In situ visualization of intracellular morphology of epidermal cells using stimulated Raman scattering microscopy

Abstract

Visualization of epidermal cells is important because the differentiation patterns of keratinocytes (KCs) are considered to be related to the functions and condition of skin. Optical microscopy has been widely used to investigate epidermal cells, but its applicability is still limited because of the need for sample fixation and staining. Here, we report our staining-free observation of epidermal cells in both tissue and culture by stimulated Raman scattering (SRS) microscopy that provides molecular vibrational contrast. SRS allowed us to observe a variety of cellular morphologies in skin tissue, including ladder-like structures in the spinous layer, enucleation of KCs in the granular layer, and three-dimensional cell column structures in the stratum corneum. We noticed that some cells in the spinous layer had a brighter signal in the cytoplasm than KCs. To examine the relevance of the observation of epidermal layers, we also observed cultured epidermal cells, including KCs at various differentiation stages, melanocytes, and Langerhans cell-like cells. Their SRS images also demonstrated various morphologies, suggesting that the morphological differences observed in tissue corresponded to the cell lineage. These results indicate the possible application of SRS microscopy to dermatological investigation of cell lineages and types in the epidermis by cellular-level analysis.

Label-free stimulated Raman scattering microscopy visualizes changes in intracellular morphology during human epidermal keratinocyte differentiation

“…Figure 2 shows typical molecular vibrational images of each epidermal layer in optical horizontal unsliced cross-sections of skin from a 70-year-old Caucasian female. Characteristic intracellular morphologies of each epidermal layer were observed without labelling, similar to the previously reported preliminary results of a 52-year-old Caucasian female at various depths 39 . In the SB and SS, nuclei were observed as a low intensity area both at 2850 cm −1 , reflecting mainly lipids (Fig.…”

“…2c), thus reflecting the existence of intercellular lipids. In addition, visualization of the SC without distinct layers was observed in some skin (as for this subject), a feature not observed in the previously reported 52-year-old subject 39 .

Figure 2Typical molecular vibrational images in horizontal cross-sections of each epidermal layer of abdominal skin of a 70-year-old subject. ( a ) Images at 2850 cm −1 mainly reflect lipids, while those at ( b ) 2930 cm −1 mainly reflect proteins.

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“…We previously reported our preliminary label-free observations of epidermal cells in both cell culture and tissue using SRS microscopy. Our results indicated the potential applications of SRS microscopy to the dermatological investigation of cell lineages and types by cellular-level analysis 39 . However, elucidation of epidermal KC differentiation has not been fully accomplished in humans even using SRS microscopy.…”

“…The power of Ti:S and YbF lasers at the input of laser scanners was 110–120 mW each, and that in the sample plane was estimated to be −60 mW each. Horizontal cross-section SRS images (80 × 80 μm; 500 × 500 pixels; transmission mode; horizontal spatial resolution of −0.5 μm) were acquired in the wavenumber range of 2800 to 3100 cm −1 , corresponding to the C–H stretching region 20,21,39 . The frame rate of the microscope was 30 frames/s.…”

Changes in facial moisture distribution and feelings of moisture/dryness among various environmental temperatures and humidities in summer and winter

“…Perhaps it would also be necessary to compare the epidermal morphology. 24,25 As the skin's environmental conditions vary depending on the area of residence, residential building, season, and clothing, our results indicate the importance of environmental conditions when skin conditions are being measured. We usually evaluate skin conditions at 23-25°C and 40%-50% RH.…”