Fig. 1: Parallel acquisition of tilt-series using beam-image shift electron cryo-tomography (BISECT).

a Schematic of the movement of ROIs (white circles) from the initial position (gray) and after stage tilting (blue). b Evolution of the targeting and defocus over a 5 × 5-hole area. The initial field of view of each area is shown as red squares. The high-magnification tracking area is shown as an orange square. Lines show the movement of the targeting obtained from cross-correlation alignment (scaled by 5 for visualization) and are color coded according to the estimated defocus for each image (large movements are attributed to errors in the cross-correlation at high-tilt and are not reflective of the actual motion of the sample). To allow for errors and discrepancies in the targeting of each hole, every BIS area is treated individually and gets its own Z-height fit. This approach makes our routines more robust to irregular specimens that strongly deviate from an ideal plane such as the bending observed in samples with thicker ice.