Cryo-FIB workflow for imaging brain tissue via in situ cryo-electron microscopy

Ning, J.; Glausier, J. R.; Hsieh, C.; Schmelzer, T.; Buck, S. A.; Franks, J.; Hampton, C. M.; Lewis, D. A.; Marko, M.; Freyberg, Z.

doi:10.1101/2023.02.11.528064

bioRxiv · 10.1101/2023.02.11.528064

Cryo-FIB workflow for imaging brain tissue via in situ cryo-electron microscopy

Ning, J.·Glausier, J. R.·Hsieh, C.·Schmelzer, T.·Buck, S. A.·Franks, J.·Hampton, C. M.·Lewis, D. A.·Marko, M.·Freyberg, Z.

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Abstract

Cryo-electron microscopy (cryo-EM) enables the study of protein complexes, cytoskeletal elements, and organelles in three dimensions without the use of chemical fixation. Most cryo-EM studies focus on vitreously frozen individual cells separated from their native tissue contexts. This reliance on imaging of single cells is primarily due to technical challenges associated with preparing fresh tissue sections at a thinness sufficient for visualization via cryo-EM. Highly heterogenous and specialized tissues, such as brain, are especially affected by this limitation as the cellular, subcellular, and synaptic milieus can significantly vary across neuroanatomical locations. To address this limitation, we established new instrumentation and a workflow that consists of: 1) high-pressure freezing of fresh brain tissue; 2) tissue trimming followed by cryo-focused ion beam milling via the H-bar approach to generate ultrathin lamellae; and 3) cryo-EM imaging. Here, we apply this workflow to visualize the fine ultrastructural details of organelles, as well as cytoskeletal and synaptic elements that comprise the cortical neuropil within fresh, unfixed mouse brain tissue. Moreover, we present initial studies that apply principles of the above workflow to the analysis of postmortem human brain tissue. Overall, our work integrates the strengths of cryo-electron microscopy and tissue-based approaches to produce a generalizable workflow capable of visualizing subcellular structures within complex tissue environments.

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Ning, J., Glausier, J. R., Hsieh, C., Schmelzer, T., Buck, S. A., Franks, J., Hampton, C. M., Lewis, D. A., Marko, M., Freyberg, Z.. 2023-02-12. Cryo-FIB workflow for imaging brain tissue via in situ cryo-electron microscopy. https://doi.org/10.1101/2023.02.11.528064

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