Phasor analysis of multiphoton spectral images distinguishes autofluorescence components of in vivo human skin.

Skin contains many autofluorescent components that can be studied using spectral imaging. We employed a spectral phasor method to analyse two photon excited autofluorescence and second harmonic generation images of in vivo human skin. This method allows segmentation of images based on spectral features. Various structures in the skin could be distinguished, including Stratum Corneum, epidermal cells and dermis. The spectral phasor analysis allowed investigation of their fluorescence composition and identification of signals from NADH, keratin, FAD, melanin, collagen and elastin. Interestingly, two populations of epidermal cells could be distinguished with different melanin content.

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). An oily vehicle was then added to the formula to improve the solubility and the photostability of the lipophilic UV filters. RESULTS: The combination of filters, i.e., terephthalylidene dicamphor sulfonic acid (TDSA), bis-ethylhexyloxyphenol methoxyphenyl triazin (BEMT), and butyl methoxy dibenzoyl methane (BMDM), obtained an elevated SPF as well as a high UVA-PF. Isopropyl lauroyl sarcosinate (ILS), a derivative of a natural amino acid (sarcosine, also known as N-methylglycine) was introduced in this formulation in order to dissolve the oil-soluble UV absorbers and to photostabilize BMDM. The new sunscreen formulation obtained with this combination is photostable and contains a reduced amount of UV filters compared to other sunscreens with the same level of efficacy. CONCLUSION: This report described the steps resulting in the formulation of a new combination of UV filters in an oily emollient, which presents a high UVA-PF (UVA-PF = 38) and a SPF 50+, is photostable, and offers good protection against UV-induced biological damage.

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. 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.

Vibrational motions associated with primary processes in bacteriorhodopsin studied by coherent infrared emission spectroscopy.

The primary energetic processes driving the functional proton pump of bacteriorhodopsin take place in the form of complex molecular dynamic events after excitation of the retinal chromophore into the Franck-Condon state. These early events include a strong electronic polarization, skeletal stretching, and all-trans-to-13-cis isomerization upon formation of the J intermediate. The effectiveness of the photoreaction is ensured by a conical intersection between the electronic excited and ground states, providing highly nonadiabatic coupling to nuclear motions. Here, we study real-time vibrational coherences associated with these motions by analyzing light-induced infrared emission from oriented purple membranes in the 750-1400 cm(-)(1) region. The experimental technique applied is based on second-order femtosecond difference frequency generation on macroscopically ordered samples that also yield information on phase and direction of the underlying motions. Concerted use of several analysis methods resulted in the isolation and characterization of seven different vibrational modes, assigned as C-C stretches, out-of-plane methyl rocks, and hydrogen out-of-plane wags, whereas no in-plane H rock was found. Based on their lifetimes and several other criteria, we deduce that the majority of the observed modes take place on the potential energy surface of the excited electronic state. In particular, the direction sensitivity provides experimental evidence for large intermediate distortions of the retinal plane during the excited-state isomerization process.

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. Image quality is significantly enhanced by the simultaneous recording of background spectra. In vivo spectral images of 224 × 224 pixels were acquired, background corrected and previewed in real RGB color in 6.5 seconds. A clear increase in melanin content in deeper epidermal layers in in vivo human skin was observed.

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. 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.

Quantification of sudden light-induced polarization in bacteriorhodopsin by optical rectification.

Upon population of its excited state, the retinal chromophore in the membrane protein bacteriorhodopsin (bR) undergoes a sudden (less than approximately 10 fs) change in dipole moment, Deltamu, that can be visualized in a direct way by optical rectification of a broadband visible femtosecond light pulse to the infrared but has not been quantified in this way. Here we show that a transparent thick AgGaS2 crystal delivers infrared radiation with the same spectral profile as bR and is a suitable reference for quantifying conversion efficiency. Using this reference, we estimate the projection of Deltamu on the membrane normal at 11 D, corresponding to the displacement of a full charge over approximately half the length of the retinal chromophore. This result may help to evaluate models describing the interplay between the initial polarization change and the subsequent isomerization of the retinal.

[Cellular phones and cancer: current status]. / Téléphonie cellulaire et cancer: où en est-on?

Evaluation of the impact of new technologies on the human body is essential in order to impose regulations to limit health risks. The appearance and evolution of cellular phones have been one of the fastest in the history of innovation. Research reported worldwide has tried to evaluate any potential link between adverse health effects and the mobile phone and its broadcasting stations. This article gives an overview of current research knowledge on the impact of radiofrequency waves on health. Epidemiologic, cellular and animal studies have been carried out, but none of them have reached definitive conclusions. Although some biological effects on cell culture have been observed, their link with human cancer development is far from established. Most of the animal studies show negative results. Epidemiologic studies lack a sufficient perspective to be able to evaluate the effect of evolving technologies used today. High levels of concern by the public have urged mobile phone operators, manufacturers and governmental authorities to finance a number of scientific projects aimed at defining adapted and effective regulations.

Resonant optical rectification in bacteriorhodopsin.

The relative role of retinal isomerization and microscopic polarization in the phototransduction process of bacteriorhodopsin is still an open question. It is known that both processes occur on an ultrafast time scale. The retinal trans-->cis photoisomerization takes place on the time scale of a few hundred femtoseconds. On the other hand, it has been proposed that the primary light-induced event is a sudden polarization of the retinal environment, although there is no direct experimental evidence for femtosecond charge displacements, because photovoltaic techniques cannot be used to detect charge movements faster than picoseconds. Making use of the known high second-order susceptibility chi(2) of retinal in proteins, we have used a nonlinear technique, interferometric detection of coherent infrared emission, to study macroscopically oriented bacteriorhodopsin-containing purple membranes. We report and characterize impulsive macroscopic polarization of these films by optical rectification of an 11-fs visible light pulse in resonance with the optical transition. This finding provides direct evidence for charge separation as a precursor event for subsequent functional processes. A simple two-level model incorporating the resonant second-order optical properties of retinal, which are known to be a requirement for functioning of bacteriorhodopsin, is used to describe the observations. In addition to the electronic response, long-lived infrared emission at specific frequencies was observed, reflecting charge movements associated with vibrational motions. The simultaneous and phase-sensitive observation of both the electronic and vibrational signals opens the way to study the transduction of the initial polarization into structural dynamics.