Thomas M. Hancewicz

Two-photon 3-D mapping of ex vivo human skin endogenous fluorescence species based on fluorescence emission spectra

Spectral resolved tissue imaging has a broad range of biomedical applications such as the minimally invasive diagnosis of diseases and the study of wound healing and tissue engineering processes. Two-photon microscopy imaging of endogenous fluorescence has been shown to be a powerful method for the quantification of tissue structure and biochemistry. While two-photon excited autofluorescence is observed ubiquitously, the identities and distributions of endogenous fluorophores have not been completely characterized in most tissues. We develop an image-guided spectral analysis method to analyze the distribution of fluorophores in human skin from 3-D resolved two-photon images. We identify five factors that contribute to most of the luminescence signals from human skin. Luminescence species identified include tryptophan, NAD(P)H, melanin, and elastin, which are autofluorescent, and collagen that contributes to a second harmonic signal.

Three-dimensional chemical imaging of skin using stimulated Raman scattering microscopy

Abstract. Stimulated Raman scattering (SRS) microscopy is used to generate structural and chemical three-dimensional images of native skin. We employed SRS microscopy to investigate the microanatomical features of skin and penetration of topically applied materials. Image depth stacks are collected at distinct wavelengths corresponding to vibrational modes of proteins, lipids, and water in the skin. We observed that corneocytes in stratum corneum are grouped together in clusters, 100 to 250 μm in diameter, separated by 10- to 25-μm-wide microanatomical skin-folds called canyons. These canyons occasionally extend down to depths comparable to that of the dermal–epidermal junction below the flat surface regions in porcine and human skin. SRS imaging shows the distribution of chemical species within cell clusters and canyons. Water is predominately located within the cell clusters, and its concentration rapidly increases at the transition from stratum corneum to viable epidermis. Canyons do not contain detectable levels of water and are rich in lipid material. Oleic acid-d34 applied to the skin surface lines the canyons down to a depth of 50 μm below the surface of the skin. This observation could have implications on the evaluation of penetration profiles of bioactive materials measured using traditional methods, such as tape-stripping.

Improved Modeling of In Vivo Confocal Raman Data Using Multivariate Curve Resolution (MCR) Augmentation of Ordinary Least Squares Models

In vivo confocal Raman spectroscopy has become the measurement technique of choice for skin health and skin care related communities as a way of measuring functional chemistry aspects of skin that are key indicators for care and treatment of various skin conditions. Chief among these techniques are stratum corneum water content, a critical health indicator for severe skin condition related to dryness, and natural moisturizing factor components that are associated with skin protection and barrier health. In addition, in vivo Raman spectroscopy has proven to be a rapid and effective method for quantifying component penetration in skin for topically applied skin care formulations. The benefit of such a capability is that noninvasive analytical chemistry can be performed in vivo in a clinical setting, significantly simplifying studies aimed at evaluating product performance. This presumes, however, that the data and analysis methods used are compatible and appropriate for the intended purpose. The standard analysis method used by most researchers for in vivo Raman data is ordinary least squares (OLS) regression. The focus of work described in this paper is the applicability of OLS for in vivo Raman analysis with particular attention given to use for non-ideal data that often violate the inherent limitations and deficiencies associated with proper application of OLS. We then describe a newly developed in vivo Raman spectroscopic analysis methodology called multivariate curve resolution-augmented ordinary least squares (MCR-OLS), a relatively simple route to addressing many of the issues with OLS. The method is compared with the standard OLS method using the same in vivo Raman data set and using both qualitative and quantitative comparisons based on model fit error, adherence to known data constraints, and performance against calibration samples. A clear improvement is shown in each comparison for MCR-OLS over standard OLS, thus supporting the premise that the MCR-OLS method is better suited for general-purpose multicomponent analysis of in vivo Raman spectral data. This suggests that the methodology is more readily adaptable to a wide range of component systems and is thus more generally applicable than standard OLS.

Two-photon 3D mapping of tissue endogenous fluorescence species based on fluoresence excitation spectra

Deep tissue imaging may have important biomedical applications in the areas of skin disease diagnosis, wound healing, and tissue engineering. For the study oftissue physiology with microscopic resolution, we used two-photon microscopic imaging based on the excitation of endogenous fluorophores. While autofluorescence is observed ubiquitously in many tissue types, the identities and distributions of these fluorophores have not been completely characterized. The different fluorescent species are expected to have different fluorescence excitation and emission spectra. Self-modeling curve resolution (SMCR) can be applied to extract spectroscopic components from two-photon images. In ex vivo human skin, we were able to acquire a four-dimensional data set (3D space + excitation spectra). We extracted the major spectral components from this data set using multivariate curve resolution and correlated these species with known tissue structures. From the SMCR analysis, it was determined that there are approximately seven factors that contribute to most of the autofluorescence from human skin. This analysis provides us with the concentration ofthe species at different depths within the skin and also with a reconstructed image of the skin due to each single factor alone. Several ofthese chemical components have been identified, such as collagen, elastin, and NAD(P)H. In addition to providing insight into tissue physiology, we are able to optimize the excitation wavelength for each biochemical species for skin imaging applications.

Two-photon 3D mapping of tisssue endogenous fluorescence species based on fluorescence emission spectra

Two-photon microscopy imaging of endogenous fluorescence has been shown to be a powerful method for the quantification of tissue structure and biochemistry. While autofluorescence is observed in many tissue types, the identities and distributions of these fluorophores have not been completely characterized. Image guided spectral analysis is being developed to aid in extracting spectroscopic components from two-photon images. This methodology is being applied to the study of human skin. In ex vivo specimens, the overall bulk emission spectrum of the skin, the layer-resolved emission spectra of the stratum corneum, stratum spinosum, basal layer, and dermis, and the emission spectra of surgically exposed dermis have been measured. From the image guided spectral analysis, it was determined that there are approximately five factors that contribute to most of the luminescence signals from human skin. The autofluorescent species identified include tryptophan, NAD(P)H, melanin (or localizing species), and elastin. The collagen matrix contributes to a second harmonic signal.

EXPRESS: Quantification of Lipid Phase Order of In Vivo Human Skin Using Attenuated Total Reflection Fourier Transform Infrared (ATR FT-IR) Spectroscopy and Multivariate Curve Resolution Analysis

A new analysis methodology utilizing multivariate curve resolution (MCR) has been successfully combined with Fourier transform infrared (FT-IR) measurement of in vivo human skin to resolve lipid phase constituents in the spectra relative to high and low chain ordering. A clinical study was performed to measure lipid order through different depths of stratum corneum of human subjects. Fourier transform IR spectra were collected through the top 10 layers of the skin on four sites on the left and right forearm of 12 individuals. Depth profiling was achieved by tape stripping to remove layers of skin with 10 successive tapes from each site. In vivo ATR FT-IR spectra were collected after removing each tape. Three isolated spectral regions were analyzed, centered around 2850 cm−1, 1460–1480 cm−1, and 730 cm−1, corresponding to stretching, scissoring, and rocking –CH2 vibrational modes, respectively. Both traditional lipid conformation analysis and MCR analysis were performed on the same spectral data. The lipid order ratio, expressed as the fraction of highly ordered orthorhombic (OR) lipids to the total lipids content (orthorhombic + hexagonal [HEX] + liquid crystal [LC]), was assessed as function of depth. Lipid order depth profiles (LODP) show an increase in order with the stratum corneum depth which can be adequately described by an exponential function for the data obtained in this study. The LODP derived from the three vibrational modes show very similar trends, although the absolute order ratios are somewhat different. The variance of the skin LODP across individuals is much greater than between sites within the same individual. The higher arm sites closer to the elbow on the left and right arm show no statistically significant difference and are recommended for use in comparative studies. The scissoring mode shows the highest sensitivity for determination of LODP value.