2024Harley 40
Citation
I. Harley, F. Mazzotta, X. Shaulli, F. Scheffold, K. Landfester, and I. Lieberwirth, “Practical considerations for plunge freezing samples over 40° C for Cryo-EM,” Micron, p. 103745, 2024.
Abstract
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.
Keywords
Links
https://www.sciencedirect.com/science/article/pii/S0968432824001628