Benchmarking the ideal sample thickness in cryo-EM

TLDR

Sample thickness effects on electron scattering in cryo‑EM had not been systematically investigated before. The study aims to investigate how increasing sample thickness affects cryo‑EM data quality. Near‑identical protein crystals were examined by microcrystal electron diffraction at three common accelerating voltages, with data analyzed using the calculated inelastic mean free path. Structures can be resolved up to twice the inelastic mean free path, while thicknesses exceeding four times the mean free path yield no coherent information, establishing practical limits for biological specimen thickness in cryo‑EM.

Abstract

Significance A systematic investigation of the effects of sample thickness on electron scattering in electron cryo-microscopy (cryo-EM) was previously not feasible. Here, methods are employed to investigate the effects of increasing sample thickness. Near identical protein crystals are used as samples, and microcrystal electron diffraction data are used to assess the effects of thickness. These experiments were conducted using the three most-common accelerating voltages in cryo-EM, and data were compared using the calculated inelastic mean free path. Structures may be determined using crystals up to twice the inelastic mean free path. No coherent information remains at thicknesses over four times the mean free path. This study provides limits for biological specimen thickness with implications for all cryo-EM methods.