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International audience; BackgroundIn vivo multiphoton imaging and automatic 3D image processing tools provide quantitative information on human skin constituents. These multiphoton‐based tools allowed evidencing retinoids epidermal effects in the occlusive patch test protocol developed for antiaging products screening. This study aimed at investigating their relevance for non‐invasive, time course assessment of retinoids cutaneous effects under real‐life conditions for one year.Materials and MethodsThirty women, 55‐65 y, applied either retinol (RO 0.3%) or retinoic acid (RA 0.025%) on one forearm dorsal side versus a control product on the other forearm once a day for 1 year. In vivo multiphoton imaging was performed every three months, and biopsies were taken after 1 year. Epidermal thickness and dermal‐epidermal junction undulation were estimated in 3D with multiphoton and in 2D with histology, whereas global melanin density and its z‐epidermal distribution were estimated using 3D multiphoton image processing tools.ResultsMain results after one year were as follows: a) epidermal thickening with RO (+30%); b) slight increase in dermal‐epidermal junction undulation with RO; c) slight decrease in 3D melanin density with RA; d) limitation of the melanin ascent observed with seasonality and time within supra‐basal layers with both retinoids, using multiphoton 3D‐melanin z‐epidermal profile.ConclusionsWith a novel 3D descriptor of melanin z‐epidermal distribution, in vivo multiphoton imaging allows demonstrating that daily usage of retinoids counteracts aging by acting not only on epidermal morphology, but also on melanin that is shown to accumulate in the supra‐basal layers with time.

There is an increasing need in cosmetic research for non-invasive, high content, skin imaging techniques offering the possibility to avoid performing invasive biopsies and to supply a maximum of information on skin state throughout a study, especially before, during and after product application. Multiphoton microscopy is one of these techniques compatible with in vitro and in vivo investigations of human skin, allowing its three-dimensional (3D) structure to be characterized with sub-μm resolution. Various intra-/extra-cellular constituents present specific endogenous two-photon excited fluorescence and second harmonic generation signals enabling a non-invasive visualization of the 3D structure of epidermal and superficial dermal layers. In association with fluorescence lifetime imaging (FLIM) and specific 3D image processing, one can extract several quantitative parameters characterizing skin constituents in terms of morphology, density and function. Multiphoton FLIM applications in cosmetic research range from knowledge to efficacy evaluation studies. Knowledge studies aim at acquiring a better understanding of appearing skin differences, for example, with aging, solar exposure or between the different skin phototypes. Evaluation studies deal with efficacy assessment of cosmetic ingredients in anti-aging or whitening domains. When using other nonlinear optics phenomena such as CARS (Coherent Anti-Stokes Raman Scattering), multiphoton imaging opens up the possibility of characterizing the cosmetic ingredients distribution inside the skin and founds application in other cosmetic domains such as hydration or antiperspirants. Developments in user-friendly, ultrasensitive, compact, multimodal imaging systems, on-the-fly data analysis and the synthesis of cosmetic ingredients with non-linear optical properties will certainly allow trespassing the todays frontiers of cosmetic applications.

There is an increasing need in cosmetic clinical research for non-invasive, high content, skin imaging techniques offering the possibility on the one hand, to avoid performing invasive biopsies, and on the other hand, to supply a maximum of information on the skin state throughout a study, especially before, during and after product application. Multiphoton microscopy is one of these techniques compatible with in vivo human skin investigations, allowing human skin three-dimensional (3D) structure to be characterized with sub-µm resolution. In association with fluores-cence lifetime imaging (FLIM) and specific 3D-image processing, one can extract several quantitative parameters characterizing skin constituents in terms of morphology, density and organization. Various intracellular and extracellular constituents present specific endogenous signals enabling a non-invasive visualization of the 3D structure of epidermal and superficial dermal layers. Multiphoton FLIM applications in the cosmetic field range from knowledge to evaluation studies. Knowledge studies aim to acquire a better knowledge of skin differences appearing with aging, solar exposure or between the different skin phototypes. Evaluation studies deal with the efficacy of cosmetic anti-aging or whitening ingredients. The goal of this chapter is not to give a literature review of multiphoton FLIM applications in cosmetic clinical research, but rather to acquaint the reader with the quantitative 3D information afforded by multi-photon FLIM imaging of human skin and its interest in cosmetic clinical research.

Background The occlusive patch test developed for assessing topical retinoids activity in human skin has been extended as a short-term screening protocol for anti-ageing agents. In this model, biopsies are performed at the end of the occlusion period for morphological and immuno-histochemistry analysis. Multiphoton microscopy is a recent non-invasive imaging technique that combined with image processing tools allows the in vivo quantification of human skin modifications. Objective To validate with gold standards of anti-ageing that are retinoids, the relevance of multiphoton microscopy for kinetic and quantitative assessment in this model. Methods Twenty women, aged 50–65 years, were enrolled. Retinol 0.3% (RO) and Retinoic acid 0.025% (RA) were applied to the dorsal photo-damaged side of their forearm under occlusive patches for 12 days. A patch alone was applied to a third area as control. Evaluation was performed at day D0, D12 (end of treatment), D18 and D32 using multiphoton microscopy. Epidermal thickness, normalized area of the dermal-epidermal junction (DEJ) and melanin density were estimated using 3D image processing tools. Results Main significant results are: Epidermal thickening at D12, D18 and D32 with RO and at D12, D18 with RA vs. baseline and vs. control. Increased DEJ undulation at D32 with RO and at D12 with RA vs. baseline and vs. control. Decreased melanin content with RO (at D12 and D18 vs. baseline and at D32 vs. baseline and vs. control) and with RA (at D12 vs. baseline). Conclusions This study shows that multiphoton microscopy associated to specific 3D image processing tools allows cutaneous effects induced by topical retinoids in this in vivo model to be non-invasively detected, quantified and followed over time. This innovative approach could be applied to the evaluation of other active compounds.

Expression levels of endoglin, αv integrin and vascular endothelial growth factor receptor 2 (VEGFR2) were investigated using targeted, contrast-enhanced ultrasonography in murine melanoma tumor models. Microvasculature and expression levels of biomarkers were investigated using specific contrast agents conjugated with biotinylated monoclonal antibodies. Ultrasound signal intensity from bound contrast agents was evaluated in two groups of mice: control mice and mice treated with sorafenib. Expression levels were analyzed by immunohistochemistry. Endoglin biomarkers were more highly expressed than αv integrin and VEGFR2. Endoglin decreased in the sorafenib group, whereas it tended to increase with time in the control group. Targeted ultrasound contrast agents may be used for non-invasive longitudinal evaluation of tumor angiogenesis during tumor growth or therapeutic treatment in preclinical studies. Endoglin protein, which plays an important role in angiogenesis, seems to be a target of interest for detection of cancer and for prediction of therapeutic efficacy.