Fig. 2: Rationale for 3D imaging and acquisition of volumetric data with FIB-SEM.

a Schematic representation of 2D imaging of a host mitochondrion where the location of the cross-section can lead to misinterpretations (green ellipsoid: bacterium). b A hypothetical 3D image where mitochondria (spheres) and bacteria (ellipsoids) randomly distributed and the true number of mitochondria-bacteria intersection (NMBI) were taken. c For the 2D imaging simulation, multiple 2D slices were taken from the same hypothetical 3D image, and the NMBIs were calculated when bacteria (green) and mitochondria (orange) overlap (blue). d Graph depicting the increase in the NMBI with more 2D cross sections (N-slices) analyzed for object density where the centrelines show the mean and transparent bands show the 95% confidence interval for n = 4 object density (d = 30, 60, 90, 120), 90 image sets per density. The dots represent the true NMBIs calculated from 3D. e Consecutive electron micrographs in 10 nm distance were generated using FIB-SEM, and f aligned to build the volumetric images. g FIB-SEM imaging quality allows distinguishing typical interaction patterns throughout the 3D images; the representative image shows a mitochondrion (M) hosting a bacterium (bac) within the mitochondrial outer membrane (arrow) (Scale bar: 500 nm, n = 1 wild-type vitellogenic tick). Source data are provided as a Source Data file.