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

Accurate determination of the image pixel size is critical for quantitative cryo-electron microscopy analyses, yet existing calibration methods remain under-utilized because installation barriers and workflow complexity discourage routine adoption. To fill in this gap, a web-based application, WebCalEM, was developed to transform specialized calibration procedures into an accessible routine practice. Micrographs of any specimen with a known crystalline lattice, such as gold or graphene-oxide, are uploaded through a standard browser, processed entirely client-side, and analyzed with real-time visualization and downloadable statistical outputs. The application is delivered as a single self-contained HTML file that runs in any modern web browser without server-side computation, a configuration well suited to isolated core-facility microscope workstations. Cross-standard consistency between gold and graphene-oxide measurements across two microscopes and ten magnification settings yields a Bland-Altman bias of -0.005% of nominal with 95% limits of agreement of [-0.30%, +0.29%]. By delivering this workflow with no local installation, WebCalEM lowers the practical barrier to documented per-dataset magnification calibration in routine cryo-EM operation. SynopsisWebCalEM is a browser-based, install-free application that performs routine cryo-EM pixel-size calibration directly from gold or graphene-oxide reflections in standard sample-support grids using sub-pixel Fourier-space peak localization; it reproduces the precision of established command-line calibration tools while removing the installation barrier and supporting retrospective per-region calibration on archived datasets.