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bioRxiv · 10.1101/2022.11.19.517200

Bridging the light-electron resolution gap with correlative cryo-SRRF and dual-axis cryo-STEM tomography

Kirchweger, P.·Mullick, D.·Sawin, P. P.·Wolf, S. G.·Elbaum, M.
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Abstract

Cryo-electron tomography (cryo-ET) is the prime method for cell biological studies in three dimensions (3D) at high resolution. We have introduced cryo-scanning transmission electron tomography (CSTET), which can access larger 3D volumes, on the scale of 1 micron, making it ideal to study organelles and their interactions in situ. Here we introduce two relevant advances: a) we demonstrate the utility of multi-color super-resolution radial fluctuation light microscopy under cryogenic conditions (cryo-SRRF), and b) we extend the use of deconvolution processing for dual-axis CSTET data. We show that cryo-SRRF nanoscopy is able to reach resolutions in the range of 100 nm, using commonly available fluorophores and a conventional widefield microscope for cryo-correlative light-electron microscopy (cryo-CLEM). Such resolution aids in precisely identifying regions of interest before tomographic acquisition and enhances precision in localizing features of interest within the 3D reconstruction. Dual-axis CSTET tilt series data and application of entropy regularized deconvolution during post-processing results in close-to isotropic resolution in the reconstruction without averaging. We show individual protein densities in a mitochondrion-ER contact in a cell region 850 nm thick. The integration of cryo-SRRF with deconvolved dual-axis CSTET provides a versatile workflow for studying unique objects in a cell.

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Kirchweger, P., Mullick, D., Sawin, P. P., Wolf, S. G., Elbaum, M.. 2022-11-19. Bridging the light-electron resolution gap with correlative cryo-SRRF and dual-axis cryo-STEM tomography. https://doi.org/10.1101/2022.11.19.517200

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