Shiseido Global Innovation Center, 2-2-1 Hayabuchi, Tsuzuki-ku, Yokohama, Kanagawa 224-0025, Japan.
The University of Tokyo, Department of Electrical Engineering and Information Systems, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan.
J Biomed Opt. 2016 Aug 1;21(8):86017. doi: 10.1117/1.JBO.21.8.086017.
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.
表皮细胞的可视化很重要，因为角朊细胞（keratinocytes，KCs）的分化模式被认为与皮肤的功能和状况有关。光学显微镜已被广泛用于研究表皮细胞，但由于需要对样本进行固定和染色，其适用性仍然有限。在这里，我们报告了我们通过受激拉曼散射（stimulated Raman scattering，SRS）显微镜对组织和培养物中的表皮细胞进行无染色观察的结果，该显微镜提供了分子振动对比。SRS 使我们能够观察到皮肤组织中各种细胞形态，包括棘层中的梯状结构、颗粒层中 KC 的去核以及角质层中的三维细胞柱结构。我们注意到棘层中的一些细胞在细胞质中的信号比 KC 更亮。为了检查观察表皮层的相关性，我们还观察了培养的表皮细胞，包括处于不同分化阶段的 KC、黑素细胞和成纤维细胞样细胞。它们的 SRS 图像也显示出各种形态，表明组织中观察到的形态差异与细胞谱系相对应。这些结果表明，SRS 显微镜可能适用于通过细胞水平分析对表皮中的细胞谱系和类型进行皮肤科研究。
