Correlative cryo-microscopy pipelines for in situ cellular studies

Area of Science:

• Cell Biology
• Structural Biology
• Microscopy

Background:

• Correlative cryo-light and electron microscopy (cryoCLEM) is crucial for visualizing cellular structures at high resolution.
• Identifying specific targets within the complex cellular environment is a challenge in cryo electron tomography (cryoET).
• Existing cryoCLEM methods often rely on cryo focused ion beam (cryo FIB) milling, which can be destructive.

Purpose of the Study:

• To present detailed protocols for two cryoCLEM approaches that avoid cryo FIB milling.
• To enable in situ studies of adherent cells at the single-cell level.
• To image fluorescently labeled cellular structures within their unperturbed cellular environment.

Main Methods:

• Developed a complete cryogenic pipeline for simultaneous fluorescence and electron imaging on frozen-hydrated samples.
• Established a hybrid cryoCLEM approach involving room temperature fluorescence imaging followed by rapid freezing and cryoEM imaging.
• Applied these methods to image structures like cell protrusions and peripheral organelles up to 500nm thick.

Main Results:

• Successfully demonstrated two cryoCLEM protocols that bypass the need for cryo FIB milling.
• Enabled high-resolution imaging of specific cellular targets in their native, unperturbed state.
• Facilitated visualization of peripheral cellular structures and organelles without sample damage.

Conclusions:

• The presented cryoCLEM protocols offer alternative, non-destructive methods for high-resolution cellular imaging.
• These approaches enhance the ability to study cellular structures in situ, particularly in peripheral regions.
• The methods are valuable for investigating fluorescently labeled targets in adherent cells without invasive thinning procedures.