Optimising the tilt increment for in situ cryo-electron tomography.

Cryo-electron tomography (cryo-ET) enables high-resolution, three-dimensional imaging of cellular structures in their native, frozen state. However, image quality is limited by a trade-off between angular sampling and radiation damage. Therefore, the choice of the angular increment during data collection is a critical parameter that affects tomogram quality and downstream analyses. Optimising this increment is challenging due to the high demands on microscope time, storage, and computation. In this study, we systematically evaluated tilt increments of 1°, 2°, 3°, 5°, and 10° using lamellae from Dictyostelium discoideum cells. We found that at a constant total electron dose, finer tilt increments (1-3°) produced better-aligned tomograms with higher signal-to-noise ratios and improved outcomes in template matching and subtomogram averaging. A 3° increment emerged as the optimal balance between data quality, alignment accuracy, dose per image, and processing efficiency. This practical recommendation supports both high-throughput and high-resolution structural studies and can guide future cryo-ET data acquisition strategies.