Sample deposition onto cryo-EM grids: from sprays to jets and back

Area of Science:

• Structural biology
• Biophysics
• Microscopy

Background:

• Single-particle cryogenic electron microscopy (cryo-EM) has advanced significantly, but sample preparation remains a bottleneck.
• Traditional blotting methods can damage delicate protein samples and increase exposure to the air-water interface.
• Existing techniques are insufficient for many protein complexes, hindering high-resolution structural determination.

Purpose of the Study:

• To evaluate blotless sample deposition methods for cryo-EM grid preparation.
• To compare different droplet generation techniques for direct sample deposition.
• To optimize spray technology for reproducible, high-quality cryo-EM grids.

Main Methods:

• Utilized gas dynamic virtual nozzles for generating small, high-velocity droplets.
• Deposited droplets directly onto cryo-EM grids.
• Investigated sprayer design and operational parameters for improved grid quality.

Main Results:

• Gas dynamic virtual nozzles enabled sufficient droplet spreading for high-resolution cryo-EM imaging.
• Demonstrated the potential of spray technology as a viable blotless alternative.
• Identified key sprayer design and operational factors for reproducible grid preparation.

Conclusions:

• Blotless sample preparation using spray technology, specifically gas dynamic virtual nozzles, overcomes limitations of traditional blotting.
• This approach is promising for improving cryo-EM grid quality and enabling high-resolution structural studies of challenging samples.
• Further optimization of spray technology can enhance reproducibility and broaden its application in cryo-EM sample preparation.