Ice thickness control and measurement in the VitroJet for time-efficient single particle structure determination

Area of Science:

• Structural Biology
• Biophysics
• Microscopy Techniques

Background:

• Vitreous ice embedding is crucial for cryo-electron microscopy (cryo-EM) structure determination.
• Accurate ice thickness control is essential but currently involves time-consuming preparatory steps.
• Optimizing ice thickness enhances data quality and efficiency in cryo-EM workflows.

Purpose of the Study:

• To develop a routine for measuring ice thickness during sample preparation for cryo-EM.
• To enable precise tuning and measurement of ice thickness within the optimal range for single particle analysis.
• To demonstrate the impact of controlled ice thickness on cryo-EM structure determination efficiency.

Main Methods:

• Integration of an optical camera into the VitroJet system for real-time ice thickness measurement.
• Characterization of pin printing parameters to assess reproducibility of ice thickness.
• Estimation of ice thickness with an error below ±20 nm for layers ranging from 0-70 nm.
• Determination of single particle structures using apoferritin at defined ice thicknesses (30 nm and 70 nm).

Main Results:

• The optical camera method accurately estimates ice thickness (error < ±20 nm) in the 0-70 nm range.
• Pin printing parameters allow reproducible median ice thickness with a standard deviation below ±11 nm up to 75 nm.
• Single particle structures of apoferritin were successfully determined at both 30 nm and 70 nm ice thicknesses.
• The study highlights the critical role of optimized ice thickness in accelerating cryo-EM structure determination.

Conclusions:

• A novel, efficient method for measuring and controlling vitreous ice thickness has been established for cryo-EM sample preparation.
• This technique significantly improves the speed and reproducibility of cryo-EM workflows.
• Precise control over ice thickness is demonstrated to be vital for time-efficient single particle structure determination.