Fromm, S. A.; Mattei, S.

Structure elucidation of biological macromolecules by single particle cryogenic electron microscopy (SPA cryo-EM) or cryogenic electron tomography (cryo-ET) relies on low-dose imaging on cryogenic transmission electron microscopes (cryo-TEMs). Routine microscope setup remains technically demanding and can be time-consuming, particularly for inexperienced or infrequent users. We present LowDoseWizard, a guided workflow implemented in SerialEM that enables rapid and standardised setup of cryo-TEM imaging conditions. From minimal user input, the workflow configures microscope optics, camera parameters and image shift settings for all low-dose imaging states, and guides the user through key daily alignment procedures including beam shift offset calibration, objective lens astigmatism correction and coma-free alignment. The workflow is organised into modular routines that can be executed sequentially or independently, while microscope-specific acquisition parameters are defined in editable configuration files, allowing flexible adaptation to different instruments without modification of the core scripts. Across user sessions on three microscopes at EMBL Heidelberg, the complete setup required on average less than 15 minutes. To assess whether predefined imaging conditions generated by the workflow are compatible with high-resolution data collection, we acquired apoferritin data on a 200 kV Glacios and a 300 kV Titan Krios. These datasets yielded reconstructions at 1.62 A and 1.09 A resolution, respectively, demonstrating that rapid, guided setup can support near-atomic and atomic-resolution single particle cryo-EM. LowDoseWizard lowers the barrier to robust cryo-TEM setup, reduces the time spent on routine parameter selection and alignment, and helps users focus on sample-specific aspects of data acquisition such as target selection. The workflow should be particularly valuable in shared instrumentation environments, where accessibility, reproducibility and efficient microscope use are critical.