WebCalEM: a browser-based tool for routine and accurate pixel-size calibration in cryo-EM

Area of Science:

• Cryo-electron microscopy
• Image analysis
• Scientific instrumentation

Background:

• Accurate pixel size determination is crucial for quantitative cryo-electron microscopy (cryo-EM).
• Existing calibration methods are often under-utilized due to installation barriers and complex workflows.
• Routine adoption of calibration procedures is hindered by practical implementation challenges.

Purpose of the Study:

• To develop an accessible web-based application, WebCalEM, for routine cryo-EM pixel size calibration.
• To transform specialized calibration procedures into an accessible practice for core facilities.
• To lower the barrier for documented per-dataset magnification calibration in cryo-EM.

Main Methods:

• Developed WebCalEM, a self-contained HTML file running client-side in any modern web browser.
• Users upload micrographs of specimens with known crystalline lattices (e.g., gold, graphene oxide).
• Real-time visualization and downloadable statistical outputs are provided without server-side computation.

Main Results:

• WebCalEM processes micrographs entirely client-side, suitable for isolated workstations.
• Cross-standard consistency was achieved between gold and graphene oxide measurements.
• Measurements across two microscopes and ten magnifications showed a Bland-Altman bias of -0.005% with limits of agreement [-0.30%, +0.29%].

Conclusions:

• WebCalEM effectively lowers practical barriers to magnification calibration in routine cryo-EM.
• The application facilitates accessible and documented per-dataset calibration.
• This tool promotes wider adoption of essential quantitative analysis practices in cryo-EM.