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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.

International audience; Human skin is constantly exposed to environmental stresses such as UV light and pollution. These agents cause oxidative stress associated with reactive oxygen species (ROS) generation, that will interfere with the normal cellular redox equilibrium. As ROS are mainly produced within mitochondria, the cellular metabolic activity could be impacted by UV light. We dynamically assessed UVA light (representing the majority of solar UV rays reaching Earth surface) effects on cellular metabolic activity of reconstructed human skin using multiphoton fluorescence lifetime imaging microscopy (FLIM). Multiphoton FLIM offers non-invasive, label-free quantitative functional information on cellular metabolic activity based on the endogenous two-photon excited fluorescence (2PEF) of NADH (reduced form of nicotinamide adenine dinucleotide) and FAD (flavine adenine dinucleotide) metabolic coenzymes. The experiments were performed in both stratum granulosum and spinosum layers (T-Skin™ model, Episkin™), before and after (30 min and 2 h) UVA exposure (20 J/cm²; 20 min exposure; 320 – 400 nm). We observed quasi similar effects in both epidermal layers after UVA exposure: • Decrease of RedOx ratio NADH / (NADH + FAD) at 30 min and 2 h; • Increase in the proportion of protein-bound NADH at 2 h, and in the proportion of free FAD as early as 30 min after UVA exposure; This study shows that the effects of UVA light on epidermis, can be non-invasively evidenced and followed overtime using NADH/FAD multiphoton FLIM imaging method. Altogether, these data suggest that epidermal cells respond to UVA light by promoting oxidative phosphorylation, the most efficient metabolic pathway for ATP production.

Depuis son introduction en 1990, la microscopie de fluorescence excitee a deux photons (Fluo-2P) s’est peu a peu imposee comme une methode incontournable d’imagerie de tissus intacts a l’echelle sub-cellulaire. En effet, la caracteristique la plus remarquable de la microscopie multiphotonique est de maintenir une resolution tridimensionnelle micrometrique lors de l’observation en profondeur d’un milieu optiquement diffusant. Combinee aux technologies de proteines-fusion (type GFP), cette approche est aujourd’hui utilisee dans de nombreux domaines, notamment en neurophysiologie. Un autre attrait de ce type d’imagerie reside dans l’utilisation possible d’autres phenomenes optiques non lineaires (c’est-a-dire impliquant l’interaction simultanee de plusieurs photons avec une molecule observee) comme source de contraste. Ainsi, les microscopies par generation de second harmonique (GSH) et par generation de troisieme harmonique (GTH) permettent egalement d’observer des milieux complexes et fournissent des informations complementaires par rapport a l’imagerie de fluorescence. Certaines structures cellulaires ou tissulaires fournissent, en effet, ce type de reponse optique sans necessiter de marquage exogene. La microscopie GSH permet, par exemple, de detecter le collagene fibrillaire et la microscopie GTH permet d’observer sans marquage le developpement embryonnaire de petits organismes.

Background/purpose 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. Methods We propose a 3D automatic segmentation method of multiphoton images which is a key step for epidermis and dermis quantification. This method, based on the morphological watershed and graph cuts algorithms, takes into account the real shape of the skin surface and of the dermal–epidermal junction, and allows separating in 3D the epidermis and the superficial dermis. Results The automatic segmentation method and the associated quantitative measurements have been developed and validated on a clinical database designed for aging characterization. The segmentation achieves its goals for epidermis–dermis separation and allows quantitative measurements inside the different skin compartments with sufficient relevance. Conclusions This study shows that multiphoton microscopy associated with specific image processing tools provides access to new quantitative measurements on the various skin components. The proposed 3D automatic segmentation method will contribute to build a powerful tool for characterizing human skin condition. To our knowledge, this is the first 3D approach to the segmentation and quantification of these original images.

International audience; Dermal fibroblasts are responsible for the generation of mechanical forces within their surrounding extracellular matrix and can be potentially targeted by anti-aging ingredients. Investigation of the modulation of fibroblast contraction by these ingredients requires the implementation of three-dimensional in situ imaging methodologies. We use multiphoton microscopy to visualize unstained engineered dermal tissue by combining second-harmonic generation that reveals specifically fibrillar collagen and two-photon excited fluorescence from endogenous cellular chromophores. We study the fibroblast-induced reorganization of the collagen matrix and quantitatively evaluate the effect of Y-27632, a RhoA-kinase inhibitor, on dermal substitute contraction. We observe that collagen fibrils rearrange around fibroblasts with increasing density in control samples, whereas collagen fibrils show no remodeling in the samples containing the RhoA-kinase inhibitor. Moreover, we show that the inhibitory effects are reversible. Our study demonstrates the relevance of multiphoton microscopy to visualize three-dimensional remodeling of the extracellular matrix induced by fibroblast contraction or other processes.

International audience; One of the main functions of dermal fibroblasts is the generation of mechanical forces within their surrounding extracellular matrix. Investigating molecules that could modulate fibroblast contraction and act as potent anti aging ingredients requires the development of three-dimensional in situ imaging methodologies for dermal substitute analysis. Here we use multiphoton microscopy in order to investigate the fibroblast-induced collagen matrix reorganization in engineered dermal tissue and to evaluate the effect of Y27632, a RhoA kinase inhibitor on dermal substitutes contraction. We observe that collagen fibrils rearrange around fibroblast with increasing density in control samples, whereas collagen fibrils show no remodeling in the samples containing the RhoA kinase inhibitor. Moreover, when the culture medium containing the inhibitor was replaced with a control medium, the dermal substitutes presented the same 3D reorganization as the control samples, which indicates that the inhibitory effects are reversible. In conclusion, our study demonstrates the relevance of multiphoton microscopy to visualize three-dimensional remodeling of the matrix induced by fibroblast contraction. © 2010 Copyright SPIE - The International Society for Optical Engineering.

International audience; Coherent x-ray diffractive imaging is a powerful method for studies on nonperiodic structures on the nanoscale. Access to femtosecond dynamics in major physical, chemical, and biological processes requires single-shot diffraction data. Up to now, this has been limited to intense coherent pulses from a free electron laser. Here we show that laser-driven ultrashort x-ray sources offer a comparatively inexpensive alternative. We present measurements of single-shot diffraction patterns from isolated nano-objects with a single 20 fs pulse from a table-top high-harmonic x-ray laser. Images were reconstructed with a resolution of 119 nm from the single shot and 62 nm from multiple shots.

International audience; We recorded one-photon excited fluorescence (1PEF) and two-photon excited fluorescence (2PEF) spectra of purified keratin from human epidermis, and determined the action cross section of this endogenous chromophore. We used this spectroscopic analysis to analyse multiphoton images of skin biopsies and assign the intrinsic fluorescence signals in the epidermis. We observed a good agreement between in situ and in vitro 2PEF spectra of keratin. This study provides a comprehensive characterization of the 2PEF signal of the keratins from the epidermis, and will be of practical interest for multiphoton imaging of the skin. © 2005 Optical Society of America

International audience; We compare second harmonic generation (SHG) to histological and immunohistochemical techniques for the visualization and scoring of collagen in biological tissues. We show that SHG microscopy is highly specific for fibrillar collagens and that combined SHG and two-photon excited fluorescence (2PEF) imaging can provide simultaneous three-dimensional visualization of collagen synthesis and assembly sites in transgenic animal models expressing GFP constructs. Finally, we propose several scores for characterizing collagen accumulation based on SHG images and appropriate for different types of collagen distributions. We illustrate the sensitivity of these scores in a murine model of renal fibrosis using a morphological segmentation of the tissue based on endogenous 2PEF signals. © 2007 Optical Society of America

International audience; The organization of collagen during fibrotic processes is poorly characterized because of the lack of appropriate methodologies. Here we show that multimodal multiphoton microscopy provides novel insights into lung fibrosis. We characterize normal and fibrotic pulmonary tissue in the bleomycin model, and show that second-harmonic generation by fibrillar collagen reveals the micrometer-scale three-dimensional spatial distribution of the fibrosis. We find that combined two-photon excited fluorescence and second-harmonic imaging of unstained lung tissue allows separating the inflammatory and fibrotic steps in this pathology, underlining characteristic features of fibroblastic foci in human Idiopathic Pulmonary Fibrosis samples. Finally, we propose phenomenological scores of lung fibrosis and we show that they unambiguously sort out control and treated mice, with a better sensitivity and reproducibility in the subpleural region. These results should be readily generalized to other organs, as an accurate method to assess extracellular matrix remodeling during fibrosis. Microsc. Res. Tech., 2007. © 2006 Wiley-Liss, Inc.

International audience; Lipid bodies have an important role in energy storage and lipid regulation. Here we show that lipid bodies are a major source of contrast in third-harmonic generation (THG) microscopy of cells and tissues. In hepatocytes, micrometer-sized lipid bodies produce a THG signal 1-2 orders of magnitude larger than other structures, which allows one to image them with high specificity. THG microscopy with approximately 1,200 nm excitation can be used to follow the distribution of lipid bodies in a variety of unstained samples including insect embryos, plant seeds and intact mammalian tissue (liver, lung). We found that epi-THG imaging is possible in weakly absorbing tissues because bulk scattering redirects a substantial fraction of the forward-generated harmonic light toward the objective. Finally, we show that the combination of THG microscopy with two-photon and second-harmonic imaging provides a new tool for exploring the interactions between lipid bodies, extracellular matrix and fluorescent compounds (vitamin A, NADH and others) in tissues.

Background: We characterize the application of multiphoton microscopy to the observation of the extracellular matrix of fresh unstained vessels. Method: Combined two-photon-excited fluorescence (2PEF) and second harmonic generation (SHG) imaging of large arteries reveals the architecture of elastin and collagen fibers in the vessel wall with remarkable specificity. Results: We present elastin/collagen imaging in unstained rat vessels at both micrometer and whole vessel scales, and we characterize the optical properties of rat carotid artery and aorta walls. We apply this method to evidence deleterious effects of residual doses of a pesticide on the vessel wall. Conclusion: This study illustrates the potential of 2PEF/ SHG microscopy for pharmacological studies in unlabeled arteries. q 2005 International Society for Analytical Cytology

International audience; We performed polarization-resolved surface second harmonic generation (SHG) experiments on thin films of collagen I and IV molecules, as well as conventional CD measurements. We found that collagen IV presents little CD and no SHG optical activity, whereas collagen I exhibits large chiroptical effects involving both one-electron and excitonic coupling mechanisms. We estimated that these chiral components enhance the SHG signal from fibrillar collagen in biological tissues by typically a factor of 2. By comparing the distinct behaviors of collagens I and IV in SHG microscopy and in surface SHG experiments, we concluded that SHG microscopy is a sensitive probe of the micrometer-scale structural organization of collagen in biological tissues. Cop. 2005 American Chemical Society.