Tf-endosome–mitochondrion interactions using 3D dSTORM. MDCK-PTR cells pulsed with AF647-Tf for 2 min at 37°C, chased for 2 min, and fixed, and OMM and/or IMM were immunolabeled with antibodies against Tom20 and OxPhos, respectively. (A) Representative double-labeled 3D dSTORM image of the z position information of AF647-Tf–endosomes (a) and AF568-labeled anti-OMM (b), color coded according to a 450–1,600-nm range; merged (c) to show Tf (green) and OMM (red). Bar, 1 µm. (B) Magnified x–y, x–z, and y–z cross sections of ROIs 1–4 (white boxes in A). ROIs 1–3 depict AF647-Tf–endosomes directly interacting with AF568-labeled anti-OMM in all three dimensions, whereas ROI 4 shows a noninteraction (gray arrows) despite being in close proximity. See Video 1. Bars, 100 nm. (C) Representative triple-labeled 3D dSTORM image of the z position information of AF568-Tf–endosomes (a), AF647-labeled anti-OMM (b), and Atto488-labeled anti-IMM (c), color coded according to a 130–800-nm range; merged (d) to show Tf (green), IMM (red), and OMM (blue). Bar, 1 µm. (D) Magnified 3D cross sections of ROIs 1–3 (white boxes in C) show direct interactions between AF568-Tf–endosomes and AF647-labeled anti-OMM, which distinctly envelopes Atto488-labeled anti-IMM. See Video 2. Bars, 100 nm.