Precise targeting for 3D cryo-correlative light and electron microscopy volume imaging of tissues using a FinderTOP

• Published in: Communications biology Vol. 6; no. 1; p. 510
• Main Authors: de Beer, Marit, Daviran, Deniz, Roverts, Rona, Rutten, Luco, Macías-Sánchez, Elena, Metz, Juriaan R, Sommerdijk, Nico, Akiva, Anat
• Format: Journal Article
• Language: English
• Published: England Nature Publishing Group 11.05.2023; Nature Publishing Group UK; Nature Portfolio
• Subjects: Biology; Confocal microscopy; Cryoelectron Microscopy - methods; Electron microscopes; Fluorescence microscopy; Freezing; Lamellae; Light microscopy; Microscopy, Confocal; Microscopy, Electron; Microscopy, Fluorescence - methods; Organoids; Scanning electron microscopy
• ISSN: 2399-3642; 2399-3642
• 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.