Work

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

Year: 2024

Type: article

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.

Source: Micron

DOI: 10.1016/j.micron.2024.103745

Authors: Iain Harley, Francesca Mazzotta, Xhorxhina Shaulli, Frank Scheffold, Katharina Landfester, Ingo Lieberwirth

Institutions: Max Planck Institute for Polymer Research, University of Fribourg

Language: English

Topic: Advanced Electron Microscopy Techniques and Applications

Subfield: Structural Biology

Field: Biochemistry, Genetics and Molecular Biology

Domain: Life Sciences

Open Access status: hybrid

Estimated APC paid: $3,310

Funders: Horizon 2020 Framework Programme, Horizon 2020, H2020 Marie Skłodowska-Curie Actions, HORIZON EUROPE Marie Sklodowska-Curie Actions