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Balu M.,Beckman Laser Institute | Kelly K.M.,University of California at Irvine | Zachary C.B.,University of California at Irvine | Harris R.M.,University of California at Irvine | And 5 more authors.
Cancer Research | Year: 2014

Monitoring of atypical nevi is an important step in early detection of melanoma, a clinical imperative in preventing the disease progression. Current standard diagnosis is based on biopsy and histopathologic examination, a method that is invasive and highly dependent upon physician experience. In this work, we used a clinical multiphoton microscope to image in vivo and noninvasively melanocytic nevi at three different stages: common nevi without dysplastic changes, dysplastic nevi with structural and architectural atypia, and melanoma. We analyzed multiphoton microscopy (MPM) images corresponding to 15 lesions (five in each group) both qualitatively and quantitatively. For the qualitative analysis, we identified the morphologic features characteristic of each group. MPM images corresponding to dysplastic nevi and melanoma were compared with standard histopathology to determine correlations between tissue constituents and morphology and to evaluate whether standard histopathology criteria can be identified in the MPM images. Prominent qualitative correlations included the morphology of epidermal keratinocytes, the appearance of nests of nevus cells surrounded by collagen fibers, and the structure of the epidermal-dermal junction. For the quantitative analysis, we defined a numerical multiphoton melanoma index (MMI) based on three-dimensional in vivo image analysis that scores signals derived from two-photon excited fluorescence, second harmonic generation, and melanocyte morphology features on a continuous 9-point scale. Indices corresponding to common nevi (0-1), dysplastic nevi (1-4), and melanoma (5-8) were significantly different (P < 0.05), suggesting the potential of the method to distinguish between melanocytic nevi in vivo. © 2014 American Association for Cancer Research.

Kaatz M.,Universitatsklinikum Jena | Konig K.,Saarland University | Konig K.,JenLab GmbH
Hautarzt | Year: 2010

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. © 2010 Springer-Verlag.

Konig K.,Saarland University | Konig K.,JenLab GmbH | Uchugonova A.,Saarland University | Gorjup E.,Fraunhofer Institute for Biomedical Engineering
Microscopy Research and Technique | Year: 2011

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. © 2010 Wiley Periodicals, Inc.

A nonlinear laser scanning microscope for flexible, noninvasive three-dimensional detection comprising a measuring head which is flexibly connected to at least one radiation source by transmission optics and can be freely positioned in space, at least one controllable tilt mirror is arranged for aligning the excitation beam in order to keep the excitation beam concentric to an aperture-limited optical element of the measuring head, a test beam which is coupled out of the excitation beam onto a spatially resolving photodetector for monitoring the center alignment of the test beam as a conjugate position to the target position of the excitation beam and directional stabilizing the excitation beam by a control unit of the tilt mirror depending on a determined deviation.

Breunig H.G.,JenLab GmbH | Tumer F.,JenLab GmbH | Konig K.,JenLab GmbH | Konig K.,Saarland University
Journal of Biophotonics | Year: 2013

Thermally-induced changes in Arabidopsis thaliana leaves were investigated with a novel cryo microscope by multiphoton, fluorescence lifetime and spectral imaging as well as micro spectroscopy. Samples were excited with fs pulses in the near-infrared range and cooled/heated in a cryogenic chamber. The results show morphological changes in the chloroplast distribution as well as a shift from chlorophyll to cell-wall fluorescence with decreasing temperature. At temperatures below -40 °C, also second harmonic generation was observed. The measurements illustrate the suitability of multiphoton imaging to investigate thermally-induced changes at temperatures used for cryopreservation as well as for basic investigations of thermal effects on plant tissue in general. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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