Intensity profile-based filtering of the IMM thickness measurements from Fig. 2 . (A) Aggregated intensity profiles for all IMM thickness measurements. The mean profiles prior to filtering are shown in orange, those after filtering in purple, with standard deviations indicated by shading. Membrane thickness is measured between paired points on opposing membrane segmentation surfaces, with their mean positions indicated by vertical lines and standard deviations shown as shaded regions. These measurement positions correspond to the segmentation boundaries. Filtering criteria: two identifiable minima separated by a central maximum; the minima are positioned between the paired points or within a 30% extension range outward (≈1–1.5 voxels extension in bin4 tomograms). (B) Summary statistics for the number of measurements before and after filtering across multiple thickness bins. (C) Intensity profiles binned by thickness ranges (before filtering in orange, after filtering in purple). (D) Spatial distribution of the filtering results. Left panel: Included measurement points are colored in purple; excluded points are shown in gray. Right panel: ATP synthase coordinates (blue) are overlaid for context. ATP synthase particle positions were obtained from a publicly available repository (Righetto et al., 2025) as determined by subtomogram averaging by Kelley et al. (2024, Preprint), with the resulting subtomogram average map (EMD-52802) used for visualization. (E and F) Spatial distribution of filtering results for the sub-3 nm (gray) and 7–8 nm (purple) thickness ranges, respectively. (G) Membrane thickness distributions prior to filtering across all measured organelle membranes for direct comparison with Fig. 2 E. (H) Minima-to-minima distances measured from tomogram-derived intensity profiles of each organelle shown in G.