Microscale Fluid Behavior during Cryo-EM Sample Blotting.

Area of Science:

• Cryo-electron microscopy sample preparation
• Vitrification techniques
• Biophysical imaging

Background:

• Blotting has been the standard for preparing aqueous samples for single-particle electron cryo-microscopy (cryo-EM) for over 30 years.
• This method involves pressing absorbent filter paper against the specimen grid to remove excess liquid before vitrification.
• However, blotting often results in vitreous ice of inconsistent thickness, hindering high-resolution imaging.

Purpose of the Study:

• To investigate the underlying reasons for inconsistent ice thickness in cryo-EM sample preparation using the standard blotting technique.
• To visualize and understand the liquid film dynamics during and after blotting.
• To identify potential improvements for more uniform sample preparation.

Main Methods:

• High-speed interference contrast microscopy was employed to observe liquid film behavior during blotting.
• The study focused on the interaction between filter paper fibers and the aqueous sample on a hydrophilic surface.
• Analysis of liquid film thickness variations before and after filter paper removal.

Main Results:

• The irregular fiber pattern of filter paper creates variable boundaries during liquid removal, leading to nonuniform film thickness.
• The liquid film's nonuniformity persists even after the filter paper is pulled away from the grid.
• These dynamic processes directly contribute to the formation of uneven vitreous ice after vitrification.

Conclusions:

• The standard blotting technique's reliance on direct filter paper contact is a primary cause of variable ice thickness in cryo-EM.
• Understanding these liquid dynamics is crucial for optimizing sample preparation protocols.
• Exploring blotting-free or indirect contact thinning methods could lead to more repeatable and uniform cryo-EM samples.