Super-Resolution Microscopy Using a Bioorthogonal-Based Cholesterol Probe Provides Unprecedented Capabilities for Imaging Nanoscale Lipid Heterogeneity in Living Cells.
Super-Resolution Microscopy Using a Bioorthogonal-Based Cholesterol Probe Provides Unprecedented Capabilities for Imaging Nanoscale Lipid Heterogeneity in Living Cells.
复制标题
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纳米级脂质异质性成像提供了前所未有的能力。
DOI：
10.1002/smtd.202100430
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发表时间：
2021
期刊：
影响因子：
12.4
通讯作者：
Lorizate M
中科院分区：
文献类型：
--
作者：
Lorizate M
尽管自脂筏概念提出以来已有20多年的工作，但由于缺乏非侵入性方法来研究其在未受干扰的活细胞中的天然纳米组织，这些纳米结构的存在仍然存在很大争议。对于用于在活细胞中以高空间和时间分辨率直接成像纳米级膜动力学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‐N3），与纳米显微镜相结合，成为一种新的强大的方法，用于在活细胞中以前所未有的分辨率直接可视化和表征脂筏。chol-N3探针模拟合成膜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结合时，chol-N3证明了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。使用这个工具，这样的subdiffraction极限域的脂质膜结构被确定，和胆固醇在活细胞的质膜中的纳米级时空组织揭示了多种胆固醇扩散模式在不同的空间定位。最后，厚器官样本的成像概述了这种新方法解决以前无法解决的基本生物学问题的潜力。
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‐N3probe mimics cholesterol in synthetic and cellular membranes without perturbation. When combined with live‐cell super‐resolution microscopy, chol‐N3demonstrates 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.
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