Emmanuelle Tancrède-Bohin

Automatic 3D segmentation of multiphoton images: a key step for the quantification of human skin.

Multiphoton microscopy has emerged in the past decade as a useful noninvasive imaging technique for in vivo human skin characterization. However, it has not been used until now in evaluation clinical trials, mainly because of the lack of specific image processing tools that would allow the investigator to extract pertinent quantitative three‐dimensional (3D) information from the different skin components.

In vivo multiphoton imaging of human skin: assessment of topical corticosteroid-induced epidermis atrophy and depigmentation

Multiphoton microscopy has emerged in the past decade as a promising tool for noninvasive skin imaging. Our aim was to evaluate the potential of multiphoton microscopy to detect topical corticosteroids side effects within the epidermis and to provide new insights into their dynamics. Healthy volunteers were topically treated with clobetasol propionate on a small region of their forearms under overnight occlusion for three weeks. The treated region of each patient was investigated at D0, D7, D15, D22 (end of the treatment), and D60. Our study shows that multiphoton microscopy allows for the detection of corticoid-induced epidermis modifications: thinning of stratum corneum compactum and epidermis, decrease of keratinocytes size, and changes in their morphology from D7 to D22. We also show that multiphoton microscopy enables in vivo three-dimensional (3-D) quantitative assessment of melanin content. We observe that melanin density decreases during treatment and almost completely disappears at D22. Moreover, these alterations are reversible as they are no longer present at D60. Our study demonstrates that multiphoton microscopy is a convenient and powerful tool for noninvasive 3-D dynamical studies of skin integrity and pigmentation.

Non‐invasive short‐term assessment of retinoids effects on human skin in vivo using multiphoton microscopy

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.

Multiphoton FLIM in cosmetic clinical research

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.