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.
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. The treated region of each patient was investigated at D0, D7, D15, D22 (end of the treatment), and D60. Our study shows that multiphoton microscopy allows for the detection of corticoid-induced epidermis modifications: thinning of stratum corneum compactum and epidermis, decrease of keratinocytes size, and changes in their morphology from D7 to D22. We also show that multiphoton microscopy enables in vivo three-dimensional (3-D) quantitative assessment of melanin content. We observe that melanin density decreases during treatment and almost completely disappears at D22. Moreover, these alterations are reversible as they are no longer present at D60. Our study demonstrates that multiphoton microscopy is a convenient and powerful tool for noninvasive 3-D dynamical studies of skin integrity and pigmentation.
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.
In vivo simultaneous collagen and elastin measurements using the multiphoton tomograph DermaInspect have been
performed in skin dermis. We showed it was possible to get simultaneous measurements of autofluorescence (AF) and
Second Harmonic Generation (SHG) with a newly developed device using 2 PMTs for time-correlated single photon
counting. Unlike elastin (AF), collagen structures are able to generate second harmonics (SHG). Comparing the images
and SHG / AF ratios recorded in the depth of the outer and inner sides of the forearm of two European female volunteers
(31 and 60 years old, respectively) shows differences in collagen and elastin fibres density. It decreases with depth in the
60 years old volunteer compared to the younger one, and the skin of younger volunteer shows more collagen.
In order to successfully integrate microdevices into functional systems, it is often important to address issues of real-time performance monitoring and control. The present study addresses some of these problems in the context of a piezoelectric-driven micropump. These devices are important for emerging areas of chemistry and medicine where reliable distribution of small quantities of fluid is required. A simple, low-cost, fibre optic interferometer has been used to measure the dynamic displacement of the micropump actuator surface. Measurements show significant differences in actuator velocity, displacement and settling time between different pumping media. In addition, transient underdamped vibration of the actuator surface was observed during the rapid excursion and recursion phases of the pump movement while pumping air. These non-contact measurements can be used to determine the open loop characteristics of the micropump and provide information for design improvement or failure analysis. However, the technique can also be used to provide continuous measurement for adaptive compensation, so that the pump performance criteria are always satisfied. To this end, an automated interference fringe counting algorithm has been developed, so that the steady-state parameters can be mapped into the closed-loop control elements in real time. The performance of this algorithm will be discussed, together with the implications for optimal control of the micropump and eventual integration of the interferometer and micropump systems.
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.