In vivo quantitative molecular absorption of glycerol in human skin using coherent anti-Stokes Raman scattering (CARS) and two-photon auto-fluorescence.

The penetration of small molecules through the human skin is a major issue for both safety and efficacy issues in cosmetics and pharmaceutic domains. To date, the quantification of active molecular compounds in human skin following a topical application uses ex vivo skin samples mounted on Franz cell diffusion set-up together with appropriate analytical methods. Coherent anti-Stokes Raman scattering (CARS) has also been used to perform active molecule quantification on ex vivo skin samples, but no quantification has been described in human skin in vivo. Here we introduce and validate a framework for imaging and quantifying the 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 was exemplified on topically applied glycerol diluted in various vehicles such as water and xanthan gel. In vivo glycerol quantitative percutaneous penetration over time was demonstrated, showing that, contrary to water, the xanthan gel vehicle acts as a film reservoir that releases glycerol continuously over time. More generally, the proposed imaging framework provides an enabling platform for establishing functional activity of topically applied products in vivo.

A jasmonic acid derivative improves skin healing and induces changes in proteoglycan expression and glycosaminoglycan structure.

BACKGROUND: Jasmonates are plant hormones that exhibit anti-cancer and anti-inflammatory properties and have therefore raised interest for human health applications. The molecular basis of these activities remains poorly understood, although increasing evidence suggests that a variety of mechanisms may be involved. Recently, we have reported that a jasmonate derivative (JAD) displayed anti-aging effects on human skin by inducing extracellular matrix (ECM) remodeling. Based on this observation, we have investigated here the effects of JAD on proteoglycans and glycosaminoglycan (GAG) polysaccharides, which are major cell-surface/ECM components and are involved in a multitude of biological processes. In parallel, we have examined the ability of JAD to promote growth factor activities and improve skin wound healing. METHODS: Proteoglycan expression was analyzed on epidermal primary keratinocytes and reconstituted skin epidermis, using electron/immunofluorescence microscopy, western blotting and flow cytometry. GAG composition was determined by disaccharide analysis. Finally, biological activities of JAD were assessed in cellulo, in FGF-7 induced migration/proliferation assays, as well as in vivo, using a suction blister model performed on 24 healthy volunteers. RESULTS: JAD was found to induce expression of major skin proteoglycans and to induce subtle changes in GAG structure. In parallel, we showed that JAD promoted FGF-7 and improved skin healing by accelerating epithelial repair in vivo. CONCLUSION: This study highlights JAD as a promising compound for investigating GAG structure-function relationships and for applications in skin cosmetic /corrective strategies. GENERAL SIGNIFICANCE: We propose here a novel mechanism, by which jasmonate derivatives may elicit biological activities in mammals.

Cell surface glycans in the human stratum corneum: distribution and depth-related changes.

During the formation of the stratum corneum (SC) barrier, the extracellular spaces of viable epidermis, rich in glycans, are filled with a highly organized lipid matrix and the plasma membranes of keratinocytes are replaced by cornified lipid envelopes. These structures comprise cross-linked proteins, including transmembrane glycoproteins and proteoglycans, covalently bound to a monolayer of cell surface ceramides. Little is known about the presence and distribution of glycans on the SC corneocytes despite their possible involvement in SC hydration, cohesion and desquamation. In this work, we visualized ultrastructurally and quantified the distribution of glycans on the surface of native and delipidated corneocytes. The cells were harvested at different depths of the SC, allowing us to define the relationship between the distribution of various glycans, proteoglycans and glycoproteins, and other changes occurring in SC. At the cell periphery, we found a correlation between the depth-related alterations of corneodesmosome glycoproteins and α-d-mannosyl and N-acetyl-d-glucosamine-labelling patterns. Elimination of the terminal sugars, α-linked fucose and α-(2,3) linked sialic acid, was less abrupt, but also the initial extent of their peripheral distribution was overall lower than that of concanavalin A and wheat germ agglutinin lectin-detected glycans. Diffuse labelling of heparan sulphate glycosaminoglycans disappeared completely from the outermost corneocytes, whereas that of several simple carbohydrates could be detected at all SC levels. Our results suggest that specific glycan distribution may participate in the progressive changes of SC, as it evolves from the SC compactum to the SC disjunctum, towards desquamation.

Quantitative 3D molecular cutaneous absorption in human skin using label free nonlinear microscopy.

Understanding the penetration mechanisms of drugs into human skin is a key issue in pharmaceutical and cosmetics research. To date, the techniques available for percutaneous penetration of compounds fail to provide a quantitative 3D map of molecular concentration distribution in complex tissues as the detected microscopy images are an intricate combination of concentration distribution and laser beam attenuation upon deep penetration. Here we introduce and validate a novel framework for imaging and reconstructing molecular concentration within the depth of artificial and human skin samples. Our approach combines the use of deuterated molecular compounds together with coherent anti-Stokes Raman scattering spectroscopy and microscopy that permits targeted molecules to be unambiguously discriminated within skin layers. We demonstrate both intercellular and transcellular pathways for different active compounds, together with in-depth concentration profiles reflecting the detailed skin barrier architecture. This method provides an enabling platform for establishing functional activity of topically applied products.

Contrast improvement of terahertz images of thin histopathologic sections.

We present terahertz images of 10 µm thick histopathologic sections obtained in reflection geometry with a time-domain spectrometer, and demonstrate improved contrast for sections measured in paraffin with water. Automated segmentation is applied to the complex refractive index data to generate clustered terahertz images distinguishing cancer from healthy tissues. The degree of classification of pixels is then evaluated using registered visible microscope images. Principal component analysis and propagation simulations are employed to investigate the origin and the gain of image contrast.

Thermal radiation scanning tunnelling microscopy.

In standard near-field scanning optical microscopy (NSOM), a subwavelength probe acts as an optical 'stethoscope' to map the near field produced at the sample surface by external illumination. This technique has been applied using visible, infrared, terahertz and gigahertz radiation to illuminate the sample, providing a resolution well beyond the diffraction limit. NSOM is well suited to study surface waves such as surface plasmons or surface-phonon polaritons. Using an aperture NSOM with visible laser illumination, a near-field interference pattern around a corral structure has been observed, whose features were similar to the scanning tunnelling microscope image of the electronic waves in a quantum corral. Here we describe an infrared NSOM that operates without any external illumination: it is a near-field analogue of a night-vision camera, making use of the thermal infrared evanescent fields emitted by the surface, and behaves as an optical scanning tunnelling microscope. We therefore term this instrument a 'thermal radiation scanning tunnelling microscope' (TRSTM). We show the first TRSTM images of thermally excited surface plasmons, and demonstrate spatial coherence effects in near-field thermal emission.

Three-dimensional fabrication of metallic nanostructures over large areas by two-photon polymerization.

An experimental protocol for the realization of three-dimensional periodic metallic micro/nanostructures over large areas is presented. Simultaneous fabrication of hundreds of three-dimensional complex polymer structures is achieved using a two-photon photopolymerization (TPP) technique combined with a microlens array. Metallization of the structures is performed through the deposition of thin and highly conductive films by electroless plating. A chemical modification of the photopolymerizable resin and the production of a hydrophobic coating on the glass surface supporting the structures are realized. This process prevents metal deposition on the substrate and restricts adhesion on polymer. Our technique can produce periodic and/or isolated metallic structures with arbitrary shape, created by more than 700 individual objects written in parallel.

Comparison of test images obtained from various configurations of scanning near-field optical microscopes.

The characteristics of a few experimental near-field optical microscopes, located in different laboratories, have been compared on the basis of their ability to image a well-defined submicrometer test object.