Correlated cryo-fluorescence and cryo-electron microscopy with high spatial precision and improved sensitivity

Area of Science:

• Structural biology
• Microscopy techniques
• Biophysics

Background:

• Correlative microscopy combines fluorescence and electron microscopy for enhanced sample analysis.
• Cryo-electron microscopy (cryo-EM) requires vitrified samples for high-resolution structural determination.
• Performing fluorescence microscopy at liquid-nitrogen temperatures is crucial for correlative cryo-EM.

Purpose of the Study:

• To adapt a cryo-light microscopy stage for high-numerical aperture objectives.
• To develop a workflow for correlative cryo-fluorescence and cryo-electron microscopy.
• To enable precise localization of fluorescently labeled targets in vitrified samples.

Main Methods:

• Adaptation of a cryo-light microscopy stage for high-numerical aperture objectives.
• Development of a correlative cryo-fluorescence and cryo-electron microscopy workflow.
• Implementation of a fiducial bead-based image correlation procedure.

Main Results:

• Achieved high-sensitivity and high-resolution fluorescence microscopy of vitrified samples.
• Successfully located fluorescent bacteriophages in cryo-electron microscopy images with ~50 nm accuracy.
• Demonstrated precise identification and localization of objects based on fluorescent signals.

Conclusions:

• The adapted cryo-light microscopy stage facilitates high-quality fluorescence imaging of vitrified samples.
• The developed correlative workflow enables accurate targeting for high-resolution structural analysis.
• This technique provides a powerful tool for precisely locating and studying specific structures in cryo-EM studies.