Abstract
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.
Highlights
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
Similar needs exist in cosmetic clinical research: we are looking for non-invasive, high content, large field of view, high resolution 3D 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: before, during and after product application
We proposed another approach based on multiphoton fluorescence lifetime imaging (FLIM), called Pseudo-FLIM, to detect melanin in the whole epidermis [20]
Summary
Skin imaging has always found a privileged position in the scientific literature, for teaching purposes or for the monitoring of the patient, owing to the direct access to the skin. Multiphoton microscopy presents several other advantages: superior imaging depth within biological tissues as IR light penetrates deeper in the skin than UV/VIS light, less photobleaching and phototoxicity as confined to the excitation volume It provides additional contrast modes with increased specificity, such as the second harmonic generation, a coherent second-order nonlinear process, forbidden in centrosymmetric media and implying no absorption phenomenon. NADH and FAD redox ratio imaging and FLIM imaging both require long acquisition times: firstly, due to the fact that an optimal excitation of these two chromophores implies a sequential imaging at ≈ 740 nm for NADH and ≈ 900 nm for FAD, secondly, because NADH and FAD imaging implies different image acquisition times due to their different mitochondrial concentration (orders of magnitude higher for NADH as compared to FAD) and lastly, because FLIM analysis requires an increased number of photons per pixel (hundreds of photons/pixel for a bi-exponential decay) For all these reasons, clinical trials dealing with multiphoton FLIM assessment of the metabolic activity in human volunteers involve mostly a single excitation wavelength, around 760 nm, convenient for NADH imaging. The clinical applications of in vivo multiphoton/FLIM microscopy range from the characterization of age-related or photoaging changes [38, 46–55]], dermatological disorders and melanoma [28, 56–71], up to the assessment of penetration and effects of pharmaceutical/cosmetic products on human skin [19, 21, 55, 72–81]
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