Despite more than 20 years of work since the lipid raft concept was proposed, the existence of these nanostructures remains highly controversial due to the lack of noninvasive methods to investigate their native nanorganization in living unperturbed cells. There is an unmet need for probes for direct imaging of nanoscale membrane dynamics with high spatial and temporal resolution in living cells. In this paper, a bioorthogonal-based cholesterol probe (chol-N₃) is developed that, combined with nanoscopy, becomes a new powerful method for direct visualization and characterization of lipid raft at unprecedented resolution in living cells. The chol-N₃ probe mimics cholesterol in synthetic and cellular membranes without perturbation. When combined with live-cell super-resolution microscopy, chol-N₃ demonstrates the existence of cholesterol-rich nanodomains of <50 nm at the plasma membrane of resting living cells. Using this tool, the lipid membrane structure of such subdiffraction limit domains is identified, and the nanoscale spatiotemporal organization of cholesterol in the plasma membrane of living cells reveals multiple cholesterol diffusion modes at different spatial localizations. Finally, imaging across thick organ samples outlines the potential of this new method to address essential biological questions that were previously beyond reach.

中文翻译：

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使用基于生物正交的胆固醇探针的超分辨率显微镜为活细胞中的纳米级脂质异质性成像提供了前所未有的能力

尽管自提出脂筏概念以来已经进行了 20 多年的工作，但由于缺乏非侵入性方法来研究它们在未受干扰的活细胞中的天然纳米结构，这些纳米结构的存在仍然存在很大争议。对于在活细胞中具有高空间和时间分辨率的纳米级膜动力学直接成像的探针存在未满足的需求。在本文中，开发了一种基于生物正交的胆固醇探针 (chol-N ₃ )，该探针与纳米镜检查相结合，成为在活细胞中以前所未有的分辨率直接可视化和表征脂筏的一种新的强大方法。chol-N ₃探针模拟合成和细胞膜中的胆固醇而不受干扰。当与活细胞超分辨率显微镜相结合时，chol-N ₃证明在静息活细胞的质膜上存在 <50 nm 的富含胆固醇的纳米结构域。使用该工具，确定了此类亚衍射极限域的脂质膜结构，并且活细胞质膜中胆固醇的纳米级时空组织揭示了不同空间定位下的多种胆固醇扩散模式。最后，对厚器官样本的成像概述了这种新方法在解决以前无法解决的基本生物学问题方面的潜力。