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.
Access to the requested content is limited to institutions that have purchased or subscribe to SPIE eBooks.
You are receiving this notice because your organization may not have SPIE eBooks access.*
*Shibboleth/Open Athens users─please
sign in
to access your institution's subscriptions.
To obtain this item, you may purchase the complete book in print or electronic format on
SPIE.org.
INSTITUTIONAL Select your institution to access the SPIE Digital Library.
PERSONAL Sign in with your SPIE account to access your personal subscriptions or to use specific features such as save to my library, sign up for alerts, save searches, etc.