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We propose a novel automatic segmentation algorithm that separates the components of human skin cells from the rest of the tissue in fluorescence data of three-dimensional scans using non-invasive multiphoton tomography. The algorithm encompasses a multi-stage merging on preprocessed superpixel images to ensure independence from a single empirical global threshold. This leads to a high robustness of the segmentation considering the depth-dependent data characteristics, which include variable contrasts and cell sizes. The subsequent classification of cell cytoplasm and nuclei are based on a cell model described by a set of four features. Two novel features, a relationship between outer cell and inner nucleus (OCIN) and a stability index, were derived. The OCIN feature describes the topology of the model, while the stability index indicates segment quality in the multi-stage merging process. These two new features, combined with the local gradient magnitude and compactness, are used for the model-based fuzzy evaluation of the cell segments. We exemplify our approach on an image stack with 200 × 200 × 100 μm3, including the skin layers of the stratum spinosum and the stratum basale of a healthy volunteer. Our image processing pipeline contributes to the fully automated classification of human skin cells in multiphoton data and provides a basis for the detection of skin cancer using non-invasive optical biopsy. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The clinical outcome of corneal collagen crosslinking (CXL) is typically evaluated several weeks after treatment. An earlier assessment of its outcome could lead to an optimization of the treatment, including an immediate re-intervention in case of failure, thereby, avoiding additional discomfort and pain to the patient. In this study, we propose two-photon imaging (TPI) as an earlier evaluation method. CXL was performed in human corneas by application of riboflavin followed by UVA irradiation. Autofluorescence (AF) intensity and lifetime images were acquired using a commercial clinically certified multiphoton tomograph prior to CXL and after 2h, 24h, 72h, and 144h storage in culture medium. The first monitoring point was determined as the minimum time required for riboflavin clearance from the cornea. As control, untreated samples and samples treated only with riboflavin (without UVA irradiation) were monitored at the same time points. Significant increases in the stroma AF intensity and lifetime were observed as soon as 2h after treatment. A depth-dependent TPI analysis showed higher AF lifetimes anteriorly corresponding to areas were CXL was most effective. No alterations were observed in the control groups. Using TPI, the outcome of CXL can be assessed non-invasively and label-free much sooner than with conventional clinical devices. [ABSTRACT FROM AUTHOR]