Multiphoton FLIM Analyses of Native and UVA-Modified Synthetic Melanins.

To better understand the impact of solar light exposure on human skin, the chemical characterization of native melanins and their structural photo-modifications is of central interest. As the methods used today are invasive, we investigated the possibility of using multiphoton fluorescence lifetime (FLIM) imaging, along with phasor and bi-exponential fitting analyses, as a non-invasive alternative method for the chemical analysis of native and UVA-exposed melanins. We demonstrated that multiphoton FLIM allows the discrimination between native DHI, DHICA, Dopa eumelanins, pheomelanin, and mixed eu-/pheo-melanin polymers.

In vivo multiphoton multiparametric 3D quantification of human skin aging on forearm and face.

Quantifying skin aging changes and characterizing its 3D structure and function in a non-invasive way is still a challenging area of research, constantly evolving with the development of imaging methods and image analysis tools. In vivo multiphoton imaging offers means to assess skin constituents in 3D, however prior skin aging studies mostly focused on 2D analyses of dermal fibers through their signals' intensities or densities. In this work, we designed and implemented multiphoton multiparametric 3D quantification tools for in vivo human skin pigmentation and aging characterization.

In vivo melanin 3D quantification and z-epidermal distribution by multiphoton FLIM, phasor and Pseudo-FLIM analyses.

Characterizing melanins in situ and determining their 3D z-epidermal distribution is paramount for understanding physiological/pathological processes of melanin neosynthesis, transfer, degradation or modulation with external UV exposure or cosmetic/pharmaceutical products. Multiphoton fluorescence intensity- and lifetime-based approaches have been shown to afford melanin detection, but how can one quantify melanin in vivo in 3D from multiphoton fluorescence lifetime (FLIM) data, especially since FLIM imaging requires long image acquisition times not compatible with 3D imaging in a clinical setup? We propose an approach combining (i) multiphoton FLIM, (ii) fast image acquisition times, and (iii) a melanin detection method called Pseudo-FLIM, based on slope analysis of autofluorescence intensity decays from temporally binned data. We compare Pseudo-FLIM to FLIM bi-exponential and phasor analyses of synthetic melanin, melanocytes/keratinocytes coculture and in vivo human skin.

Simultaneous NAD(P)H and FAD fluorescence lifetime microscopy of long UVA-induced metabolic stress in reconstructed human skin.

Solar ultraviolet longwave UVA1 exposure of human skin has short-term consequences at cellular and molecular level, leading at long-term to photoaging. Following exposure, reactive oxygen species (ROS) are generated, inducing oxidative stress that might impair cellular metabolic activity. However, the dynamic of UVA1 impact on cellular metabolism remains unknown because of lacking adequate live imaging techniques.

In vivo multiphoton imaging for non-invasive time course assessment of retinoids effects on human skin.

Background: In 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.

Imaging and quantifying drug delivery in skin - Part 2: Fluorescence andvibrational spectroscopic imaging methods.

Understanding the delivery and diffusion of topically-applied drugs on human skin is of paramount importance in both pharmaceutical and cosmetics research. This information is critical in early stages of drug development and allows the identification of the most promising ingredients delivered at optimal concentrations to their target skin compartments. Different skin imaging methods, invasive and non-invasive, are available to characterize and quantify the spatiotemporal distribution of a drug within ex vivo and in vivo human skin.

Imaging and quantifying drug delivery in skin - Part 1: Autoradiography and mass spectrometry imaging.

In this two-part review we present an up-to-date description of different imaging methods available to map the localization of drugs on skin as a complement of established ex-vivo absorption studies. This first part deals with invasive methods which are grouped in two classes according to their underlying principles: i) methods using radioactivity such as autoradiography and ii) mass spectrometry methods such as MALDI and SIMS. For each method, a description of the principle is given along with example applications of imaging and quantifying drug delivery in human skin.

Multicolor two-photon imaging of endogenous fluorophores in living tissues by wavelength mixing.

Two-photon imaging of endogenous fluorescence can provide physiological and metabolic information from intact tissues. However, simultaneous imaging of multiple intrinsic fluorophores, such as nicotinamide adenine dinucleotide(phosphate) (NAD(P)H), flavin adenine dinucleotide (FAD) and retinoids in living systems is generally hampered by sequential multi-wavelength excitation resulting in motion artifacts. Here, we report on efficient and simultaneous multicolor two-photon excitation of endogenous fluorophores with absorption spectra spanning the 750-1040 nm range, using wavelength mixing.

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

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.

New combination of ultraviolet absorbers in an oily emollient increases sunscreen efficacy and photostability.

Introduction: It is now recognized that to adequately protect skin from sun damage, sunscreens require a photostable combination of ultraviolet (UV) filters with a suitable level of UVA protection. The total amount of UV filters should be as low as possible to avoid adverse skin reactions, potential environmental impact, and to ensure acceptable texture for better application and usage.

Methods: A synergistic combination of UV filters was selected to obtain a high sun protection factor (SPF) and UVA protection factor (UVA-PF).

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.

Fast nonlinear spectral microscopy of in vivo human skin.

An optimized system for fast, high-resolution spectral imaging of in vivo human skin is developed and evaluated. The spectrograph is composed of a dispersive prism in combination with an electron multiplying CCD camera. Spectra of autofluorescence and second harmonic generation (SHG) are acquired at a rate of 8 kHz and spectral images within seconds.

Multiphoton microscopy of engineered dermal substitutes: assessment of 3-D collagen matrix remodeling induced by fibroblast contraction.

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.

Three-dimensional investigation and scoring of extracellular matrix remodeling during lung fibrosis using multiphoton microscopy.

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.

[Second- and third-harmonic generation microscopies for the structural imaging of intact tissues].

One principal advantage of multiphoton excitation microscopy is that it preserves its three-dimensional micrometer resolution when imaging inside light-scattering samples. For that reason two-photon-excited fluorescence microscopy has become an invaluable tool for cellular imaging in intact tissue, with applications in many fields of physiology. This success has driven increasing interest in other forms of nonlinear microscopy that can provide additional information on cells and tissues, such as second- (SHG) and third- (THG) harmonic generation microscopies.

Imaging lipid bodies in cells and tissues using third-harmonic generation microscopy.

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.

Chiroptical effects in the second harmonic signal of collagens I and IV.

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.