Vibrational microscopy and imaging of skin: from single cells to intact tissue

Abstract

Vibrational microscopy and imaging offer several advantages for a variety of dermatological applications, ranging from studies of isolated single cells (corneocytes) to characterization of endogenous components in intact tissue. Two applications are described to illustrate the power of these techniques for skin research. First, the feasibility of tracking structural alterations in the components of individual corneocytes is demonstrated. Two solvents, DMSO and chloroform/methanol, commonly used in dermatological research, are shown to induce large reversible alterations (alpha-helix to beta-sheet) in the secondary structure of keratin in isolated corneocytes. Second, factor analysis of image planes acquired with confocal Raman microscopy to a depth of 70 microm in intact pigskin, demonstrates the delineation of specific skin regions. Two particular components that are difficult to identify by other means were observed in the epidermis. One small region was formed from a conformationally ordered lipid phase containing cholesterol. In addition, the presence of nucleated cells in the tissue (most likely keratinocytes) was revealed by the spectral signatures of the phosphodiester and cytosine moieties of cellular DNA.

Fig. 1

Vibrational microscopy of human corneocytes. a IR point mode spectrum (1,150–3,600 cm⁻¹ region) acquired using a 40 μm² aperture of a single corneocyte isolated from the third sequential tape strip applied to human forearm skin. The inset shows the optical image of the corneocyte (bar = 10 μm). b Raman spectrum (800–1,720 cm⁻¹ region) of a similarly isolated corneocyte displaying bands characteristic of the lipid and protein (keratin) components

Fig. 2

Solvent-induced conformational changes in the keratin of single corneocytes. a and b IR and Raman spectra, respectively, of an isolated corneocyte prior to and following immersion in DMSO, and after an overnight rehydration period (top to bottom, respectively). c and d IR and Raman spectra, respectively, of a corneocyte prior to and following C/M treatment, and after an overnight rehydration period (top to bottom, respectively). In the IR spectra (a and c), α-helical and β-sheet components of the Amide I (ca. 1,650 cm⁻¹) and II (ca. 1,500–1,550 cm⁻¹) bands are noted. In the Raman spectra (b and d), α-helical and β-sheet components of the Amide I are marked along with disordered constituents at 1,685 cm⁻¹. One component of the Amide III band is marked at 1,240 cm⁻¹

Fig. 3

a An optical micrograph of a microtomed 5-μm-thick pigskin section acquired using the PerkinElmer Spotlight (bar = 10 μm). The results of factor analysis in the 800–1,150 cm⁻¹ region conducted on a confocal Raman map acquired from intact pigskin. b The five distinct factor loadings generated by the ISys score segregation algorithm (see Materials and methods) are offset and labeled Factor 1–5. c The spatial distribution of factor scores for each of the loadings as marked. Dark blue indicates the lowest score with green, yellow, orange, and red indicative of progressively higher scores. Factor loadings and score images have been assigned to different micro regions in skin as described in the text

Fig. 4

Averaged Raman spectra from within the stratum corneum (top) and lipid (bottom) regions as noted in Fig. 3b and c as Factor 1 and 2, respectively. a The 590–790 cm⁻¹ region with bands assigned to cholesterol marked at 605 and 700 cm⁻¹. b The 800–1,380 cm⁻¹ region displays several bands (1,058, 1,130, and 1,296 cm⁻¹) characteristic for ordered (all-trans) lipid acyl chains. c The CH stretching region (2,800–3,030 cm⁻¹) with methylene symmetric (2,850 cm⁻¹) and asymmetric (2,880 cm⁻¹) stretching modes noted. The frequencies and relative intensity of the bands are consistent with well-ordered lipid acyl chains

Fig. 5

The results of factor analysis conducted in two separate spectral regions for a confocal Raman map of pigskin after stratum corneum removal by tape stripping. Both analyses were performed allowing the generation of only two factors. In each case, one factor contained a spectral feature specific to DNA and the other mapped in a fairly uniform way throughout the epidermal region. a Factor score image for the factor-labeled nuclei shown in (b) with red corresponding to the highest score and dark blue to the lowest. b Factor loadings for the 600–820 cm⁻¹ region with a distinctive vibrational band at approximately 785 cm⁻¹ assigned to cytosine. c Factor loadings for the 800–1,150 ⁻¹ region with a second characteristic DNA vibrational band due to the phosphodiester backbone stretching mode, at about 1,090 cm⁻¹ noted