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

Area of Science:

• Cell Biology
• Microscopy
• Biophysics

Background:

• Traditional cell imaging requires chemical fixation and staining, which can introduce artifacts.
• Three-dimensional (3D) visualization of vitrified cells offers a path to preserving native cellular structures.
• Subcellular complex visualization is crucial for understanding cellular function.

Purpose of the Study:

• To develop and validate an integrated imaging pipeline for multi-scale 3D visualization of vitrified cells.
• To demonstrate the pipeline's capability in revealing subcellular structures and organelle distribution.
• To assess the potential for large-scale phenotypic studies of frozen-hydrated specimens.

Main Methods:

• Integration of three imaging modalities: cryo-fluorescence confocal microscopy, volume cryo-focused ion beam scanning electron microscopy, and transmission cryo-electron tomography.
• Cryogenic temperature imaging of the same specimen.
• Proof-of-concept benchmark using heat-shocked yeast cells.

Main Results:

• Successful 3D visualization of organelles and subcellular structures in whole yeast cells.
• Detailed ultrastructure of protein inclusions and their recruitment of fluorescently-labeled chaperone Hsp104.
• Demonstration of integrated imaging across different scales.

Conclusions:

• The developed pipeline enables artifact-free 3D visualization of vitrified cells at multiple scales.
• This workflow can be applied to various cell types for studying healthy and diseased conditions.
• The method facilitates large-scale phenotypic studies of frozen-hydrated specimens.