vEM is essential for the analysis of mitochondrial intrusion sites. (A) 3D-rendered examples of mitochondria containing intrusion sites, with their interior cavities colorized by the category of intrusion. (B) Quantitative analysis comparing the membrane volume of mitochondria in the presence or absence of mitochondrial intrusions. (C) Bar chart showing percentage of the mitochondrial population by their number of intrusions, with plot markers indicating expected values calculated by a Poisson distribution of the same average. (D and F) Quantitative analyses of each intrusion cavity by category, showing their (D) total interior volume, (E) radius of the largest inscribed sphere they could enclose, and (F) the length of their major ellipsoidal axis. (G) Statistical modeling of Buffon’s needle problem, showing the probability of detecting an intact mitochondrial intrusion (solid line) or only half an intrusion (dashed line) when an intrusion is present, as a function of the Z-depth (“d”) in a hypothetical FIB-SEM dataset or TEM section. Plot markers in B indicate value for individual mitochondria, and in D–F indicate value for individual intrusion cavities. The interquartile range for each chart is indicated by the box, the median is indicated by the horizontal lines, and the minimum and maximum are indicated by the whiskers. Scale bars: Major markers on the scale grid surrounding mitochondria are 1,000-nm apart, and minor markers are 500-nm apart.