Ultra-stable, photon-efficient cryogenic super-resolution fluorescence imaging for visualizing vitrified biological samples with molecular-scale resolution

摘要 低温电子显微镜(CryoEM)和低温电子断层扫描(CryoET)是最强大的 用于可视化处于接近自然状态的大分子组装及其 细胞内的功能上下文；然而，这种无区别/非特定的对比 技术通常使识别感兴趣区域和特定的分子靶标变得复杂 高分辨率的低温EM/低温ET数据集。超分辨(SR)荧光方法有 精致的分子特异性，但SR技术仅实现了有限的空间分辨率 (~20-60 nm半高宽)在低温下对玻璃化生物样品成像时。这个 拟议的项目将创造用于成像的新的超稳定、光子效率的低温SR方法 玻璃化生物标本，具有真正的分子尺度分辨率(&lt；2-3 nm半高宽)，关闭 接近(亚)纳米分辨率的低温电子显微镜/低温电子冷凝器。空间分辨率的提高 将被用来进一步发展强大的相关光电子显微镜 (Clem)方法，最终将能够阐明许多关键 细胞内的大分子过程。

ABSTRACT Cryogenic electron microscopy (CryoEM) and cryo-electron tomography (CryoET) are powerful techniques for visualizing macromolecular assemblies at a near-native state and in their functional context inside cells; however the undiscriminating/non-specific contrast of such techniques often complicates identifying regions of interest and specific molecular targets in high-resolution cryoEM/cryoET datasets. Super-resolution (SR) fluorescence methods have exquisite molecular specificity, but SR techniques have only achieved limited spatial resolutions (~20-60 nm FWHM) when imaging vitrified biological samples at cryogenic temperatures. The proposed project will create new ultra-stable, photon efficient cryogenic SR methods for imaging vitrified biological specimens with true molecular-scale resolution (<2-3 nm FWHM), closing the gap towards the (sub-)nanometer resolution of cryoEM/cryoET. The increased spatial resolution will be leveraged for further development of powerful correlative light-electron microscopy (CLEM) approaches that will ultimately enable elucidating the structural basis for many critical macromolecular processes inside cells.