Visualization by FIB-SEM of perforated endolysosomes in iNs and primary mouse neurons. (A–C) Orthogonal views, each averaged from three consecutive planes, and surface renderings of iNs visualized by FIB-SEM, acquired at 5 × 5 × 5 nm resolution. Samples were prepared by high-pressure freezing and freeze substitution. Scale bars: 200, 500, 1,000 nm. A and B show representative examples from two different iNs with perforated endolysosomes, each exhibiting a small nanopore in the limiting membrane, highlighted by the arrowheads. Data from 10 somas revealed 5 compromised endolysosomes and 3 early endosomes out of a total of 91 endolysosomes and 147 early endosomes examined. Inspection of limiting membranes in 13 endosomes from 37 volumetric cross-sections of different neurites did not reveal nanopores. C shows a representative endolysosome from parental iPSCs, intact and lacking nanopores. (D) The top three panels show single-plane views of three perforated endolysosomes, highlighted by the arrowheads, from three different mouse CA1 pyramidal neurons, prepared by chemical fixation and imaged by volumetric FIB-SEM at 5.4 × 5.4 × 16 nm resolution (Sheu et al., 2022). The bottom panel shows a zoomed-out view of the region containing the upper-left panel. Scale bars: 400, 4,000 nm. (E) Representative examples of nanopores in the limiting membranes of two endolysosomes (left and center panels) and one early endosome (right panel) from somas of three different iNs. Perforation outlines were manually traced with single pixels using LabKit, with voxel resolution of 5 nm. The longest and shortest inner dimensions of the perforations were measured in orthogonal views using FIJI. For visualization, voxels were enlarged during 3D rendering in Imaris to clearly depict the perforations.