Two-photon, excitation fluorescent microscopy featuring autofluorescence or immunofluorescence, combined with optical clearance using a transparency-enhancing technique, allows deep imaging of three-dimensional (3D) skin structures. However, it remains difficult to obtain high-quality images of individual cells or 3D structures. We combined a new dye with a transparency-enhancing technology and performed high-quality structural analysis of human epidermal structures, especially the acrosyringium. Human fingertip skin samples were collected, formalin-fixed, embedded in both frozen and paraffin blocks, sliced, stained with propidium iodide, optically cleared using a transparency-enhancing technique, and stained with a new fluorescent, solvatochromic pyrene probe. Microscopy revealed fine skin features and detailed epidermal structures including the stratum corneum (horny layer), keratinocytes, eccrine sweat glands, and peripheral nerves. Three-dimensional reconstruction of an entire acrosyringium was possible in one sample. This new fluorescence microscopy technique yields high-quality epidermal images and will aid in histopathological analyses of skin disorders.

具有自发荧光或免疫荧光功能的两光子激发荧光显微镜，结合使用透明增强技术的光学清除率，可以对三维（3D）皮肤结构进行深度成像。但是，仍然难以获得单个细胞或3D结构的高质量图像。我们将一种新型染料与增强透明度的技术相结合，并对人的表皮结构（尤其是丙烯醛）进行了高质量的结构分析。收集人的指尖皮肤样品，福尔马林固定，包埋在冷冻和石蜡块中，切片，用碘化丙啶染色，使用透明增强技术进行光学清除，并用新的荧光增溶剂性pyr探针染色。显微镜检查显示出良好的皮肤特征和详细的表皮结构，包括角质层（角质层），角质形成细胞，内分泌汗腺和周围神经。可以在一个样品中对整个丙烯腈进行三维重建。这项新的荧光显微镜技术可产生高质量的表皮图像，并有助于皮肤疾病的组织病理学分析。