Endosome-mitochondria proximity increases chelatable, mitochondrial iron. Alexa Green 488 transferrin (green) and RPA (red) were imaged simultaneously with a dualcam system and 2 Cascade 512B cameras. (A) The white box shows a region where a transferrin containing endosome appears to contact a mitochondria, enlarged in (B). (C) Pseudo color images of the red signal to more clearly illustrate the decrease in intensity of the signal following contact with the iron containing endosome. An “uncontacted” mitochondrion whose intensity does not decrease with time is denoted by black arrows. (D) Enlarged images of the region outlined by the white box in C. Images are an average of 10 image frames, corrected for background intensity and were smoothed with a 2 × 2 pixel low pass filter. Scale bar is 1 μm. (E) Plot of the intensity versus time for mitochondria that either made contact with endosomes (open circles) or control mitochondria that did not contact endosomes (closed circles). Error bars represent standard error of the mean. Data were collected and smoothed as described in “Materials and methods; Data analysis”.