Practical considerations for plunge freezing samples over 40 °C for Cryo-EM

Area of Science:

• Structural biology
• Biophysics
• Materials science

Background:

• Cryo-electron microscopy (cryo-EM) allows sample examination in native, hydrated states via vitrification.
• Visualizing temperature-responsive samples under functional conditions requires specialized cryo-EM techniques.
• Plunge-freezing at elevated temperatures (>40 °C) presents challenges like condensation and temperature instability.

Purpose of the Study:

• To address challenges in plunge-freezing temperature-responsive samples at elevated temperatures.
• To provide practical strategies for minimizing condensation and temperature fluctuations.
• To improve sample preservation and grid reproducibility for cryo-EM analysis.

Main Methods:

• Preheating essential equipment used in the plunge-freezing process.
• Reducing humidity within the blotting chamber.
• Minimizing blotting times during sample preparation.

Main Results:

• Developed and demonstrated practical strategies for plunge-freezing samples at elevated temperatures.
• Successfully minimized condensation and reduced temperature fluctuations.
• Achieved improved sample preservation and grid reproducibility.

Conclusions:

• Effective strategies for plunge-freezing temperature-responsive samples at elevated temperatures are crucial for cryo-EM.
• Optimized sample preparation enhances structural insights into dynamic biological and material systems.
• The presented methods facilitate the study of temperature-induced structural transitions, exemplified by poly(N-isopropylacrylamide) microgels.