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bioRxiv · 10.1101/2022.07.31.502212

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

de Beer, M.·Daviran, D.·Roverts, R.·Rutten, L.·Sanchez, e. M.·Metz, J.·Sommerdijk, n.·Akiva, A.
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Abstract

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.

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de Beer, M., Daviran, D., Roverts, R., Rutten, L., Sanchez, e. M., Metz, J., Sommerdijk, n., Akiva, A.. 2022-08-02. Precise targeting for 3D cryo-correlative light and electron microscopy volume imaging of tissues using a FinderTOP. https://doi.org/10.1101/2022.07.31.502212

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