Introduction Technological developments in infrared and Raman microscopic imaging have permitted several new types of biomedical applications. As demonstrated elsewhere in this volume, the diagnosis of pathological states in a wide range of tissues is now feasible, and is being pursued vigorously in laboratories around the world. The vast volume of spectral data generated from imaging experiments permits sophisticated multivariate statistical analysis of data and enables the experimentalist to discern tissue regions where spectral signatures from pathological states differ from those of healthy tissue. 1 2 4 The first example uses both IR and Raman microscopy to evaluate structural changes in the keratin of single cells (corneocytes) induced by treatment with dimethyl sulfoxide (DMSO) or chloroform/methanol (C/M). These solvents have been widely used in dermatology for studies of permeation enhancement and for extracting lipid from the stratum corneum (SC), respectively. The characterization of DMSO-induced structural changes in keratin will aid in the determination of DMSO permeation pathways and in the evaluation of SC barrier recovery. Assessing C/M’s effect on keratin in isolated corneocytes including the reversibility of potential structural changes, will facilitate the delineation of solvent treatment effects from that of lipid removal. In the second example, we demonstrate that confocal Raman microscopy along with multivariate (factor) analysis provides the detection of particular skin regions (stratum corneum, epidermis, dermis) as well as endogenous sub-areas (keratinocytes, lipid inclusions) in the epidermis. The availability of spectra–structure correlations permits molecular structure information to be deduced from either the spectra or from the factor loadings. Materials and methods Preparation of corneocytes 2 2 Corneocytes were placed on substrates that were directly dipped in pure DMSO for about 1 min. Separate aliquots of harvested corneocytes were transferred to glass vials using 5 mL C/M (2:1 v/v). Non-covalently bound lipids were extracted for 68 h while stirring at room temperature in sealed vials. Corneocytes, collected by centrifugation, were further washed 3 times using fresh C/M before the final suspension was placed on the appropriate substrate. Samples were dried overnight under vacuum prior to spectroscopic measurements. No vibrational bands from residual solvent were observed in corneocyte spectra. Subsequently, corneocytes from both DMSO and C/M treatment were rehydrated on the solid substrates by exposure to 100% relative humidity overnight before a final set of spectra were acquired. Intact porcine skin preparation 2 IR microspectroscopy of corneocytes 4 2 −1 −1 Raman microspectroscopy 5 6 −1 −1 −1 5 Data analysis S S −1 S X S S Results and discussion Solvent-induced conformational changes in the keratin of single cells 7 8 3 −1 −1 1 −1 6 2 5 8 5 9 10 Fig. 1 a −1 2 inset bar b −1  2 2 −1 −1 11 −1 11 Fig. 2 a b top bottom c d top bottom −1 −1 b d −1 −1  12 2 −1 −1 13 14 8 −1 13 14 −1 15 α 16 17 2 −1 −1 −1 12 C/M solutions are commonly used to extract lipids from isolated SC and intact skin samples, but solvent effects on the structure of the remaining constituents have not been detailed. The utility of vibrational spectroscopy for evaluation of keratin secondary structure after lipid removal is somewhat hampered by the native ceramide contribution to the Amide I and II band prior to solvent exposure. In SC samples it is therefore difficult to accurately access the contribution ceramides make to Amide I and II band intensities and position. In the current work, this concern is mostly overcome by acquiring spectra of isolated corneocytes in which the ceramide contribution to the Amide modes is substantially reduced. 2 −1 −1 −1 2 −1 13 −1 2 −1 −1 −1 12 12 8 The importance of the current experiments lies in the fact, that in studies of supposedly native protein structures in the SC, C/M is often used to remove lipid components. As shown in the current work, this procedure is not innocuous, but can induce irreversible changes in protein structure in corneocytes. Confocal Raman microscopy: delineation of skin regions from factor analysis Z λ λ Materials and methods 5 18 19 20 3 Materials and methods 21 3 3 3 4 3 −1 4 9 22 −1 trans 2 −1 2 −1 −1 4 23 3 4 3 Fig. 3 a bar −1 b Materials and methods Factor 1–5 c Dark blue green yellow orange red  Fig. 4 top bottom 3 a −1 −1 b −1 −1 trans c −1 −1 −1  3 −1 24 25 5 3 5 −1 24 25 5 5 5 Fig. 5 a b red dark blue b −1 −1 c −1 −1  Conclusions 3 3 5 2 Finally, it is feasible to begin to examine biochemical heterogeneity at a single cellular level. Issues such as the relationship of protein structure (either native or solvent-modified) to hydration levels or to the levels of natural moisturizing factors, in single cells may be profitably probed. In addition, changes in the spectra of cells from pathological states may be examined.