Electron Microscopy Center, Radboud Technology Center Microscopy, Radboud University Medical Center, Nijmegen, The Netherlands.
Department of Medical Biosciences, Radboud University Medical Center, Nijmegen, The Netherlands.
Commun Biol. 2023 May 11;6(1):510. doi: 10.1038/s42003-023-04887-y.
Cryo-correlative light and electron microscopy (cryoCLEM) is a powerful strategy to high resolution imaging in the unperturbed hydrated state. In this approach fluorescence microscopy aids localizing the area of interest, and cryogenic focused ion beam/scanning electron microscopy (cryoFIB/SEM) allows preparation of thin cryo-lamellae for cryoET. However, the current method cannot be accurately applied on bulky (3D) samples such as tissues and organoids. 3D cryo-correlative imaging of large volumes is needed to close the resolution gap between cryo-light microscopy and cryoET, placing sub-nanometer observations in a larger biological context. Currently technological hurdles render 3D cryoCLEM an unexplored approach. Here we demonstrate a cryoCLEM workflow for tissues, correlating cryo-Airyscan confocal microscopy with 3D cryoFIB/SEM volume imaging. Accurate correlation is achieved by imprinting a FinderTOP pattern in the sample surface during high pressure freezing, and allows precise targeting for cryoFIB/SEM volume imaging.
冷冻相关的光镜和电子显微镜（cryoCLEM）是一种在未受干扰的水合状态下进行高分辨率成像的强大策略。在这种方法中，荧光显微镜有助于定位感兴趣的区域，而低温聚焦离子束/扫描电子显微镜（cryoFIB/SEM）允许制备用于 cryoET 的薄 cryo 薄片。然而，当前的方法不能准确地应用于体积较大（3D）的样品，如组织和类器官。需要对大体积进行 3D 冷冻相关成像，以缩小 cryo- 光镜和 cryoET 之间的分辨率差距，将亚纳米级观察置于更大的生物学背景下。目前，技术障碍使得 3D cryoCLEM 成为一种未探索的方法。在这里，我们展示了一种用于组织的 cryoCLEM 工作流程，将 cryo-Airyscan 共聚焦显微镜与 3D cryoFIB/SEM 体积成像相关联。通过在高压冷冻过程中在样品表面压印 FinderTOP 图案来实现精确的相关性，这允许对 cryoFIB/SEM 体积成像进行精确的靶向。
