Ultra-stable super-resolution fluorescence cryo-microscopy for correlative light and electron cryo-microscopy

Area of Science:

• Biophysics
• Microscopy
• Cell Biology

Background:

• Correlative light and electron microscopy (CLEM) is vital for cellular research.
• Dedicated cryo-fluorescence microscopes (cryo-FM) are essential for cryo-CLEM.
• Previous cryo-FMs faced limitations in stability for long-term imaging.

Purpose of the Study:

• To develop and characterize an ultra-stable super-resolution cryo-fluorescence microscope (cryo-FM).
• To assess the thermal and mechanical stability of the new cryo-FM system.
• To demonstrate its suitability for advanced cryo-CLEM applications.

Main Methods:

• Construction of an ultra-stable cryo-fluorescence microscope.
• Measurement of temperature fluctuations over 10 hours (<0.06 K).
• Assessment of 3D mechanical drift over 5 hours (<200 nm).
• Demonstration of super-resolution imaging with single molecule localization accuracy (~13.0 nm).

Main Results:

• The cryo-FM system demonstrated exceptional thermal stability (<0.06 K over 10 h).
• Exceptional mechanical stability was achieved (<200 nm 3D drift over 5 h).
• High super-resolution imaging performance was confirmed with ~13.0 nm localization accuracy.

Conclusions:

• The developed cryo-FM offers superior stability for demanding microscopy techniques.
• This system is ideal for long-term observations in single molecule localization microscopy (SMLM).
• It significantly advances capabilities for cryogenic super-resolution correlative light and electron microscopy (csCLEM).