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.

The application of standard two-way curve resolution methods is reported for analysis of three-way Raman image data. Two current curve resolution methods are described: principal factor multivariate curve resolution (PF-MCR), which uses principal factor analysis (PFA) combined with varimax rotation and alternating least-squares optimization (ALS), and orthogonal projection multivariate curve resolution (OP-MCR), which uses a Gram–Schmidt modified orthogonal projection approach (OPA) followed by ALS. The OP-MCR technique is shown to be an extremely rapid method of analysis producing results equivalent to those of PF-MCR in one-third to one-fourth the time. The results from MCR analysis using either method provide the number of chemical species present in the sample, the spectrum of each species for identification, and the concentration image for each species. The additional benefit of image noise reduction also results from the MCR techniques. A brief description of the theory is presented followed by analysis and comparison of results for two real Raman image data. A discussion is given addressing the rapid analysis aspects of OP-MCR and the relative merits and drawbacks of the technique in comparison to PF-MCR. The use of data subsampling is also discussed as a way of decreasing analysis time without loss in accuracy or performance.

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Spectroscopic images are singular chemical measurements that enclose chemical and spatial information about samples. Resolution of spectroscopic images is focused on the recovery of the pure spectra and distribution maps of the image constituents from the sole raw spectroscopic measurement. In image resolution, constraints are generally limited to non-negativity and the spatial information is generally not used. Local rank analysis methods have been adapted to describe the local spatial complexity of an image, providing specific pixel information. This local rank information combined with reference spectral information allows the identification of absent compounds in pixels with low compound overlap. The introduction of this information in the resolution process under the form of constraints helps to increase the performance of the resolution method and to decrease the ambiguity linked to the final solutions.

A near infrared (NIR) multispectral imaging method for measuring skin hydration has been applied in a clinical study for estimating skin hydration effects of skin moisturizers and cleansers. The method has been compared to the commercially available, standard electrical methods for evaluating changes in skin hydration based on conductance and capacitance measurements. All of the instrumental methods have been compared to the visual assessment of skin dryness. It has been shown that the NIR imaging method is capable of detecting changes in skin hydration induced by skin moisturizers and cleansers. A large positive hydration change upon treatment by a moisturizing cream was easily detected by all three instrumental methods and by the expert visual assessment of dryness. The imaging technique is rapid, noncontact and noninvasive, and has the additional important advantage of showing the degree of hydration as a function of location, for rapid assessment of change in hydration. There was a clear difference between the instrumental methods when the induced changes were not as great as that from the moisturizing cream. The imaging technique showed more sensitive discrimination between treatments and control, and strong correlation to visual appearance of dryness. (c) 2005 Society of Photo-Optical Instrumentation Engineers.

Abstract. Stimulated Raman scattering (SRS) microscopy is used to generate structural and chemical threedimensional 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-d 34 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.