DT criterion to determine Tf-endosome–mitochondria interactions. (A, a–c) Representative confocal z stack image of MDCK-PTR cells pulsed with AF647-Tf (red) for 2 min, chased, fixed, and immunolabeled with AF555 anti-OMM (green) and AF488 anti-IMM (cyan). (d–f) Corresponding Imaris-based 3D-rendered images depicting OMM and IMM by surface and early Tf-endosomes by spots. Bars, 10 µm. (B, a–c) Magnified ROIs (dotted boxes in A, d–f) where the Tf-endosome under consideration (yellow spots) was in close proximity to the OMM (a) and subsequently validated by DT = 0. Upon deselecting the OMM and selecting the IMM surface render, the Tf-endosome showed failed interaction with the IMM of the same mitochondrion (b), also confirmed by a DT > 0 value with respect to the IMM surface; merged image (c). (d–f) Magnified ROIs (solid boxes in A, d–f) where another Tf-endosome (yellow spots) was identified to be in close proximity to both OMM (d) and IMM (e). Bars, 1 µm. The brightness and contrast of images have been enhanced equally across each panel to aid visualization. (C) Percentage of Tf-endosomes detected in close proximity to OMM or IMM per z stack of an image field (four to seven cells per field).