LowDoseWizard: rapid and standardized setup of low-dose cryo-TEM imaging in SerialEM

Area of Science:

• Structural Biology
• Microscopy Techniques
• Biophysics

Background:

• Single-particle cryogenic electron microscopy (SPA cryo-EM) and cryogenic electron tomography (cryo-ET) require low-dose imaging on cryogenic transmission electron microscopes (cryo-TEMs).
• Routine cryo-TEM setup is technically demanding and time-consuming, especially for novice users.

Purpose of the Study:

• To introduce LowDoseWizard, a guided workflow within SerialEM for rapid and standardized cryo-TEM setup.
• To simplify and accelerate the optimization of microscope optics, camera parameters, and alignment procedures for low-dose imaging.

Main Methods:

• Implementation of a guided workflow in SerialEM for automated microscope setup and daily alignment.
• Modular routines for sequential or independent execution, with editable configuration files for instrument flexibility.
• Testing the workflow on Glacios and Titan Krios microscopes at EMBL Heidelberg.

Main Results:

• Complete cryo-TEM setup averaged less than 15 minutes across multiple user sessions and microscopes.
• High-resolution reconstructions of apoferritin were achieved at 1.62 Å (200 kV Glacios) and 1.09 Å (300 kV Titan Krios).
• Demonstrated compatibility of workflow-generated conditions with near-atomic and atomic-resolution SPA cryo-EM data collection.

Conclusions:

• LowDoseWizard significantly lowers the barrier to entry for robust cryo-TEM setup.
• The workflow reduces time spent on routine parameter selection and alignment, allowing users to focus on sample-specific tasks.
• LowDoseWizard enhances accessibility, reproducibility, and efficiency in shared cryo-TEM facilities.