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

Cryo-correlative light and electron microscopy (cryoCLEM) has evolved as a powerful strategy to high resolution imaging in the unperturbed hydrated state, and is now the number one technique for the 3D structural analysis of proteins in their cellular context using cryo-electron tomography (cryoET). In this approach fluorescence microscopy aids in localizing the area of interest, and cryogenic focused ion beam / scanning electron microscopy (cryoFIB/SEM) is used to prepare thin lamellae for cryoET. While cryoCLEM is now routinely applied to thin on grid lamellae extracted from plunge frozen 2D cell cultures, it cannot address questions related to more complex systems such as tissues and organoids. Here, 3D cryo correlative imaging of large volumes can close the resolution gap between cryo-light microscopy and cryoET, placing the sub-nanometer observations in a larger biological context. Until now, a few technological hurdles made 3D cryoCLEM a challenging, and unexplored approach. Here we demonstrate a deformation free cryoCLEM workflow for tissues, where we correlate 3D cryo-Airyscan confocal microscopy (cryoCACM) 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 of regions for cryoFIB/SEM volume imaging and cryo-lift out of lamella for cryoTEM.