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bioRxiv · 10.1101/2020.05.21.107771

Multi-Scale 3D Cryo-Correlative Microscopy for Vitrified Cells

Wu, G.-H.·Mitchell, P. G.·Galaz-Montoya, J. G.·Hecksel, C. W.·Sontag, E. M.·Gangadharan, V.·Marshman, J.·Mankus, D.·Bisher, M. E.·Lytton-Jean, A. K.·Frydman, J.·Czymmek, K.·Chiu, W.
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Abstract

Three-dimensional (3D) visualization of vitrified cells can uncover structures of subcellular complexes without chemical fixation or staining. Here, we present a pipeline integrating three imaging modalities to visualize the same specimen at cryogenic temperature at different scales: cryo-fluorescence confocal microscopy, volume cryo-focused ion beam scanning electron microscopy, and transmission cryo-electron tomography. Our proof-of-concept benchmark revealed the 3D distribution of organelles and subcellular structures in whole heat-shocked yeast cells, including the ultrastructure of protein inclusions that recruit fluorescently-labelled chaperone Hsp104. Since our workflow efficiently integrates imaging at three different scales and can be applied to other types of cells, it could be used for large-scale phenotypic studies of frozen-hydrated specimens in a variety of healthy and diseased conditions with and without treatments.

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Wu, G.-H., Mitchell, P. G., Galaz-Montoya, J. G., Hecksel, C. W., Sontag, E. M., Gangadharan, V., Marshman, J., Mankus, D., Bisher, M. E., Lytton-Jean, A. K., Frydman, J., Czymmek, K., Chiu, W.. 2020-05-22. Multi-Scale 3D Cryo-Correlative Microscopy for Vitrified Cells. https://doi.org/10.1101/2020.05.21.107771

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