Fig. 3: Effects of backside amorphous and striated areas on STA resolution.

a TEM overview of a lamella prepared from high-pressure frozen E. coli described in Fig. 1 (n = 52 lamellae). Amorphous areas are visible only on the backside of the lamella (red arrows), not on the front near the GIS layer (white arrows). White dashed square indicates area shown in (b). Scalebar: 2 μm. b TEM image of the amorphous area on the backside of the lamella, in the area shown in the white dashed square in (a). A distinct striated pattern of alternating high- and low electron-dense material is visible at the back of the lamella (red double-headed arrow), with an area without any remaining biological contrast of typically 0.5 to 1.5 μm in length (white double-headed arrow), which often contains small cracks (black arrows). The area without biological contrast is delineated by a striated pattern (blue arrows), weaker in contrast than at the very back of the lamella. Scalebar: 500 nm. c Tilt-image of a bacterium, where the contrast of the bacterium abruptly disappears at the border of amorphous area without biological contrast (blue arrows). Scalebar: 100 nm. n = 15. d Tomographic slice of a bacterium near the area without biological contrast (blue arrows), with ribosomes clearly visible directly adjacent to this border (white dashed circles). Scalebar: 100 nm. n = 15. e Enlarged TEM overview of the lamella shown in (a). For each ribosome (orange circle) the distance to the amorphous layer (white dashed line) was determined by taking the distance of the corresponding tomogram (white dashed squares) to the amorphous layer and offsetting it with the ribosome position in the tomogram. Ribosomes were then grouped based on their distance to the amorphous layer in 1 μm bins (black dashed lines). Scalebar: 1 μm. f B-factor plot for all ribosome distance groups from the amorphous layer on the backside of the lamellae, in 1 μm groups. Resolution is plotted on square inverse scale and particle number is plotted on log_e scale, with resolutions reported as mean ± SE (n = 3 technical replicates of the B-factor analysis). Source data are provided as a Source Data File.