We introduce and validate a framework for imaging and quantifying active molecule penetration into human skin in vivo. Our approach combines nonlinear imaging microscopy modalities, such as two-photon excited auto-fluorescence (TPEF) and coherent anti-Stokes Raman scattering (CARS), together with the use of deuterated active molecules. The imaging framework is exemplified on topically applied glycerol diluted in various vehicles such as water and xanthan gel. In vivo glycerol quantitative percutaneous penetration over time is demonstrated, showing that, contrary to water, xanthan gel vehicle acts as a film reservoir that releases glycerol continuously over time.
KEYWORDS: Skin, Collagen, In vivo imaging, 3D image processing, Second-harmonic generation, Fluorescence lifetime imaging, Multiphoton microscopy, Image segmentation, In vitro testing, 3D acquisition, CARS tomography
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.
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.
Nonlinear microscopies, including two-photon excited autofluorescence (TPEF) and coherent anti-Stokes Raman scattering (CARS), were used to study individual human sweat pore morphology and topically applied antiperspirant salt penetration inside sweat pore, in vivo on human palms. Sweat pore inner morphology in vivo was imaged up to the depth of 100 μm by TPEF microscopy. The 3D penetration and distribution of “in situ calcium carbonate” (isCC), an antiperspirant salt model, was investigated using CARS microscopy.
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.
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.
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