Uemura Takumi, Kawakami Ryosuke, Seki Hitomi, Yoshida Satoshi, Murakami Masamoto, Imamura Takeshi, Shingo Hadano, Watanabe Shigeru, Niko Yosuke
Research and Education Faculty, Multidisciplinary Science Cluster, Interdisciplinary Science Unit, Kochi University 2-5-1 Akebono-cho, Kochi-shi Kochi 780-8520 Japan
Department of Molecular Medicine for Pathogenesism, Graduate School of Medicine, Ehime University Shitsukawa Toon Ehime 791-0295 Japan.
Chem Sci. 2025 Jun 20. doi: 10.1039/d5sc03047a.
Fluorescent plasma membrane probes are indispensable tools for biological studies, enabling the visualization of the fine structure and dynamics of plasma membranes, and, by extension, the overall morphology of living cells. However, their use has been mostly limited to imaging cultured cells or fixed tissue slices. Indeed, few probes have been optimized for visualizing cellular morphologies in intact tissues or organs. Here, we report a new bright squaraine-based membrane probe, dSQ12AQ, which incorporates two anionic anchor groups (sulfonate and long alkyl chain) to ensure high water dispersibility without precipitation-even at concentrations exceeding 10 mg mL. This highly concentrated probe solution was intravenously administered to living mice without the need for dimethyl solfoxide or other solubilizing agents. Combined with two-photon microscopy, dSQ12AQ enabled clear visualization of whole-cell morphology , allowing dynamic imaging of flowing, rolling, and/or remaining stationary blood cells in the bone marrow vasculature. Moreover, dSQ12AQ extravasated from blood vessels, enabling further staining and visualization of cells in the perivascular bone marrow region. This extravasation was also observed in the hind paw skin, enabling clear visualization of keratinocytes in the epidermis, as well as fibroblasts and eccrine sweat duct cells in the dermis. These results highlight the potential of dSQ12AQ as a valuable tool for studies of various cellular processes and for investigation of refractory or poorly understood diseases and their treatments.
荧光质膜探针是生物学研究中不可或缺的工具，可实现质膜精细结构和动态变化的可视化，进而实现活细胞整体形态的可视化。然而，它们的应用大多局限于对培养细胞或固定组织切片进行成像。实际上，很少有探针针对完整组织或器官中细胞形态的可视化进行优化。在此，我们报告一种基于方酸菁的新型明亮膜探针dSQ12AQ，它含有两个阴离子锚定基团（磺酸根和长烷基链），即使在浓度超过10 mg/mL时也能确保高水分散性而不沉淀。这种高浓度的探针溶液无需二甲基亚砜或其他增溶剂即可静脉注射给活体小鼠。结合双光子显微镜，dSQ12AQ能够清晰地可视化全细胞形态，从而对骨髓脉管系统中流动、滚动和/或静止的血细胞进行动态成像。此外，dSQ12AQ从血管外渗，能够进一步对血管周围骨髓区域的细胞进行染色和可视化。在后爪皮肤中也观察到了这种外渗现象，从而能够清晰地可视化表皮中的角质形成细胞以及真皮中的成纤维细胞和外分泌汗腺细胞。这些结果突出了dSQ12AQ作为研究各种细胞过程以及难治性或了解甚少的疾病及其治疗方法的宝贵工具的潜力。
