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

Cryo-EM is now an established tool for examining samples in their native, hydrated states-a leap made possible by vitrification. Utilising this sample preparation method to directly visualise temperature-responsive samples allows for deeper insights into their structural behaviours under functional conditions. This requires samples to be plunge-frozen at elevated temperatures and presents additional challenges, including condensation within the blotting chamber and difficulties in maintaining a stable sample temperatures. Here, we address these challenges and suggest practical strategies to minimise condensation and reduce temperature fluctuations during the plunge-freezing of samples at elevated temperatures (>40 °C). By preheating equipment and reducing chamber humidity and blotting times, we can improve sample preservation and grid reproducibility. These considerations are then demonstrated on poly(N-isopropylacrylamide) microgels, which exhibit a volume phase transition due to temperature changes.