Local variations in membrane thickness correspond to the presence of membrane-associated proteins. (A) Representative membrane surface reconstruction of mitochondria colored by IMM membrane thickness. (B) Enlarged images of the corresponding boxed regions shown in A reveal the local membrane thickness and the colocalized membrane-associated macromolecules (outlined by dashed lines). Scale bars = 25 nm. (C) Line scan profiles along the green dashed arrow line in B of membrane-associated macromolecule patches show double Gaussian peaks (orange dashed line) corresponding to IMM (orange arrows), along with additional peaks representing protein macromolecules (green arrows). (D) Averaged line scan density profile across all ATP synthase patches (blue) shows a distinct peak compared with randomized patches (green).