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

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-labeled 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.

Keywords: Airyscan microscopy; Hsp104 chaperone; Saccharomyces cerevisiae (yeast); amyloid; cryo mill and view (cryoMAV).; cryo-correlative light and electron microscopy (cryoCLEM); cryo-electron tomography (cryoET); protein aggregation; protein misfolding; volume cryo-focused ion bean scanning electron microscopy (cryoFIB-SEM).