg: Spectrally-resolved Fluorescence Imaging of Skin after Topical ALA-Administration, LASER 95, München 94. K. König (invited): Two-Photon Excited Autofluorescence in CHO Cells and Motile Spermatozoa. Third International Symposium on Innovative Fluorescence Methodologies in Biochemistry and Medicine. July 30-Aug 2, 1995, Kaanapali, Maui, Hawaii, USA 95. L. T. Norvang, E. J. Fisherstrand, K. König, B. Bakken, D. Grini, Standahl, T. E. Milner, M. W. Berns, J. S. Nelson, L. O. Svaasand: Comparison between reflectance spectra obtained with an integrating sphere and a fiber-optic collection system. BIOS Sept. 1995, Barcelona 96. K. König, P. So, W. W. Mantulin, E. Gratton, M. W. Berns, B. J. Tromberg: Two-Photon Excited Cellular Autofluorescence Induced by CW and Femtosecond NIR Microirradiation. BIOS Sept. 1995, Barcelona 97. K. König, T. Krasieva, E. Bauer, U. Fiedler, M. W. Berns, B. J. Tromberg: UVA-Induced Oxidative Stress Probed by Autofluorescence Imaging, Cloning Assay, and Comet Assay. BIOS Sept. 1995, Barcelona 98. K. König (invited): NAD(P)H- and Porphyrin-Attributed Autofluorescence for Medical Diagnosis. Laser 95. Charleston, Dez. 95, South Carolina, USA 99. K. König (invited): Continuous Wave Induced Two-Photon Excitation in Living Cells. SPIE. Jan. 1996. San Jose, Kalifornien 100. K. König: Multiphotonen-Effekte in der Lichtfalle. 60. Physikertagung der DPG. 11.-15.3.1996, Jena. (Interview mit dem Deutschlandfunk zu Fragen der Optischen Mikromanipulation von Vitalzellen anlässlich der Physikertagung, 14.3.96 (Sendung 15.3.96)) 101. K. König, P. So, W.W. Mantulin, M.W. Berns, B.J. Tromberg, E. Gratton: Two-photon excited fluorescence in living cells. (Poster) Tripartite Meeting der Anatomischen Gesellschaft (91. Versammlung), Anatomical Society of Great Britain and Ireland, Nederlandse Anatomen Vereniging. 24.3.-27.3.1996, Jena. 102. K. König (invited): Two-photon excitation in a single motile sperm cell confined in an optical trap. Annual Meeting German Society for Cell Biology. 24.3.-28.3.1996, Hamburg. 103. K. König, P. So, W.W. Mantulin, E. Gratton: Cell damage in Two-Photon Microscopes. BiOS Sept 1996, Wien 104. K. König, L. Svaasand, Y. Tadir, B. Tromberg, M.W. Berns: Optical determination of motility forces in human spermatozoa with laser tweezers. BiOS Sept 1996, Wien 105. K. König: Two-photon femtosecond microscopy in living cells. 6th International Conference on Laser Applications in Life Sciences (LALS), Sept 1996, Jena 106. K. König, T. Krasieva, B. Tromberg, U. Diedler, E. Bauer, M.W. Berns, K.O. Greulich. UVA-induced oxidative stress in living cells. (Poster). 6th International Conference on Laser Applications in Life Sciences (LALS), Sept 1996, Jena 107. K.J. Halbhuber, K. König, Ch. Scheven, A. Aschoff, H. Feuerstein. Visualization of enzymatic primary reaction products by laser scanning microscopy. (Poster). 6th International Conference on Laser Applications in Life Sciences (LALS), Sept 1996, Jena 108. K. König, U. Simon, K.-J. Halbhuber. Three-Dimensional Two-Photon Femtosecond Fluorescence Microscopy using a modified Confocal Laser Scanning Microscope. 166. WE-Heraeus-Seminar: Multiphoton Photochemistry in Biological Systems. 28.-30.10.1996, Bad Honnef 109. K. König, S. Kimel, L.O. Svaasand, B. Tromberg, T. Krasieva, M.W. Berns, P. So, W.W. Mantulin, E. Gratton, K.J. Halbhuber: Cell damage in UVA and cw/femtosecond NIR microscopes. SPIE Photonics West Febr. 1997, San Jose, California 110. K. König (Gastredner): Zweiphotonenanregung vitaler Zellen mittels CW und Femtosekunden-NIR-Mikroskopie. 5. Kolloquium des DFG-Schwerpunktprogramms: Neue mikroskopische Techniken für Biologie und Medizin. 4.-6.3.1997, Rostock 111. König (Gastredner): Eigenfluoreszenz-Spektroskopie an lebenden Zellen: NADH als Bioindikator für Lichtstress. 1. Rostocker Biosystemtechnik-Symposium des Innovationskollegs: Komplexe und zelluläre Sensorsysteme. 6.-7.3.1997, Rostock 112. K. König, K.-J. Halbhuber: Zwei-Photonen-Femtosekundenmikroskopie vitaler Zellen. 13th International Congress LASER 97. Munich, June 1997. 113. K. König, H. Oehring, K.-J. Halbhuber, U. Fiedler, E. Bauer, K.O. Greulich. Comet assay, cloning assay, histochemistry, light- and electrom microscopy on one preselected cell. SPIE-EUROPTO'97, San Remo, 4.-8.9.97. 114. K. König. Laser tweezers as novel nonlinear tools in cell and biomolecule diagnostics. SPIE-EUROPTO'97, San Remo, 4.-8.9.97. 115. K. König, K.-J. Halbhuber. Multiphoton femtosecond microscopy of living cells. XXXIX. Symposium of the Society for Histochemistry, Jena, 24.-27.9.97, abstract: Histochem. Cell. Biol. 108(1997)276. 116. K. König, P. So, W. Mantulin, M. Berns, B. Tromberg, E. Gratton. Two-photon excited fluorescence in living cells. XXXIX. Symposium of the Society for Histochemistry, Jena, 24.-27.9.97, abstract: Histochem. Cell. Biol. 108(1997)277. 117. K. König, M. Schüler, K.-J. Halbhuber. Autofluorescence of cells and tissues as a diagnsotic tool. XXXIX. Symposium of the Society for Histochemistry, Jena, 24.-27.9.97, abstract: Histochem. Cell. Biol. 108(1997)277. 118. K. König. Two-photon effects in living cells. International Practical Course on Modern Laser Microscopy.Jena, 18.-21.11.1997. 119. K. König. How safe is the gamete micromanipulation by laser tweezers? Photonics West. SPIE-Tagung. 23.1.-30.1.1998, San Jose, California. 120. K. König, M. Teschke, W. Pfister, H. Meyer: Photodynamically inactivation of propionibacterium acnes. Photonics West. SPIE-Tagung. 23.1.-30.1.1998, San Jose, California. 121. K. König. Photoproduct formation during porphyrin PDT. First World Congress of Photomedicine in Gynecology. 19.-21.2.98. Zurich. 122. K. König. Cell damage in UV and Multiphoton NIR microscopes. Scanning 98. 9.-12.5.98, Baltimore. 123. K. König,P. So, W.W. Mantulin, B. Tromberg, E. Gratton. Two-Photon Excitation in Living Cells. Deutsche Gesellschaft für Biophysik. Jahrestagung. 21.-23.9.1998, Frankfurt/M. 124. K.-J. Halbhuber, R. Krieg, K. König. Fluorescence laser microscopical demonstration of endogenous and immunobond phosphatase activity using metallo-azo dye complexes. 40. Symposium of the Society of Histochemistry. 22.-25.9.1998, Giessen. 125. K. König, P. Fischer. I. Riemann, K.-J. Halbhuber. Multiphoton-excited fluorescence in histochemistry. 40. Symposium of the Society of Histochemistry. 22.-25.9.1998, Giessen. 126. K. König, P. Fischer. I. Riemann, K.-J. Halbhuber. Femtosecond versus picosecond multiphoton microscopy. 23.-29.1.1999, San Jose, California. 127. K. König. Cloning assay thresholds on cells exposed to ultrafast laser pulses. 23.-29.1.1999, San Jose, California. 128. K. König, P. Fischer. I. Riemann, K.-J. Halbhuber. PDT by non-resonant two-photon excitation. 23.-29.1.1999, San Jose, California. 129. K. König. Nahe Infrarot Laserscanningmikroskopie an vitalen Zellen. 10. Deutscher MTA-Kongress. 10.-12.3.1999, Mannheim. 130. K. König. Plenarvortrag: Cellular response to ultrashort laser pulses in multiphoton microscopes. Focus on Microscopy 1999. 11.-15.4.1999, Heidelberg. 131. K. König. Femtosecond multiphoton microscopy for cell and tissue diagnostics. Optical Society of America (OSA) Topical Meeting at Laser 99. 14.-16.6.1999. München. 132. K. König. Multiphoton near infrared microscopy: optical trapping, 4D microscopy, and nanosurgery. Third Conference on Fluorescence Microscopy and Fluorescent Probes. 20.-23.6.1999. Prag. Tschechische Republik. 133. K. König. Intracellular nanosurgery with compact femtosecond laser. 18. Congress of the International Commission for Optics (ICO XVIII): Optics for the next Millenium. 02.-06.8.1999, San Francisco, USA. 134. K. König. Anwendung optischer Mikroskopien in den Biowissenschaften. W.E. Heraeus Fereinkurs: Moderne Fernfeld- und Nahfeld- Mikroskopien. 20.9-01.10.1999, Chemnitz. 135. K. König, I. Riemann, P. Fischer, H. Oehring, Th. Becker, K.-J. Halbhuber. Apoptosis induced by continuous wave ultraviolet and red radiation as well as by near infrared femtosecond laser pulses. 41^st Symposium of the International Society for Histochemistry. 22.9.-25.9.1999, Gargellen, Austria. 136. K.-J. Halbhuber, K. König. 41^st Symposium of the International Society for Histochemistry. 22.9.-25.9.1999, Gargellen, Austria. 137. Oehring, K. König. (Poster). 41^st Symposium of the International Society for Histochemistry. 22.9.-25.9.1999, Gargellen, Austria. 138. K. König, I. Riemann, W. Fritsche, P. Fischer, K.-J. Halbhuber. Intracellular nanosurgery with femtosecond laser pulses. Jahrestagung der Deutschen Gesellschaft für Biophysik. 03.-06.9.1999, Ulm. 139. K. König. Multiphoton femtosecond laser microscopy for 4D fluorescence diagnostics and nanosurgery of living cells.. XI International Symposium "Ultrafast Phenomena in Spectroscopy (UPS '99)", 25.-29.10.1999, Taipei, Taiwan, ROC. 140. K. König. Intracellular nanosurgery using near infrared femtosecond laser pulses. Focus on Microscopy 2000 (FOM2000). 09.-13.4.2000, Shirahama, Japan. 141. K. König. Intracellular nanosurgery using near infrared femtosecond laser pulses. OPTO und IRS². 9-11.5.2000, Erfurt. 142. K. König, I. Riemann, W. Fritzsche, U. Tirlapur, P. Fischer, K.J. Halbhuber. Nanochirurgie mittels NIR-Femtosekundenlaser-Lasermikoskopie. 101. Tagung der Deutschen Gesellschaft für angewandte Optik. 13.-17.7.2000, Jena (mit Beteiligung von Japan, Österreich, Schweiz). 143. K. König, I. Riemann, A. Göhlert, P. Fischer, T. Liehr, I.F. Loncarevic, U. Claussen, K.J. Halbhuber. Multiphoton Multicolor FISH. SPIE-EBiOS 2000, 04.-08.7.2000, Amsterdam. 144. K. König. Multiphoton Microscopy to study macromolecules in living cells. Gordon Research Conference on Macromoleuclar Organization and Cell Function, 06.-11.8.2000, Oxford, U.K. 145. K. König, C. Peuckert, I. Riemann, U. Wollina. Non-invasive 3D optical biopsy of human skin with NIR femtosecond laser pulses for diagnosis of dermatological disorders. 3rd World Conference on Cellular and Molecular Biology, 08.-13.10.2000, Jena. 146. K. König, U. Tirlapur, C. Peuckert, I. Riemann, A. Bergmann, W. Becker. Time-resolved two-photon fluorescence imaging of living cells and tissues. 3rd World Conference on Cellular and Molecular Biology, 08.-13.10.2000, Jena. 147. C. Peuckert, I. Riemann, U. Wollina, K. König. (Poster) Remission microscopy with near femtosecond laser pulses for 3D in vivo imaging of human skin. 3rd World Conference on Cellular and Molecular Biology, 08.-13.10.2000, Jena. 148. C. Peuckert, I. Riemann, K. König. Two-photon induced autofluorescence of in vivo human skin with femtosecond laser pulses - a novel imaging tool of high spatial, spectral and temporal resolution. 3rd World Conference on Cellular and Molecular Biology, 08.-13.10.2000, Jena. 149. A. Göhlert, I. Riemann, T. Liehr, I.F. Lomcarevic, U. Claussen, K.H. Halbhuber, K. König. (Poster) Multiphoton multicolor FISH (MM-FISH): A verstaile technique to detect specific sequences within single DNA molecules. 3rd World Conference on Cellular and Molecular Biology, 08.-13.10.2000, Jena. 150. M. Rothammel, M. Teschke, W. Pfister, K.J. Halbhuber, K. König. (Poster) Inactivation of Porphyromonas gingivalis by red light evaluated by laser scanning microscopy. 3rd World Conference on Cellular and Molecular Biology, 08.-13.10.2000, Jena. 151. I. Riemann, A. Göhlert, U. Claussen, K.J. Halbhuber, K. König. Three dimensional imaging of specific DNA sequences in cells and tissues by multiphoton multicolor FISH using near infrared femtosecond laser pulses. 3rd World Conference on Cellular and Molecular Biology, 08.-13.10.2000, Jena. 152. U.K. Tirlapur, K. König. Multiphoton microscopy in plant cells. 3rd World Conference on Cellular and Molecular Biology, 08.-13.10.2000, Jena. 153. N. Rubtsov, I. Riemann, V. Trifonov, T. Karamysheva, T. Liehr, U. Claussen, K. König. (Poster)Chromosome microdissection using NIR femtosecond laser pulses and generation of band specific DNA-libraries with DOP-PCR. 3rd World Conference on Cellular and Molecular Biology, 08.-13.10.2000, Jena. 154. M. Wiederhold, U.K. Tirlapur, K. König, S. Russwurm (Poster). Association of procalcitonin in HEPG2 cells studied with confocal laser scanning microscopy. 3rd World Conference on Cellular and Molecular Biology, 08.-13.10.2000, Jena. 155. U.K. Tirlapur, K. König, R. Krieg, K.J. Halbhuber. Femtosecond NIR laser pulses elicit generation of ROS in marsupial cells leading to apoptosis-like cell death. 3rd World Conference on Cellular and Molecular Biology, 08.-13.10.2000, Jena. 156. M. Wiederholt, I. Riemann, S. Russwurm, K. König. (Poster). The uptake of fluoresceinisothiocyanate-lipopoly-saccharide by cultivated human mononuclear cells using two-photon fluorescence microscopy. 3rd World Conference on Cellular and Molecular Biology, 08.-13.10.2000, Jena. 157. K. König, U. Tirlapur, I. Riemann, W. Fritzsche. Nanosurgery of chromosomes, living cells and tissues with femtosecond laser pulses in the near infrared. 3rd World Conference on Cellular and Molecular Biology, 08.-13.10.2000, Jena. 158. D. Volkmann, U.K. Tirlapur, K. König, T. Mori, T. Fujiwara, F. Baluska. Plant plasmodesmata:myosin VIII-supported celll-to-cell channels for macromolecular trafficking. 3rd World Conference on Cellular and Molecular Biology, 08.-13.10.2000, Jena. 159. K. König. Diagnosis and knocking out of genomic regions with multiphoton microscopes.SPIE-Photonics West 2001. San Jose, California. 160. K. König. Time-resolved multiphoton microscopy. First symposium on FRET and FLIM: Advanced fluorescence techniques for biological imaging. 08.-10.6.2001. Austin, Texas, USA. 161. K.König. Multiphoton microscopy of cells and tissues. European Conference on Biomedical Optics. Laser 2001. 17.-20.6.2001, München. 162. K. König, O. Krauss, I. Riemann, D. Schweitzer. Poster. Corneal surgery with 80 MHz nanojoule femtosecond laser pulses in the near infrared. European Conference on Biomedical Optics. Laser 2001. 17.-20.6.2001, München. 163. K. König. Optical biopsy with femtosecond laser pulses: in vivo skin diagnostics and 3D gene imaging with submicron resolution. 47^th Annual Paediatric Pathology Society Meeting. 13.-15.9.2001. Warsaw, Poland. 164. I. Riemann, A. Göhlert, T. Liehr, I.F. Loncarevic, U. Claussen, K.J. Halbhuber, K. König. Poster. 3D imaging of specific DNA sequences in cells, tissues and within single DNA molecules with multiphoton multicolor FISH (MM-FISH). 43^rd Symposium of the Society for Histochemistry. 26.-29.9.2001, Vienna, Austria. 165. R. Krieg, U.K. Tirlapur, K. König, C. Peuckert, K.J. Halbhuber. Poster. In situ localization of femtosecond near-infrared laser induced ROS with a new NLO fluorochrome after two-photon excitation at 800 nm. 43^rd Symposium of the Society for Histochemistry. 26.-29.9.2001, Vienna, Austria. 166. K. König. Möglichkeiten der intrastromalen Hornhautchirurgie mit Femtosekunden-Laserpulsen. 99. Tagung der Deutschen Ophthalmologischen Gesellschaft (internationale Beteiligung). 29.9.-02.10.2001, Berlin. 167. K. König. Optical tomography of human skin with subcellular spatial and picosecond time resolution using intense near infrared femtosecond laser pulses. 3^rd International Symposium Lasers in Dermatology. 01.-02.10.2001, Ulm. 168. K. König, U. Wollina, I. Riemann, C. Peuckert, K.J. Halbhuber, V. Fünfstück, T.W. Fischer, P. Elsner. Optical tomography of human skin with subcellular spatial and picosecond time resolution. SPIE-Photonics West 2002. 19.-25.1.2002, San Jose, California. 169. K. König, I. Riemann, O. Krauss, W. Fritzsche. Nanodissection of human chromosomes and ultraprecise eye surgery with nanojoule near infrared femtosecond laser pulses. SPIE-Photonics West 2002. 19.-25.1.2002, San Jose, California. 170. K. König. Fluorescence Lifetime Imaging. Focus on Microscopy 2002. April 2002. Kaohsiung, Taiwan. 171. K. König, O. Krauss, I. Riemann. Optical Tomography of the Eye by Multiphoton Autofluorescence and SHG Imaging. 100. Tagung der Deutschen Ophthalmologischen Gesellschaft (internationale Beteiligung). 26.9.-29.9.2001, Berlin. 172. O. Krauss, I. Riemann, K. König. Intrastromal surgery of the cornea with 80 MHz nanojoule femtosecond laser pulses in the near infrared. 100. Tagung der Deutschen Ophthalmologischen Gesellschaft (internationale Beteiligung). 26.9.-29.9.2001, Berlin. 173. K. König. Photonics West 2003, Jan. 174. K. König, FOM2003. April 12-19, 2003. Genua, Italy. 175. K. König, Skin. May 21-24, 2003. Hamburg, Germany. 176. K. König. Biomedical Optics, Laser2003, June 24-25, 2003. Munic, Germany. 177. K. König, July 21-23, 2003. Banff, Canada. 178. K. König. Imaging and manipulation on a micron scale. Microscopes, Laser Tweezers, Multiphoton Effects. Graduate Summer Scholl Bio-Photonics `03, June 15-21, 2003, Ven, Sweden 179. K. König. Femtosecond Lasers in Dermatology. The 87th OSA Annual Meeting: Frontiers in Optics. Laser Science XIX. Oct 5-9, 2003, Arizona, Florida 180. I. Riemann, E. Dimitrow, P. Fischer, A. Reif, M. Kaatz, P. Elsner, K. König. High resolution multiphoton tomography of human skin in vivo and in vitro. SPIE-Photonics West 2004, 24.-29.01.2004, San José, USA. 181. K. König: Multiphoton Fluorescence/SHG imaging of cells, skin and heart tissue. Berkeley 182. W. Becker, A. Bergmann, G. Biscotti, K. König. High-Speed FLIM Data Acquisition by Time-Correlated Single Photon Counting. SPIE-Photonics West 2004, 24.-29.01.2004, San José, USA. 183. K. Schenke-Layland, I. Riemann, U. A. Stock, K. König. Non-invasive multiphoton imaging of cardiovascular structures using NIR femtosecond laser scanning microscopy. SPIE-Photonics West 2004, 24.-29.01.2004, San José, USA. 184. A. Czaki, G. Maubach, F. Garwe, A. Bochmann, K. König, W. Fritzsche. Poster: Metal nanoparticles for nanooptics and nanoanalytics. SPIE-Photonics West 2004, 24.-29.01.2004, San José, USA. 185. M. Kaatz, J. Fluhr, E. Dimitrow, P. Elsner, I Riemann, J. Kobow, K. König. Monitoring of the accumulation and pharmacokinetics of fluorescent dyes througth the stratum corneum by high-resolution multiphoton tomography. Stratum Corneum, May 19, 2004. 186. I. Riemann, K. König. High Resolution Multiphoton Tomography of the Epidermis with Submicron Resolution. Stratum Corneum, May 19, 2004. 187. K. König: Femtosecond laser application in biotechnology and medicine. The 5^th International Symposium on Laser Precision Microfabrication, 11.-14. Mai 2004 in Nara, Japan (2004). 188. K. König. 1) Photodamage during light microscopy. 2) Multiphoton microscopy. International Summer School, Vancouver, Canada 189. T. Velten, H. Schuck, I. Riemann, F. Bauerfeld, D. Sauer, K. König. Time-Resolved and Spectrally-Resolved 5D Multiphoton Microscopy for Analysis and Nanoprocessing of Biological and Non-Biological Materials. LANE 2004, 21.-24.09.2004, Erlangen, Germany. 190. I. Riemann, K. König. High-resolution multiphoton tomography of human skin in vivo and in vitro. Photonics Europe, 26.-30. April 2004, Straßburg, France. 191. K. Schenke-Layland, F. Opitz, I. Riemann, U.A. Stock. Multiphoton imaging of cardiovascular structures. Photonics Europe, 26.-30. April 2004, Straßburg, France. 192. V. Ulrich, P. Fischer, E. Haupt, I. Riemann, K. König. Spectral Imaging and fluorescence lifetime imaging with a compact. Photonics Europe, 26.-30. April 2004, Straßburg, France. 193. K. König, F. Fischer, P. Fischer, S. Puschmann, I. Riemann, V. Ulrich, R. Wepf. Characterization of multiphoton laser scanning microscope optical parameters. Photonics Europe, 26.-30. April 2004, Straßburg, France. 194. K. König, G. Maubach, A. Csaki, W. Fritzsche. Specific cutting of DNA with NIR fs-laser. Photonics Europe, 26.-30. April 2004, Straßburg, France. 195. K. König, F. Garwe, O. Krauss, B. Wang, W. Fritzsche, I. Riemann. Nanoprocessing of DNA, cells and tissues. Photonics Europe, 26.-30. April 2004, Straßburg, France. 196. B. Wang, I. Riemann, H. Schubert, K. König. Intraocular optical tomography of the cornea in vitro and in vivo. Photonics Europe, 26.-30. April 2004, Straßburg, France. 197. K. Schenke-Layland, I. Riemann, V. Ulrich, F. Opitz, U.A. Stock, K. König. Multiphoton Imaging of collagen and elastin of native and tissue-engineered heart valves. 183. K. König, F. Garwe, O. Krauss, B. Wang, W. Fritzsche, I. Riemann. Nanoprocessing of DNA, cells and tissues. Photonics Europe, 26.-30. April 2004, Straßburg, France. 198. B. Wang, I. Riemann, H. Schubert, K. König. Intraocular optical tomography of the cornea in vitro and in vivo. Photonics Europe, 26.-30. April 2004, Straßburg, France. 199. G. Ehrlich, T. Boneva, I. Riemann, R. Wetzke, K. König. Investigation of PI3Kgamma association with p101by MP-FLIM in living cells. 183. K. König, F. Garwe, O. Krauss, B. Wang, W. Fritzsche, I. Riemann. Nanoprocessing of DNA, cells and tissues. Photonics Europe, 26.-30. April 2004, Straßburg, France. 200. K. König. Transfection and nanosurgery with 80 MHz femtosecond lasers. Photonics Europe, 26.-30. April 2004, Straßburg, France. 201. K. König. Multiphoton microscopy and tomography of human skin. FOM2004, Philadelphia, USA 202. K. König. Lasertechnik: konventionell und photodynamisch. Internet-Übertragung. Zurich, Switzerland 203. K. König, I. Riemann, A. Ehlers, J. Kobow. In vivo non-invasive multiphoton tomography of human skin with subcellular resolution to detect bio- and chemohazards. SPIE Conference on Defence, Oct 10, 2004, London, UK 204. K. König. In vivo confocal microscopy of the skin and its application in cosmetology. International Society for Bioengineering and the Skin. Oct 28-30, 2004, Orlando, FL, USA 205. K. König. Multiphoton laser microscopy with compact femtosecond lasers. Biomaterials 2004, Nov 4-5, 2004, Erfurt, Germany 206. K. König. Plenary talk sponsered by Coherent Inc. In vivo multiphoton tomography of skin cancer. The 8^th International Conference on Optics Within Life Sciences (OWLS8), Nov 28- Dec 1, 2004, Melbourne, Australia 207. K. König. Multiphoton tomography with subcellular resolution. First Scientific Skin Workshop, Jan 14-15, 2005, Reyjakvik, Iceland 208. K. König. In vivo multiphoton time-resolved fluorescence/SHG imaging in human skin. Berkeley 209. A. Czaki, G. Maubach, F. Garwe, A. Steinbrück, K. König, W. Fritzsche. A novel DNA restriction technology based on laser pulse energy conversion on sequence-specific bound metal nanoparticles. Photonics West, Jan 22-27, 2005, San Jose, USA 210. W. Fritzsche, A. Czaki, A. Steinbrück, F. Garwe, K. König, M. Raschke. Metal nanoparticles as passive and active tools for bioanalytics. Photonics West, Jan 22-27, 2005, San Jose, USA 211. T. Anhut, K. Hassler, T. Lasser, K. König, R. Rigler. Fluorescence CorrelationSpectroscopy on dielectric surfaces in total internal reflection geometries. Photonics West, Jan 22-27, 2005, San Jose, USA 212. B. Wang, I. Riemann, H. Schubert, S. Kirste, K. König. In vivo animal follow-up studies on intrastromal surgery with near infrared nanojoule femtosecond laser pulses. Poster. Photonics West, Jan 22-27, 2005, San Jose, USA 213. K. König, I. Riemann, H. Schuck, D. Sauer, T. Velten, R. LeHarzic. Time-resolved and spectrally resolved 5D multiphoton microscopy for analysis and nanoprocessing of materials. Photonics West, Jan 22-27, 2005, San Jose, USA. 214. I. Riemann, A. Ehlers, A. Reif, J. Kobow, K. König. In vivo multiphoton tomography of skin as a tool to study the effects of topically applied probes and UV exposure. Photonics West, Jan 22-27, 2005, San Jose, USA 215. I. Riemann, T. Anhut, F. Stracke, R. LeHarzic, K. König. Multiphoton nanosurgery in cells and tissues. Photonics West, Jan 22-27, 2005, San Jose, USA 216. I. Riemann, E. Dimitrow, M. Kaatz, J. Fluhr, P. Elsner, J. Kobow, K. König. In vivo multiphoton tomography of inflammatory tissue and melanoma. Photonics West, Jan 22-27, 2005, San Jose, USA 217. K. König, B. Wang, I. Riemann, J. Kobow. Cornea surgery with nanojoule femtosecond laser pulses. Photonics West, Jan 22-27, 2005, San Jose, USA 218. W. Becker, K. König. Fluorescence lifetime images and correlation spectra obtained by multi-dimensional TCSPC. Photonics West, Jan 22-27, 2005, San Jose, USA 219. K. König. From a physics point of view: Are femtosecond lasers safe for ophthalmic applications? 6^th International Congress of Wavefront Sensing&Optimized Refractive Corrections. Feb 11-13, 2005. Athens, Greece

Tagungsvorträge

1. E. Knoth, H. Kneipp, M. Rentsch, K. König: Kupfer- und Golddampflaser zur bronchiolog. Verwendung. Symposium "Neue Diagnostik- und Therapieverfahren in der Bronchol. (1986) Bad Berka

2. V. Bockhorn, W. Dietel, K. König, U. Krause: Erste Erfahrungen im Forschungsprojekt: Laserinduzierte Fluoreszenz- und Photochemotherapie des Harnblasencarcinoms. III. Symposium: Methoden und Ergebnisse tierexperimenteller Arbeiten (1986) Jena

3. V. Bockhorn, W. Dietel, K. König: Fluoreszenzdiagnostik und Photochemotherapie maligner Tumoren. Workshop mit internationaler Beteiligung: Laseranwendung in der Medizin (1987) Dresden

4. L.P. Löbe, U. Krause, P. Lotz, W. Dietel, K. König, H. Schubert: Photodynamische Tumortherapie im HNO-Gebiet. Prinzip und erste experimentelle Ergebnisse. 34. Gemeinschaftstagung der med.-wissensch. Gesellschaft für HNO-Heilkunde (1987) Rostock

5. V. Bockhorn, W. Dietel, K. König, A. Werth, H. Schubert: Laserinduzierte Fluoreszenz und Photochemotherapie von Tumoren. (Poster) VII. Onkologie-Symposium (1987) Jena

6. K. König, W. Dietel: Fluorescence Investigations on Animal Tumors. (Poster) 5. Symposium Optical Spectroscopy (1988) Eisenach

7. K. König, E. Welsch, H.G. Walther: Photoacoustic absorption measurements on marked tissue.(Poster) Fifth Symposium Optical Spectroscopy (1988) Eisenach

8. I. Bugiel, K. König, H. Wabnitz: Fluoreszenzmikroskopische Untersuchungen an Zellen mit sub-ns Zeitauflösung. PDT-Schule (1988) Berlin

9. W. Dietel, K. König: Laserinduzierte in-vivo Fluoreszenz tierexperimenteller Tumoren. PDT-Schule (1988) Berlin

10. K. König, V. Bockhorn, W. Dietel, H. Schubert: Photochemotherapie tierexperimenteller Tumoren. PDT-Schule (1988) Berlin

11. E. Knoth, H. Kneipp, M. Rentsch, K. König: Experimentelle PDT mittels Kupfer- und Golddampflaser. PDT-Schule (1988) Berlin

12. E. Knoth, K. König, W. Grassme, W. Dietel: HpD-induzierte Tumorfluoreszenz. PDT-Schule (1988) Berlin

13. U. Krause, P. Lotz, L.P. Löbe, K. König, H. Schubert: Versuche zur Darstellung von HpD und seines Einsatzes zur PDT an Mäuse-Ascites-Tumoren. PDT-Schule (1988) Berlin

14. U. Krause, L.P. Löbe, P. Lotz, J. Barth, H. Schubert, K. König: Die Wirkung klassischer Strahlungsquellen in der photodynamischen HpD-Therapie von malignen Geschwülsten. 35. Gemeinschaftstagung der med.-wissensch. Gesellschaft für HNO-Heilkunde (1988) Rostock

15. L.P. Löbe, U. Krause, P. Lotz, K. König, W. Dietel, J. Barth: Photodynamic therapy with the HpD-Halle II. Results of animal experiments. International Conference: Photodynamic Therapy and Medical Laser Application (1988) London Proceedings: Vortrag 141

16. U. Krause, L.P. Löbe, P. Lotz, K. König, H. Schubert, J. Barth: Tierexperimentelle Untersuchungen zur PDT mit dem HpD-Typ Halle. 4. Photodynamisches Kolloquium mit intern. Beteiligung der AG Photodermatologie der Gesellsch. für Dermatologie der DDR (1988) Wilthen

17. L.P. Löbe, U. Krause, P. Lotz, K. König, H. Schubert, J. Barth: Photodynamische Tumortherapie im HNO-Gebiet mit einem Hämatoporphyrinderivat. III. Symposium über Tumorforschung in der HNO-Heilkunde (1988) Mannheim

18. W. Dietel, K. König: Laser stimulated in vivo Fluorescence of animal tumors. Second Intern. Conference on Laser Scattering Spectroscopy of Biological Objects. (1988) Pecs, Hungaria

19. V. Bockhorn, W. Dietel, K. König, H. Schubert: Fluoreszenzdiagnostik und PCT oberflächlicher Tumoren. XIX. Urologenkongress der DDR (1988) Erfurt

20. W. Dietel, K. König: Photobleaching of the HpD fluorescence and photoproduct formation in-vivo and in solution. Third Congress of the European Society for Photobiology. (Poster) (1989) Budapest

21. K. König, H. Schneckenburger, A. Rück, S. Auchter: Photoproduct formation of endogeneous protoporphyrin and its photodynamic activity. SPIE: Future Trends in Biomedical Applications of Lasers (Poster) (1991) Berlin, Proceedings vol. 1525, 412-419

22. K. König, A. Rück, S. Auchter, W. Strauss, H. Schneckenburger: Photoinduced reactions of porphyrin photosensitizers. (A) HpD. Laser in Medicine (Laser 91) (1991) München

23. W. Strauss, A. Rück, T. Köllner, K. König, H. Schneckenbur-ger: Photoinduced reactions of porphyrin photosensitizers. (B) Hydrophile Tetraphenylporphyrines. Laser in Medicine (Laser 91) (1991) München

24. C. Westphal, K. König, J. Krauter, G. Heil: Morphological changes of human leukaemic cells during photodynamic therapy. Jahrestagung der Deutschen und Österreichischen Gesellsch. für Hämatologie und Onkologie (1991) Innsbruck.

25. K. König, E. Welsch, H.G. Walther: Photoacoustic absorption measurements on tumor tissue stained with the photosensitizer Methylene Blue. Fourth Congress of the European Society for Photobiology (Poster) (1991) Amsterdam

26. K. König, H. Schneckenburger, H. Meier, A. Rück, R. Kaufmann: Laserinduced in-vivo autofluorescence of the human skin caused on the porphyrin production of Propionibacterium acnes. Fourth Congress of the European Society for Photobiology (Poster) (1991) Amsterdam

27. K. König, R. Fischer, Puhl, A. Rück, R. Steiner: Selective Excimer ablation of intervertebral discs using fluorescence spectroscopy. 2nd Conference on Methods and Applications of Fluorescence Spectroscopy (Poster) (1991) Graz

28. K. König, H. Schneckenburger, H. Meier, A. Rück, R. Kaufmann: Laserinduced in-vivo autofluorescence of the human skin caused on the porphyrin production of Propionibacterium acnes. 2nd Conference on Methods and Applications of Fluorescence Spectroscopy (Poster) (1991) Graz

29. R. Fischer, K. König, Puhl, A. Rück, R. Steiner: Selective Excimer ablation of intervertebral discs using fluorescence spectroscopy. 1st Conference of the European Orthopaedic Research Society (EORS) (11.-12.11.1991) Paris

30. R. Fischer, K. König, W. Puhl: Selektive Ablation von Bandscheibengewebe mit dem Excimer-Laser mittels Fluoreszenz-spektroskopie - eine in vitro Untersuchung. 40. Jahrestagung der Vereinigung Süddeutscher Orthopäden e.V. (30.4.-3.5.1992) Baden-Baden

31. A. Rück, H. Schneckenburger, K. König, C. Westphal-Frösch, K. Kunzi-Rapp, W. Strauss, H. Wald: Observation of light-induced intracellular reactions and photodynamic organelle destruction of cationic and anionic mesotetraphenylporphyrins. Gordon Conference, New Hampshire, 1992

32. K. König, H. Meyer: Photodynamische Aktivität von Methylenblau. Photodermatologie-Kongreß (1992) Düsseldorf

33. K. König, H. Meyer, H. Schneckenburger, A. Rück: Fluoreszenzverhalten und photodynamische Wirksamkeit von Propionibacterium acnes. Photodermatologie-Kongreß (1992) Düsseldorf

34. A. Rück, H. Schneckenburger, W. Strauß, C. Westphal-Frösch, K. König, K. Kunzi-Rapp: Detektion von intrazellulären photodyna-mischen Reaktionen mit Hilfe hochauflösender mikroskopischer Techniken. Jahreskongreß der Deutschen Gesellschaft für Lasermedizin (1992) Münster, Abstract: Laser in Medicine and Surgery 8(1992)127

35. W.H. Boehncke, K. König, A. Rück, R. Kaufmann, W. Sterry: Proliferationsinhibition einer menschlichen T-Zell-Linie durch Photodynamische- und PUVA-Therapie. Jahreskongreß der Deutschen Gesellschaft für Lasermedizin (1992) Münster, Abstract: Laser in Medicine and Surgery 8(1992)93

36. K. König, A. Rück, W. Strauß, H. Schneckenburger, H. Wabnitz: Photobleaching and Photoproduct Formation of HPD. International Conference Photodynamic Therapy and Medical Laser Applications (1992) Mailand, Abstract: Lasers in Medical Science 7(1992)251

37. K. König, F. Genze, E. Reich, R. Miller, A. Rück, d. Repassy: PDT of tumor-bearing mice using liposome-delivered texaphyrins. International Conference Photodynamic Therapy and Medical Laser Applications (1992) Mailand, Abstract: Lasers in Medical Science 7(1992)268

38. K. König, J. Hemmer, H. Schneckenburger: Laser-induced autofluorescence of squamous cell carcinoma. International Conference Photodynamic Therapy and Medical Laser Applications (1992) Mailand, Abstract: Lasers in Medical Science 7(1992)233

39. K. König, H. Schneckenburger, A. Rück, H. Meyer: Fluorescence diagnosis and photodynamic therapy of acne vulgaris. International Conference Photodynamic Therapy and Medical Laser Applications (1992) Mailand, Abstract: Lasers in Medical Science 7(1992)291

40. A. Rück, K. König, W. Strauss, H. Schneckenburger: Light-induced reactions of cationic and anionic meso-tetraphenylporphyrins in-vitro and in-vivo probed by different spectroscopic techniques. International Conference Photodynamic Therapy and Medical Laser Applications (1992) Mailand, Abstract: Lasers in Medical Science 7(1992)280

41. R. Fischer, K. König, W. Puhl, A. Rück, R. Steiner: Selective Ablation of Intervertebral Discs using Fluorescence Spectroscopy - an in-vitro Examination. 3rd World Congress of the International Society for Low Power Laser Application in Medicine. Bologna, 9.-12.9.1992

42. A. Rück, H. Schneckenburger, C. Westphal-Frösch, K. Kunzi-Rapp, K. König, W. Strauss: Direct observation of photodynamically induced organelle destruction probed by video-enhanced contrast microscopy and fiber optical spectroscopy. 11th International Congress on Photobiology (1992) Kyoto (Abstract V 09-13)

43. E. Reich, K. Miller, D. Repassy, K. König, A. Rück, R. Bachor: Liposome-administered tetrametyhlhematoporphyrin (TMHP) as a photodynamic agent for bladder tumor cells. 11th International Congress on Photobiology (1992) Kyoto (Abstract P201)

44. R. Bachor, E.Reich, K. Miller, A. Rück, K. König, R. Hautmann: Phototoxicity of aminolevulinic acid (ALA) in vitro. 11th International Congress on Photobiology (1992) Kyoto (Abstract V 17-02)

45. K. König, E. Reich, F. Genze, R. Miller: In-vivo fluorescence behaviour and photodynamic activity of liposomal Tetramethyl-Hematoporphyrin (TMHP). 11th International Congress on Photobiology (1992) Kyoto (Abstract P 144)

46. K. König, A. Rück, F. Genze, E. Reich, K. Miller, R. Bachor Distribution and photodynamic activity of ALA-induced endogeneous protoporphyrin. 11th International Congress on Photobiology (1992) Kyoto (Abstract P 145)

47. K. König, H. Schneckenburger, A. Rück, E. Reich, K.Miller, R. Bachor: On-line fluorescence spectroscopy during photodynamic therapy with ALA-induced endogeneous porphyrin. 11th International Congress on Photobiology (1992) Kyoto (Abstract V 09-12)

48. H. Schneckenburger, K. König, P. Gessler, I. Pavenstädt-Grupp: Laser-induzierte Autofluoreszenz für die Diagnostik. Jahreskongreß der Deutsch. Gesellsch. für Lasermedizin 1992, Münster, Abstract: Laser in Medicine and Surgery 8(1992)128

49. W. Strauss, K. König, K. Miller, W. Mohr, A. Rück, H. Schneckenburger, R. Steiner: Meso-Tetra(4-carboxyphenyl)porphyrin-Fluoreszenzverhalten und photodynamische Therapie an xenotransplantierten Blasentumoren. Jahreskongreß der Deutsch. Gesellsch. für Lasermedizin 1992, Münster, Abstract: Laser in Medicine and Surgery 8(1992)135

50. K. König, H. Schneckenburger, A. Rück, R. Steiner, E. Reich, K. Miller, R. Bachor: On-line Fluoreszenzspektroskopie während der PDT nach ALA-Applikation. Jahreskongreß der Deutsch. Gesellsch. für Lasermedizin 1992, Münster, Abstract: Laser 92. Reihe Medizin. Eds.: G.H. Willital, M. Maragakis, R.R. Lehmann. Verlag Shaker, Aachen 1992, Seite 22

52 K. König, A. Rück, F. Genze, E. Reich, K. Miller, R. Bachor: Pharmakokinetik und photodynamische Aktivität von ALA-induziertem Protoporphyrin IX. Jahreskongreß der Deutsch. Gesellsch. für Lasermedizin 1992, Münster, Abstract: Laser 92, Seite 241 (s.o.)

53. K. König, E. Reich, F. Genze, K. Miller: In-vivo Fluoreszenzverhalten und photodynamische Wirkung von Tetramethyl-Hämatoporphyrin. Jahreskongreß der Deutsch. Gesellsch. für Lasermedizin 1992, Münster, Abstract: Laser 92, Seite 242 (s.o.)

54. R. Fischer, K. König, W. Puhl: Möglichkeiten und Grenzen der percutanen Nukleotomie mit dem Excimer Laser - Ergebnisse von in-vitro Untersuchungen. Kongreß der Deutsch. Gesellsch. für Orthopädie und Traumatologie. Mannheim, 16.-20.9.1992

55. W.H. Boehncke, K. König, A. Rück, K. Kaltoft, R. Kaufmann, W. Sterry: Proliferationsinhibition einer menschlichen T-Zell Linie durch photodynamische und PUVA Therapie. Lasermedizin 8(1992)93

56. K. König, R. Hibst, H. Schneckenburger, G. Flemming: Laserinduced autofluorescence of caries. SPIE (Januar 1993) Los Angeles

57. K. König, H. Schneckenburger, A. Rück, H. Wald, R. Steiner:

Autofluorescence of cells and tissue. SPIE (Januar 1993) Los Angeles

58. R. Kaufmann, W.H. Boehncke, K. König, R. Hibst: Comparitive study of Q-switched Nd:YAG- and Alexandrite laser treatment of tattoos. 13th annual meeting of the American Society for Laser Medicine and Surgery (April 1993) New Orleans

59. K. König (invited), H. Schneckenburger, A. Rück: The Nature and Role of Protoporphyrin Photoproducts: In Vitro and In Vivo. 21st Annual Meeting of the American Society for Photobiology (26.-30.6.93) Chicago, abstract: Photochem. Photobiol. 57(1993)76S-77S

60. W.H. Boehncke, K. König, A. Rück, K. Kaltoft, R. Kaufmann, W. Sterry: Inhibitory effects of photodynamic therapy on malignant transformed T cells suggest its potential in the treatment of cutaneous T cell lymphomas. Kongress der Arbeitsgemeinschaft Dermatol. Forschung, ADF. Mainz, 1992. Abstract: Arch Dermatol Res 285(1993)64-65.

61. R. Kaufmann, W-H Boehncke, K. König, R. Hibst: Comparative study of Q-switched Nd:YAG- and alexandrite laser treatment of tattoos. Congress of the American Society of Lasermedicine, New Orleans, 1993. Abstract: Laser Surg Med 1993, Suppl 5:54

62. W.H. Boehncke, K. König, W. Scheffold, W. Sterry: Application of photodynamic therapy in psoriasis. Tricontinental Meeting, Kyoto, 1993. Abstract: J Invest Dermatol 101(1993)465.

63. W. Sterry, K. König, A. Rück, R. Kaufmann, W-H Boehncke: Application of photodynamic therapy in mycosis fungoides. Tricontinental Meeting, Kyoto, 1993. Abstract: J Invest Dermatol 101(1993)464

64. W-H Boehncke, K. König, W. Scheffold, W. Sterry. Photodynamic therapy in psoriasis. Meeting of the Skin Pharmacology Society, Hongkong, 1993. Abstract: Skin Pharmacol, in press

65. W-H Boehncke, K. König, A. Rück, K. Kaltoft, R. Kaufmann, W. Sterry: Proliferationsinhibition einer menschlichen T-Zell Linie durch photodynamische und PUVA Therapie. Kongress der Deutsch. Gesellschaft für Lasermedizin, Münster. Abstract: Lasermedizin 8(1992)93

66. K. König, R. Hibst, H. Meyer, G. Flemming, H. Schneckenburger: Laser-induced autofluorescence of carious regions of human teeth and caries-involved bacteria. International Symposium on Biomedical Optics Europe '93, 1993, Sept 1-5, Budapest, abstract:

67. K. König, A. Kienle, W-H. Boehncke, R. Kaufmann, A. Rück, T. Meier, R. Steiner: Photodynamic tumour therapy and on-line fluorescence spectroscopy after ALA administration using 633 nm-light as therapeutic and fluorescence excitation radiation. International Symposium on Biomedical Optics Europe '93, 1993, Sept 1-5, Budapest, abstract:

68. K. König, G. Beck, R. Steiner: International Symposium on Biomedical Optics Europe '93, 1993, Sept 1-5, Budapest, abstract:

69. K. König, W-H. Boehncke, A. Rück, R. Kaufmann, R. Steiner, W. Sterry: Photodynamic effects on T-cells and skin lesions of a patient with mycosis fungoides using porphyrin photosensitizers. International Symposium on Biomedical Optics Europe '93, 1993, Sept 1-5, Budapest, abstract:

70. H. Schneckenburger, M. Gschwend, K. König, A. Rück, R. Sailer, W. Strauss: Subcellular distribution of photodynamic photosensitizers. International Symposium on Biomedical Optics Europe '93, 1993, Sept 1-5, Budapest, abstract:

71. K. König, K. Kunzi-Rapp, H. Schneckenburger, A. Rück: Natural and ALA stimulated autofluorescence and PDT of xenotransplanted tumors in the CAM of chicken embryos.(Poster), International Congress on Photobiology, Marburg, 1994, Sept., abstract: V-10/P 10

72. R. Sailer, W. Strauss, K. König, A. Rück, R. Steiner: Fluorescence properties and photodynamic inactivation of the gram-negative bacterium Pseudomonas aeruginosa in comparison to the amount of endogenous porphyrins. International Congress on Photobiology, Marburg, 1994, Sept., abstract: V-10/p15

73. H. Schneckenburger, K. König, T. Dienersberger, M. Gschwend: Time-gated microscopic imaging and spectroscopy in photobiology and medical diagnosis. International Congress on Photobiology, Marburg, 1994, Sept., abstract: VI-6/01

74. A. Rück, M. Gschwend, K. König, H. Schneckenburger, R. Sailer, W. Strauss: Photoproducts and formation of molecular species during PDT, International Congress on Photobiology, Marburg, 1994, Sept., abstract: V-2/03

75. H. Schneckenburger, K. König, T. Dienersberger, M. Gschwend: Time-gated microscopic imaging and spectroscopy in photobiology and diagnostics. UPS'93, 1993, Sept 26-30, Vilnius

76. A. Rück, W.H. Boehncke, K. König, K. Kunzi-Rapp, T. Meier, R. Kaufmann: Polychromatic vs. monochromatic PDT-comparative effects in vitro and in vivo. 10th Congress, International Society for Laser Surgery and Medicine, 1993, Nov 12-15, Bangkok, Thailand

77. K. König (invited), H. Schneckenburger: Laser-Induced Autofluorescence for Medical Diagnosis. Congress on Fluorescence. Prag, Febr. 1994

78. K. König, H. Schneckenburger, H. Walt, T. Leemann, M.T. Wyss-Desserich, A. Rück, B. Tromberg: Microscopic Studies on ALA-incubated tumor cells and tumor spheroids. 1994, Los Angeles, Jan 22-29, abstract: 2133-40

79. K. König, H. Schneckenburger, J. Hemmer, B. Tromberg, R. Steiner: In-vivo fluorescence detection and imaging of porphyrin-producing bacteria in the human skin and in the oral cavity for diagnosis of acne vulgaris, caries, and squamous cell carcinoma. SPIE 1994, Los Angeles, Jan 22-29, abstract: 2135-16

80. K. König, H. Schneckenburger: Laser-induced dental caries and plaque diagnosis on patients by sensitive autofluorescence spectroscopy and time-gated video imaging. Preliminary studies. SPIE 1994, Los Angeles, Jan 22-29, abstract: 2128-64

81. K. König, H. Schneckenburger, A. Rück, R. König: Studies on Porphyrin Photoproducts in Solution, Cells, and Tumor Tissue. SPIE 1994, Los Angeles, Jan 22-29, abstract: 2133-39

82. K. König, H. Schneckenburger, H. Boehncke, R. Hibst: In vivo fluorescence spectroscopy and imaging of ALA-induced endogenous porphyrins in skin after Er:YAG ablation of stratum corneum. SPIE 1994, Los Angeles, Jan 22-29, abstract: 2128-38

83. A. Rück, M. Gschwend, W. Strauss, K. König, R. Sailer, H. Schneckenburger: Photoproducts and new spectral bands during PDT probed by cw and time-gated microscopy and spectroscopy. SPIE 1994, Los Angeles, Jan 22-29, abstract:

84. H. Schneckenburger, K. König, K. Kunzi-Rapp, M. Gschwend: Zeitaufgelöste Untersuchungen zur Intrazellulären Verteilung und Licht-induzierten Reaktion von ALA-induziertem Protoporphyrin. 1. Münchner ALA-Workshop, 17./18.3.94, München.

85. K. König (invited): Laser-induced autofluorescence in medicine. International Conference: Lasers & Applications. Advances in science, medicine and technology, NILES 1994, Cairo, March 26-30, 1994

86. BJ. Tromberg, TB. Krasieva, SJ. Madsen, K. König: Principles of Laser Based Diagnostics. 14th Annual meeting of the American Society for Laser Medicine and Surgery, Ontario, April 8-10, 1994, abstract 305, Lasers Surg Med, Suppl 6, 1994, 58

87. K. König, T. Krasieva, B. Tromberg: Spectrally-resolved fluorescence imaging of porphyrin-producing bacteria in human skin. CLEO, May 8-13, 1994, Anaheim, abstract: CTuA3 in "Biomedical Optical Imaging"

88. BJ. Tromberg, C. Chapman, K. König, MW. Berns, Y. Liu, G. Sonek: Microirradiation effects on cells. Gordon Conference, New Hampshire, July, 1994

89. K. König , Y. Liu, GJ. Sonek, MW. Berns, BJ. Tromberg: Photoinduced modifications of cells in an optical trap. BIOS Europe '94, Lille, Sept 6-10, 1994, abstract: 2329-40

90. S. Kimel, K. König, MW. Berns: Photodynamic effects on human and chicken erythrocytes. SPIE-Proceedings, BIOS Europe '94, Lille, Sept 6-10, 1994, abstract: 2329-46

91. K. König (invited): Fluorescence imaging and spectroscopy of motile sperm cells and CHO cells in an optical trap, SPIE '95, San Jose, Febr 4-10, 1995, abstract:

92. K. König, BJ Tromberg, MW Berns: One- and Two Photon Excited Fluorescence of Motile Cells in the Optical Trap, LASER 95, München

93. K. König, P. Fergin, MW. Berns, BJ. Tromberg: Spectrally-resolved Fluorescence Imaging of Skin after Topical ALA-Administration, LASER 95, München

94. K. König (invited): Two-Photon Excited Autofluorescence in CHO Cells and Motile Spermatozoa. Third International Symposium on Innovative Fluorescence Methodologies in Biochemistry and Medicine. July 30-Aug 2, 1995, Kaanapali, Maui, Hawaii, USA

95. L. T. Norvang, E. J. Fisherstrand, K. König, B. Bakken, D. Grini, Standahl, T. E. Milner, M. W. Berns, J. S. Nelson, L. O. Svaasand: Comparison between reflectance spectra obtained with an integrating sphere and a fiber-optic collection system. BIOS Sept. 1995, Barcelona

96. K. König, P. So, W. W. Mantulin, E. Gratton, M. W. Berns, B. J. Tromberg: Two-Photon Excited Cellular Autofluorescence Induced by CW and Femtosecond NIR Microirradiation. BIOS Sept. 1995, Barcelona

97. K. König, T. Krasieva, E. Bauer, U. Fiedler, M. W. Berns, B. J. Tromberg: UVA-Induced Oxidative Stress Probed by Autofluorescence Imaging, Cloning Assay, and Comet Assay. BIOS Sept. 1995, Barcelona

98. K. König (invited): NAD(P)H- and Porphyrin-Attributed Autofluorescence for Medical Diagnosis. Laser 95. Charleston, Dez. 95, South Carolina, USA

99. K. König (invited): Continuous Wave Induced Two-Photon Excitation in Living Cells. SPIE. Jan. 1996. San Jose, Kalifornien

100. K. König: Multiphotonen-Effekte in der Lichtfalle. 60. Physikertagung der DPG. 11.-15.3.1996, Jena. (Interview mit dem Deutschlandfunk zu Fragen der Optischen Mikromanipulation von Vitalzellen anlässlich der Physikertagung, 14.3.96 (Sendung 15.3.96))

101. K. König, P. So, W.W. Mantulin, M.W. Berns, B.J. Tromberg, E. Gratton: Two-photon excited fluorescence in living cells. (Poster) Tripartite Meeting der Anatomischen Gesellschaft (91. Versammlung), Anatomical Society of Great Britain and Ireland, Nederlandse Anatomen Vereniging. 24.3.-27.3.1996, Jena.

102. K. König (invited): Two-photon excitation in a single motile sperm cell confined in an optical trap. Annual Meeting German Society for Cell Biology. 24.3.-28.3.1996, Hamburg.

103. K. König, P. So, W.W. Mantulin, E. Gratton: Cell damage in Two-Photon Microscopes. BiOS Sept 1996, Wien

104. K. König, L. Svaasand, Y. Tadir, B. Tromberg, M.W. Berns: Optical determination of motility forces in human spermatozoa with laser tweezers. BiOS Sept 1996, Wien

105. K. König: Two-photon femtosecond microscopy in living cells. 6th International Conference on Laser Applications in Life Sciences (LALS), Sept 1996, Jena

106. K. König, T. Krasieva, B. Tromberg, U. Diedler, E. Bauer, M.W. Berns, K.O. Greulich. UVA-induced oxidative stress in living cells. (Poster). 6th International Conference on Laser Applications in Life Sciences (LALS), Sept 1996, Jena

107. K.J. Halbhuber, K. König, Ch. Scheven, A. Aschoff, H. Feuerstein. Visualization of enzymatic primary reaction products by laser scanning microscopy. (Poster). 6th International Conference on Laser Applications in Life Sciences (LALS), Sept 1996, Jena

108. K. König, U. Simon, K.-J. Halbhuber. Three-Dimensional Two-Photon Femtosecond Fluorescence Microscopy using a modified Confocal Laser Scanning Microscope. 166. WE-Heraeus-Seminar: Multiphoton Photochemistry in Biological Systems. 28.-30.10.1996, Bad Honnef

109. K. König, S. Kimel, L.O. Svaasand, B. Tromberg, T. Krasieva, M.W. Berns, P. So, W.W. Mantulin, E. Gratton, K.J. Halbhuber: Cell damage in UVA and cw/femtosecond NIR microscopes. SPIE Photonics West Febr. 1997, San Jose, California

110. K. König (Gastredner): Zweiphotonenanregung vitaler Zellen mittels CW und Femtosekunden-NIR-Mikroskopie. 5. Kolloquium des DFG-Schwerpunktprogramms: Neue mikroskopische Techniken für Biologie und Medizin. 4.-6.3.1997, Rostock

111. König (Gastredner): Eigenfluoreszenz-Spektroskopie an lebenden Zellen: NADH als Bioindikator für Lichtstress. 1. Rostocker Biosystemtechnik-Symposium des Innovationskollegs: Komplexe und zelluläre Sensorsysteme. 6.-7.3.1997, Rostock

112. K. König, K.-J. Halbhuber: Zwei-Photonen-Femtosekundenmikroskopie vitaler Zellen. 13th International Congress LASER 97. Munich, June 1997.

113. K. König, H. Oehring, K.-J. Halbhuber, U. Fiedler, E. Bauer, K.O. Greulich. Comet assay, cloning assay, histochemistry, light- and electrom microscopy on one preselected cell. SPIE-EUROPTO'97, San Remo, 4.-8.9.97.

114. K. König. Laser tweezers as novel nonlinear tools in cell and biomolecule diagnostics. SPIE-EUROPTO'97, San Remo, 4.-8.9.97.

115. K. König, K.-J. Halbhuber. Multiphoton femtosecond microscopy of living cells. XXXIX. Symposium of the Society for Histochemistry, Jena, 24.-27.9.97, abstract: Histochem. Cell. Biol. 108(1997)276.

116. K. König, P. So, W. Mantulin, M. Berns, B. Tromberg, E. Gratton. Two-photon excited fluorescence in living cells. XXXIX. Symposium of the Society for Histochemistry, Jena, 24.-27.9.97, abstract: Histochem. Cell. Biol. 108(1997)277.

117. K. König, M. Schüler, K.-J. Halbhuber. Autofluorescence of cells and tissues as a diagnsotic tool. XXXIX. Symposium of the Society for Histochemistry, Jena, 24.-27.9.97, abstract: Histochem. Cell. Biol. 108(1997)277.

118. K. König. Two-photon effects in living cells. International Practical Course on Modern Laser Microscopy.Jena, 18.-21.11.1997.

119. K. König. How safe is the gamete micromanipulation by laser tweezers? Photonics West. SPIE-Tagung. 23.1.-30.1.1998, San Jose, California.

120. K. König, M. Teschke, W. Pfister, H. Meyer: Photodynamically inactivation of propionibacterium acnes. Photonics West. SPIE-Tagung. 23.1.-30.1.1998, San Jose, California.

121. K. König. Photoproduct formation during porphyrin PDT. First World Congress of Photomedicine in Gynecology. 19.-21.2.98. Zurich.

122. K. König. Cell damage in UV and Multiphoton NIR microscopes. Scanning 98. 9.-12.5.98, Baltimore.

123. K. König,P. So, W.W. Mantulin, B. Tromberg, E. Gratton. Two-Photon Excitation in Living Cells. Deutsche Gesellschaft für Biophysik. Jahrestagung. 21.-23.9.1998, Frankfurt/M.

124. K.-J. Halbhuber, R. Krieg, K. König. Fluorescence laser microscopical demonstration of endogenous and immunobond phosphatase activity using metallo-azo dye complexes. 40. Symposium of the Society of Histochemistry. 22.-25.9.1998, Giessen.

125. K. König, P. Fischer. I. Riemann, K.-J. Halbhuber. Multiphoton-excited fluorescence in histochemistry. 40. Symposium of the Society of Histochemistry. 22.-25.9.1998, Giessen.

126. K. König, P. Fischer. I. Riemann, K.-J. Halbhuber. Femtosecond versus picosecond multiphoton microscopy. 23.-29.1.1999, San Jose, California.

127. K. König. Cloning assay thresholds on cells exposed to ultrafast laser pulses. 23.-29.1.1999, San Jose, California.

128. K. König, P. Fischer. I. Riemann, K.-J. Halbhuber. PDT by non-resonant two-photon excitation. 23.-29.1.1999, San Jose, California.

129. K. König. Nahe Infrarot Laserscanningmikroskopie an vitalen Zellen. 10. Deutscher MTA-Kongress. 10.-12.3.1999, Mannheim.

130. K. König. Plenarvortrag: Cellular response to ultrashort laser pulses in multiphoton microscopes. Focus on Microscopy 1999. 11.-15.4.1999, Heidelberg.

131. K. König. Femtosecond multiphoton microscopy for cell and tissue diagnostics. Optical Society of America (OSA) Topical Meeting at Laser 99. 14.-16.6.1999. München.

132. K. König. Multiphoton near infrared microscopy: optical trapping, 4D microscopy, and nanosurgery. Third Conference on Fluorescence Microscopy and Fluorescent Probes. 20.-23.6.1999. Prag. Tschechische Republik.

133. K. König. Intracellular nanosurgery with compact femtosecond laser. 18. Congress of the International Commission for Optics (ICO XVIII): Optics for the next Millenium. 02.-06.8.1999, San Francisco, USA.

134. K. König. Anwendung optischer Mikroskopien in den Biowissenschaften. W.E. Heraeus Fereinkurs: Moderne Fernfeld- und Nahfeld- Mikroskopien. 20.9-01.10.1999, Chemnitz.

135. K. König, I. Riemann, P. Fischer, H. Oehring, Th. Becker, K.-J. Halbhuber. Apoptosis induced by continuous wave ultraviolet and red radiation as well as by near infrared femtosecond laser pulses. 41^st Symposium of the International Society for Histochemistry. 22.9.-25.9.1999, Gargellen, Austria.

136. K.-J. Halbhuber, K. König. 41^st Symposium of the International Society for Histochemistry. 22.9.-25.9.1999, Gargellen, Austria.

137. Oehring, K. König. (Poster). 41^st Symposium of the International Society for Histochemistry. 22.9.-25.9.1999, Gargellen, Austria.

138. K. König, I. Riemann, W. Fritsche, P. Fischer, K.-J. Halbhuber. Intracellular nanosurgery with femtosecond laser pulses. Jahrestagung der Deutschen Gesellschaft für Biophysik. 03.-06.9.1999, Ulm.

139. K. König. Multiphoton femtosecond laser microscopy for 4D fluorescence diagnostics and nanosurgery of living cells.. XI International Symposium "Ultrafast Phenomena in Spectroscopy (UPS '99)", 25.-29.10.1999, Taipei, Taiwan, ROC.

140. K. König. Intracellular nanosurgery using near infrared femtosecond laser pulses. Focus on Microscopy 2000 (FOM2000). 09.-13.4.2000, Shirahama, Japan.

141. K. König. Intracellular nanosurgery using near infrared femtosecond laser pulses. OPTO und IRS². 9-11.5.2000, Erfurt.

142. K. König, I. Riemann, W. Fritzsche, U. Tirlapur, P. Fischer, K.J. Halbhuber. Nanochirurgie mittels NIR-Femtosekundenlaser-Lasermikoskopie. 101. Tagung der Deutschen Gesellschaft für angewandte Optik. 13.-17.7.2000, Jena (mit Beteiligung von Japan, Österreich, Schweiz).

143. K. König, I. Riemann, A. Göhlert, P. Fischer, T. Liehr, I.F. Loncarevic, U. Claussen, K.J. Halbhuber. Multiphoton Multicolor FISH. SPIE-EBiOS 2000, 04.-08.7.2000, Amsterdam.

144. K. König. Multiphoton Microscopy to study macromolecules in living cells. Gordon Research Conference on Macromoleuclar Organization and Cell Function, 06.-11.8.2000, Oxford, U.K.

145. K. König, C. Peuckert, I. Riemann, U. Wollina. Non-invasive 3D optical biopsy of human skin with NIR femtosecond laser pulses for diagnosis of dermatological disorders. 3rd World Conference on Cellular and Molecular Biology, 08.-13.10.2000, Jena.

146. K. König, U. Tirlapur, C. Peuckert, I. Riemann, A. Bergmann, W. Becker. Time-resolved two-photon fluorescence imaging of living cells and tissues. 3rd World Conference on Cellular and Molecular Biology, 08.-13.10.2000, Jena.

147. C. Peuckert, I. Riemann, U. Wollina, K. König. (Poster) Remission microscopy with near femtosecond laser pulses for 3D in vivo imaging of human skin. 3rd World Conference on Cellular and Molecular Biology, 08.-13.10.2000, Jena.

148. C. Peuckert, I. Riemann, K. König. Two-photon induced autofluorescence of in vivo human skin with femtosecond laser pulses - a novel imaging tool of high spatial, spectral and temporal resolution. 3rd World Conference on Cellular and Molecular Biology, 08.-13.10.2000, Jena.

149. A. Göhlert, I. Riemann, T. Liehr, I.F. Lomcarevic, U. Claussen, K.H. Halbhuber, K. König. (Poster) Multiphoton multicolor FISH (MM-FISH): A verstaile technique to detect specific sequences within single DNA molecules. 3rd World Conference on Cellular and Molecular Biology, 08.-13.10.2000, Jena.

150. M. Rothammel, M. Teschke, W. Pfister, K.J. Halbhuber, K. König. (Poster) Inactivation of Porphyromonas gingivalis by red light evaluated by laser scanning microscopy. 3rd World Conference on Cellular and Molecular Biology, 08.-13.10.2000, Jena.

151. I. Riemann, A. Göhlert, U. Claussen, K.J. Halbhuber, K. König. Three dimensional imaging of specific DNA sequences in cells and tissues by multiphoton multicolor FISH using near infrared femtosecond laser pulses. 3rd World Conference on Cellular and Molecular Biology, 08.-13.10.2000, Jena.

152. U.K. Tirlapur, K. König. Multiphoton microscopy in plant cells. 3rd World Conference on Cellular and Molecular Biology, 08.-13.10.2000, Jena.

153. N. Rubtsov, I. Riemann, V. Trifonov, T. Karamysheva, T. Liehr, U. Claussen, K. König. (Poster)Chromosome microdissection using NIR femtosecond laser pulses and generation of band specific DNA-libraries with DOP-PCR. 3rd World Conference on Cellular and Molecular Biology, 08.-13.10.2000, Jena.

154. M. Wiederhold, U.K. Tirlapur, K. König, S. Russwurm (Poster). Association of procalcitonin in HEPG2 cells studied with confocal laser scanning microscopy. 3rd World Conference on Cellular and Molecular Biology, 08.-13.10.2000, Jena.

155. U.K. Tirlapur, K. König, R. Krieg, K.J. Halbhuber. Femtosecond NIR laser pulses elicit generation of ROS in marsupial cells leading to apoptosis-like cell death. 3rd World Conference on Cellular and Molecular Biology, 08.-13.10.2000, Jena.

156. M. Wiederholt, I. Riemann, S. Russwurm, K. König. (Poster). The uptake of fluoresceinisothiocyanate-lipopoly-saccharide by cultivated human mononuclear cells using two-photon fluorescence microscopy. 3rd World Conference on Cellular and Molecular Biology, 08.-13.10.2000, Jena.

157. K. König, U. Tirlapur, I. Riemann, W. Fritzsche. Nanosurgery of chromosomes, living cells and tissues with femtosecond laser pulses in the near infrared. 3rd World Conference on Cellular and Molecular Biology, 08.-13.10.2000, Jena.

158. D. Volkmann, U.K. Tirlapur, K. König, T. Mori, T. Fujiwara, F. Baluska. Plant plasmodesmata:myosin VIII-supported celll-to-cell channels for macromolecular trafficking. 3rd World Conference on Cellular and Molecular Biology, 08.-13.10.2000, Jena.

159. K. König. Diagnosis and knocking out of genomic regions with multiphoton microscopes.SPIE-Photonics West 2001. San Jose, California.

160. K. König. Time-resolved multiphoton microscopy. First symposium on FRET and FLIM: Advanced fluorescence techniques for biological imaging. 08.-10.6.2001. Austin, Texas, USA.

161. K.König. Multiphoton microscopy of cells and tissues. European Conference on Biomedical Optics. Laser 2001. 17.-20.6.2001, München.

162. K. König, O. Krauss, I. Riemann, D. Schweitzer. Poster. Corneal surgery with 80 MHz nanojoule femtosecond laser pulses in the near infrared. European Conference on Biomedical Optics. Laser 2001. 17.-20.6.2001, München.

163. K. König. Optical biopsy with femtosecond laser pulses: in vivo skin diagnostics and 3D gene imaging with submicron resolution. 47^th Annual Paediatric Pathology Society Meeting. 13.-15.9.2001. Warsaw, Poland.

164. I. Riemann, A. Göhlert, T. Liehr, I.F. Loncarevic, U. Claussen, K.J. Halbhuber, K. König. Poster. 3D imaging of specific DNA sequences in cells, tissues and within single DNA molecules with multiphoton multicolor FISH (MM-FISH). 43^rd Symposium of the Society for Histochemistry. 26.-29.9.2001, Vienna, Austria.

165. R. Krieg, U.K. Tirlapur, K. König, C. Peuckert, K.J. Halbhuber. Poster. In situ localization of femtosecond near-infrared laser induced ROS with a new NLO fluorochrome after two-photon excitation at 800 nm. 43^rd Symposium of the Society for Histochemistry. 26.-29.9.2001, Vienna, Austria.

166. K. König. Möglichkeiten der intrastromalen Hornhautchirurgie mit Femtosekunden-Laserpulsen. 99. Tagung der Deutschen Ophthalmologischen Gesellschaft (internationale Beteiligung). 29.9.-02.10.2001, Berlin.

167. K. König. Optical tomography of human skin with subcellular spatial and picosecond time resolution using intense near infrared femtosecond laser pulses. 3^rd International Symposium Lasers in Dermatology. 01.-02.10.2001, Ulm.

168. K. König, U. Wollina, I. Riemann, C. Peuckert, K.J. Halbhuber, V. Fünfstück, T.W. Fischer, P. Elsner. Optical tomography of human skin with subcellular spatial and picosecond time resolution. SPIE-Photonics West 2002. 19.-25.1.2002, San Jose, California.

169. K. König, I. Riemann, O. Krauss, W. Fritzsche. Nanodissection of human chromosomes and ultraprecise eye surgery with nanojoule near infrared femtosecond laser pulses. SPIE-Photonics West 2002. 19.-25.1.2002, San Jose, California.

170. K. König. Fluorescence Lifetime Imaging. Focus on Microscopy 2002. April 2002. Kaohsiung, Taiwan.

171. K. König, O. Krauss, I. Riemann. Optical Tomography of the Eye by Multiphoton Autofluorescence and SHG Imaging. 100. Tagung der Deutschen Ophthalmologischen Gesellschaft (internationale Beteiligung). 26.9.-29.9.2001, Berlin.

172. O. Krauss, I. Riemann, K. König. Intrastromal surgery of the cornea with 80 MHz nanojoule femtosecond laser pulses in the near infrared. 100. Tagung der Deutschen Ophthalmologischen Gesellschaft (internationale Beteiligung). 26.9.-29.9.2001, Berlin.

173. K. König. Photonics West 2003, Jan.

174. K. König, FOM2003. April 12-19, 2003. Genua, Italy.

175. K. König, Skin. May 21-24, 2003. Hamburg, Germany.

176. K. König. Biomedical Optics, Laser2003, June 24-25, 2003. Munic, Germany.

177. K. König, July 21-23, 2003. Banff, Canada.

178. K. König. Imaging and manipulation on a micron scale. Microscopes, Laser Tweezers, Multiphoton Effects. Graduate Summer Scholl Bio-Photonics `03, June 15-21, 2003, Ven, Sweden

179. K. König. Femtosecond Lasers in Dermatology. The 87th OSA Annual Meeting: Frontiers in Optics. Laser Science XIX. Oct 5-9, 2003, Arizona, Florida

180. I. Riemann, E. Dimitrow, P. Fischer, A. Reif, M. Kaatz, P. Elsner, K. König. High resolution multiphoton tomography of human skin in vivo and in vitro. SPIE-Photonics West 2004, 24.-29.01.2004, San José, USA.

181. K. König: Multiphoton Fluorescence/SHG imaging of cells, skin and heart tissue. Berkeley

182. W. Becker, A. Bergmann, G. Biscotti, K. König. High-Speed FLIM Data Acquisition by Time-Correlated Single Photon Counting. SPIE-Photonics West 2004, 24.-29.01.2004, San José, USA.

183. K. Schenke-Layland, I. Riemann, U. A. Stock, K. König. Non-invasive multiphoton imaging of cardiovascular structures using NIR femtosecond laser scanning microscopy. SPIE-Photonics West 2004, 24.-29.01.2004, San José, USA.

184. A. Czaki, G. Maubach, F. Garwe, A. Bochmann, K. König, W. Fritzsche. Poster: Metal nanoparticles for nanooptics and nanoanalytics. SPIE-Photonics West 2004, 24.-29.01.2004, San José, USA.

185. M. Kaatz, J. Fluhr, E. Dimitrow, P. Elsner, I Riemann, J. Kobow, K. König. Monitoring of the accumulation and pharmacokinetics of fluorescent dyes througth the stratum corneum by high-resolution multiphoton tomography. Stratum Corneum, May 19, 2004.

186. I. Riemann, K. König. High Resolution Multiphoton Tomography of the Epidermis with Submicron Resolution. Stratum Corneum, May 19, 2004.

187. K. König: Femtosecond laser application in biotechnology and medicine. The 5^th International Symposium on Laser Precision Microfabrication, 11.-14. Mai 2004 in Nara, Japan (2004).

188. K. König. 1) Photodamage during light microscopy. 2) Multiphoton microscopy. International Summer School, Vancouver, Canada

189. T. Velten, H. Schuck, I. Riemann, F. Bauerfeld, D. Sauer, K. König. Time-Resolved and Spectrally-Resolved 5D Multiphoton Microscopy for Analysis and Nanoprocessing of Biological and Non-Biological Materials. LANE 2004, 21.-24.09.2004, Erlangen, Germany.

190. I. Riemann, K. König. High-resolution multiphoton tomography of human skin in vivo and in vitro. Photonics Europe, 26.-30. April 2004, Straßburg, France.

191. K. Schenke-Layland, F. Opitz, I. Riemann, U.A. Stock. Multiphoton imaging of cardiovascular structures. Photonics Europe, 26.-30. April 2004, Straßburg, France.

192. V. Ulrich, P. Fischer, E. Haupt, I. Riemann, K. König. Spectral Imaging and fluorescence lifetime imaging with a compact. Photonics Europe, 26.-30. April 2004, Straßburg, France.

193. K. König, F. Fischer, P. Fischer, S. Puschmann, I. Riemann, V. Ulrich, R. Wepf. Characterization of multiphoton laser scanning microscope optical parameters. Photonics Europe, 26.-30. April 2004, Straßburg, France.

194. K. König, G. Maubach, A. Csaki, W. Fritzsche. Specific cutting of DNA with NIR fs-laser. Photonics Europe, 26.-30. April 2004, Straßburg, France.

195. K. König, F. Garwe, O. Krauss, B. Wang, W. Fritzsche, I. Riemann. Nanoprocessing of DNA, cells and tissues. Photonics Europe, 26.-30. April 2004, Straßburg, France.

196. B. Wang, I. Riemann, H. Schubert, K. König. Intraocular optical tomography of the cornea in vitro and in vivo. Photonics Europe, 26.-30. April 2004, Straßburg, France.

197. K. Schenke-Layland, I. Riemann, V. Ulrich, F. Opitz, U.A. Stock, K. König. Multiphoton Imaging of collagen and elastin of native and tissue-engineered heart valves. 183. K. König, F. Garwe, O. Krauss, B. Wang, W. Fritzsche, I. Riemann. Nanoprocessing of DNA, cells and tissues. Photonics Europe, 26.-30. April 2004, Straßburg, France.

198. B. Wang, I. Riemann, H. Schubert, K. König. Intraocular optical tomography of the cornea in vitro and in vivo. Photonics Europe, 26.-30. April 2004, Straßburg, France.

199. G. Ehrlich, T. Boneva, I. Riemann, R. Wetzke, K. König. Investigation of PI3Kgamma association with p101by MP-FLIM in living cells. 183. K. König, F. Garwe, O. Krauss, B. Wang, W. Fritzsche, I. Riemann. Nanoprocessing of DNA, cells and tissues. Photonics Europe, 26.-30. April 2004, Straßburg, France.

200. K. König. Transfection and nanosurgery with 80 MHz femtosecond lasers. Photonics Europe, 26.-30. April 2004, Straßburg, France.

201. K. König. Multiphoton microscopy and tomography of human skin. FOM2004, Philadelphia, USA

202. K. König. Lasertechnik: konventionell und photodynamisch. Internet-Übertragung. Zurich, Switzerland

203. K. König, I. Riemann, A. Ehlers, J. Kobow. In vivo non-invasive multiphoton tomography of human skin with subcellular resolution to detect bio- and chemohazards. SPIE Conference on Defence, Oct 10, 2004, London, UK

204. K. König. In vivo confocal microscopy of the skin and its application in cosmetology. International Society for Bioengineering and the Skin. Oct 28-30, 2004, Orlando, FL, USA

205. K. König. Multiphoton laser microscopy with compact femtosecond lasers. Biomaterials 2004, Nov 4-5, 2004, Erfurt, Germany

206. K. König. Plenary talk sponsered by Coherent Inc. In vivo multiphoton tomography of skin cancer. The 8^th International Conference on Optics Within Life Sciences (OWLS8), Nov 28- Dec 1, 2004, Melbourne, Australia

207. K. König. Multiphoton tomography with subcellular resolution. First Scientific Skin Workshop, Jan 14-15, 2005, Reyjakvik, Iceland

208. K. König. In vivo multiphoton time-resolved fluorescence/SHG imaging in human skin. Berkeley

209. A. Czaki, G. Maubach, F. Garwe, A. Steinbrück, K. König, W. Fritzsche. A novel DNA restriction technology based on laser pulse energy conversion on sequence-specific bound metal nanoparticles. Photonics West, Jan 22-27, 2005, San Jose, USA

210. W. Fritzsche, A. Czaki, A. Steinbrück, F. Garwe, K. König, M. Raschke. Metal nanoparticles as passive and active tools for bioanalytics. Photonics West, Jan 22-27, 2005, San Jose, USA

211. T. Anhut, K. Hassler, T. Lasser, K. König, R. Rigler. Fluorescence CorrelationSpectroscopy on dielectric surfaces in total internal reflection geometries. Photonics West, Jan 22-27, 2005, San Jose, USA

212. B. Wang, I. Riemann, H. Schubert, S. Kirste, K. König. In vivo animal follow-up studies on intrastromal surgery with near infrared nanojoule femtosecond laser pulses. Poster. Photonics West, Jan 22-27, 2005, San Jose, USA

213. K. König, I. Riemann, H. Schuck, D. Sauer, T. Velten, R. LeHarzic. Time-resolved and spectrally resolved 5D multiphoton microscopy for analysis and nanoprocessing of materials. Photonics West, Jan 22-27, 2005, San Jose, USA.

214. I. Riemann, A. Ehlers, A. Reif, J. Kobow, K. König. In vivo multiphoton tomography of skin as a tool to study the effects of topically applied probes and UV exposure. Photonics West, Jan 22-27, 2005, San Jose, USA

215. I. Riemann, T. Anhut, F. Stracke, R. LeHarzic, K. König. Multiphoton nanosurgery in cells and tissues. Photonics West, Jan 22-27, 2005, San Jose, USA

216. I. Riemann, E. Dimitrow, M. Kaatz, J. Fluhr, P. Elsner, J. Kobow, K. König. In vivo multiphoton tomography of inflammatory tissue and melanoma. Photonics West, Jan 22-27, 2005, San Jose, USA

217. K. König, B. Wang, I. Riemann, J. Kobow. Cornea surgery with nanojoule femtosecond laser pulses. Photonics West, Jan 22-27, 2005, San Jose, USA

218. W. Becker, K. König. Fluorescence lifetime images and correlation spectra obtained by multi-dimensional TCSPC. Photonics West, Jan 22-27, 2005, San Jose, USA

219. K. König. From a physics point of view: Are femtosecond lasers safe for ophthalmic applications? 6^th International Congress of Wavefront Sensing&Optimized Refractive Corrections. Feb 11-13, 2005. Athens, Greece

Internat. Seminare
1. "Time-resolved and time-gated spectroscopy and imaging in cancer diagnosis" Hamamatsu, Japan, September1992 2. "Laser-induced fluorescence of ALA-induced endogenous porphyrins" Wellman Labs/Harvard University, Boston, USA, Januar 1993 3. "Photodynamic Tumor Therapy" Universitätsspital Zürich, Schweiz, Mai 1993 4. "Laser-induced diagnosis and imaging of dental caries" Ravenswood Hospital, Chicago, USA, Juni 1993 5. "Photodynamic activity of porphyrin photoproducts" Royal Military College, Kingston, Canada, Juni 1993 6. "Laser-Induced Autofluorescence" Beckman Laser Institute, Irvine, California, USA, Oktober 1993 7. "Cell Damage Induced by Near Infrared Microbeams" Dept. of Physics, LFD, Univ. of Illinois, Urbana-Champaign, USA, 11.8.95 8. "Cell Damage in Two-Photon Microscopes" Firma Spectra Physics, Mountain View, California, USA, Dezember 1995 9. "Multi-Photon Excitation in Living Cells" Lawrence Livermore National Laboratory, Livermore, California, USA, 26. Januar 1996 10. "Cell Damage by NIR microbeams" Firma Coherent, Santa Clara, California, USA, 30. Januar 1996 11. "Two-Photon Excitation in Living Cells by CW and Femtosecond NIR laser beams" Beckman Laser Institute, Irvine, California, USA, 1. Februar 1996 12. "Femtosecond Two-Photon Microscopy" Karolinska Institute (Prof. Rigler), Stockholm, August 1997 13. "Two-Photon Excitation in Living Cells" Massachusetts Institute of Technology (MIT), Cambridge, USA, 5.2.1998 14. "3D Mikroskopie mit gepulstem NIR Laser" Universität Zürich, 23.6.98 15. "Near infrared lasers for optical trapping, three dimensional data storage/imaging, and nanosurgery". Massachusetts Institute of Technology (MIT), Cambridge, USA, 17.9.98. 16. „Nanosurgery of chromosomes“ Beckman Laser Institute, Irvine, California, USA, 16. August 1999 17. „Multiphoton Microscopy: Fluorescence imaging and Nanosurgery" Laser Medical Center, Portland, Oregon, USA, 23. August 1999 18. „Femtosecond Lasers in Medicine and Biotechnology“ Academy of Sciences, Hanoi, Vietnam, April 2002 19. „Multiphoton Effects for 5D Diagnostics and Nanoprocessing of biomolecules“ MIT, Cambridge/Boston, USA, 04.07.2002 20. „Nanosurgery with Femtosecond Lasers“ Beckman Laser Institute and Medical Clinic of UCI, Irvine, USA, 17.07.2002 21. „Optical Gene Transfer with Femtosecond Lasers“ CalTech, Pasadena, USA, 19.07.2002 22. “Femtosecond laser microscopes for targeted transfection” National Institute on Deafness and Other Communication Disorders, NIH, Rockville, Maryland, USA, March 2, 2003 23. “Femtosecond lasers in nanobiotechnology, cell biology and medicine. Firma , Forster City, CA, USA 23. “Near Infrared femtosecond laser pulses for optical gene transfer, gene diagnostics and multiphoton tomography of skin cancer”. Tel Aviv, Israel, 15. April 2004 24. „Femtosecond lasers in nanobiotechnology and biomedicine”. Tel Aviv, Israel, 18. April 2004

Internat. Seminare

1. "Time-resolved and time-gated spectroscopy and imaging in cancer diagnosis" Hamamatsu, Japan, September1992

2. "Laser-induced fluorescence of ALA-induced endogenous porphyrins" Wellman Labs/Harvard University, Boston, USA, Januar 1993

3. "Photodynamic Tumor Therapy" Universitätsspital Zürich, Schweiz, Mai 1993

4. "Laser-induced diagnosis and imaging of dental caries" Ravenswood Hospital, Chicago, USA, Juni 1993

5. "Photodynamic activity of porphyrin photoproducts" Royal Military College, Kingston, Canada, Juni 1993

6. "Laser-Induced Autofluorescence" Beckman Laser Institute, Irvine, California, USA, Oktober 1993

7. "Cell Damage Induced by Near Infrared Microbeams" Dept. of Physics, LFD, Univ. of Illinois, Urbana-Champaign, USA, 11.8.95

8. "Cell Damage in Two-Photon Microscopes" Firma Spectra Physics, Mountain View, California, USA, Dezember 1995

9. "Multi-Photon Excitation in Living Cells" Lawrence Livermore National Laboratory, Livermore, California, USA, 26. Januar 1996

10. "Cell Damage by NIR microbeams" Firma Coherent, Santa Clara, California, USA, 30. Januar 1996

11. "Two-Photon Excitation in Living Cells by CW and Femtosecond NIR laser beams" Beckman Laser Institute, Irvine, California, USA, 1. Februar 1996

12. "Femtosecond Two-Photon Microscopy" Karolinska Institute (Prof. Rigler), Stockholm, August 1997

13. "Two-Photon Excitation in Living Cells" Massachusetts Institute of Technology (MIT), Cambridge, USA, 5.2.1998

14. "3D Mikroskopie mit gepulstem NIR Laser" Universität Zürich, 23.6.98

15. "Near infrared lasers for optical trapping, three dimensional data storage/imaging, and nanosurgery". Massachusetts Institute of Technology (MIT), Cambridge, USA, 17.9.98.

16. „Nanosurgery of chromosomes“ Beckman Laser Institute, Irvine, California, USA, 16. August 1999

17. „Multiphoton Microscopy: Fluorescence imaging and Nanosurgery" Laser Medical Center, Portland, Oregon, USA, 23. August 1999

18. „Femtosecond Lasers in Medicine and Biotechnology“ Academy of Sciences, Hanoi, Vietnam, April 2002

19. „Multiphoton Effects for 5D Diagnostics and Nanoprocessing of biomolecules“ MIT, Cambridge/Boston, USA, 04.07.2002

20. „Nanosurgery with Femtosecond Lasers“ Beckman Laser Institute and Medical Clinic of UCI, Irvine, USA, 17.07.2002

21. „Optical Gene Transfer with Femtosecond Lasers“ CalTech, Pasadena, USA, 19.07.2002

22. “Femtosecond laser microscopes for targeted transfection” National Institute on Deafness and Other Communication Disorders, NIH, Rockville, Maryland, USA, March 2, 2003

23. “Femtosecond lasers in nanobiotechnology, cell biology and medicine. Firma , Forster City, CA, USA

23. “Near Infrared femtosecond laser pulses for optical gene transfer, gene diagnostics and multiphoton tomography of skin cancer”. Tel Aviv, Israel, 15. April 2004

24. „Femtosecond lasers in nanobiotechnology and biomedicine”. Tel Aviv, Israel, 18. April 2004

Patente
1. W. Dietel, K. König: Anordung zur Photochemotherapie, Phototherapie und Fluoreszenzdiagnostik. DD 254139 2. K. König, W. Dietel: Verfahren zur Herstellung eines Arzneimittels für die thermische Behandlung von tumorösem Gewebe. DD 259351 3. K. König, V. Bockhorn, W. Dietel: Verfahren zur Herstellung eines Arzneimittels zur photochemotherapeutischen Behandlung, gleichzeitiger Diagnose und Grading-Bestimmung. DD 272033 4. R. König, J. Lademann, K. König: Anordnung zur Photochemotherapie und Fluoreszenzdiagnsotik. DD 262363 5. K. König. Verfahren zur Herstellung eines Arzneimittels für die photodynamische Behandlung von Tumorerkrankungen. DD 286507 6. R. Hibst, K. König: Einrichtung zum Erkennen von Karies an Zähnen. Europäisches Patent DE/14.01.92/ DE 42 00 741. Anmeldetag: 13.1.93. 7. K. König, B. Tromberg, DS. Nelson, M. Berns: Photochemical Management of Acne vulgaris. Disclosure. University of California. Jan. 1994 8. P. Wilder-Smith, K. König, C. Wilder-Smith, M.W. Berns: Photodynamically Inactivation of Helicobacter Pylori. Disclosure. University of California. Oct. 1994 9. K. König. Anordnung zur optischen Mikromanipulation, Analyse und Bearbeitung von Objekten. Patentanmeldung: Nr. 19719344.7 vom 7.5.97. 10. K. König. Verfahren zur optischen Bearbeitung von Zellstrukturen und Biomolekülen. Patentanmeldung: Nr. 19719345.5 vom 7.5.97. 11. K. König. Verfahren zur optischen 3D Speicherung von Daten. Patentanmeldung Nr. 19754254.9 vom 6.12.97. 12. K. König, K.-J. Halbhuber, I. Riemann, P. Fischer. Verfahren zur optischen Anregung von Fluorophor-markierter DNA und RNA. Patentanmeldung Nr. 19935766.8 vom 29.7.99. 13. Teuchner, Leupold, Ehlert, König. Fluorophor für die Multi-Photonen-Laser-Scanning-Mikroskopie. Patentanmeldung Nr. 19939706.6 vom 18.8.99. 14. König, Teschke. Anordnung zur Inaktivierung von Mikroorganismen im Mund- und Rachenraum. Patentanmeldung Nr. 10002913.2 vom 18.1.00. 15. B.J.F. Wong, K. König, B.J. Tromberg. Spectrally resolved imaging of the paranasal sinuses during near infrared transillumination. Disclosure. University of California. 2000. 16. K. König. Laseranordnung und Verfahren zur optischen Untersuchung und Bearbeitung von Haut und Hautanhangsgebilden. Anmeldung am 14.9.2000.

Patente

1. W. Dietel, K. König: Anordung zur Photochemotherapie, Phototherapie und Fluoreszenzdiagnostik. DD 254139

2. K. König, W. Dietel: Verfahren zur Herstellung eines Arzneimittels für die thermische Behandlung von tumorösem Gewebe. DD 259351

3. K. König, V. Bockhorn, W. Dietel: Verfahren zur Herstellung eines Arzneimittels zur photochemotherapeutischen Behandlung, gleichzeitiger Diagnose und Grading-Bestimmung. DD 272033

4. R. König, J. Lademann, K. König: Anordnung zur Photochemotherapie und Fluoreszenzdiagnsotik. DD 262363

5. K. König. Verfahren zur Herstellung eines Arzneimittels für die photodynamische Behandlung von Tumorerkrankungen. DD 286507

6. R. Hibst, K. König: Einrichtung zum Erkennen von Karies an Zähnen. Europäisches Patent DE/14.01.92/ DE 42 00 741. Anmeldetag: 13.1.93.

7. K. König, B. Tromberg, DS. Nelson, M. Berns: Photochemical Management of Acne vulgaris. Disclosure. University of California. Jan. 1994

8. P. Wilder-Smith, K. König, C. Wilder-Smith, M.W. Berns: Photodynamically Inactivation of Helicobacter Pylori. Disclosure. University of California. Oct. 1994

9. K. König. Anordnung zur optischen Mikromanipulation, Analyse und Bearbeitung von Objekten. Patentanmeldung: Nr. 19719344.7 vom 7.5.97.

10. K. König. Verfahren zur optischen Bearbeitung von Zellstrukturen und Biomolekülen. Patentanmeldung: Nr. 19719345.5 vom 7.5.97.

11. K. König. Verfahren zur optischen 3D Speicherung von Daten. Patentanmeldung Nr. 19754254.9 vom 6.12.97.

12. K. König, K.-J. Halbhuber, I. Riemann, P. Fischer. Verfahren zur optischen Anregung von Fluorophor-markierter DNA und RNA. Patentanmeldung Nr. 19935766.8 vom 29.7.99.

13. Teuchner, Leupold, Ehlert, König. Fluorophor für die Multi-Photonen-Laser-Scanning-Mikroskopie. Patentanmeldung Nr. 19939706.6 vom 18.8.99.

14. König, Teschke. Anordnung zur Inaktivierung von Mikroorganismen im Mund- und Rachenraum. Patentanmeldung Nr. 10002913.2 vom 18.1.00.

15. B.J.F. Wong, K. König, B.J. Tromberg. Spectrally resolved imaging of the paranasal sinuses during near infrared transillumination. Disclosure. University of California. 2000.

16. K. König. Laseranordnung und Verfahren zur optischen Untersuchung und Bearbeitung von Haut und Hautanhangsgebilden. Anmeldung am 14.9.2000.

Zuletzt bearbeitet am 08.10.2018