Fig. 4. Electrical discontinuity of mitochondria, demonstrated by asynchronous depolarisation events induced by TMRE plus laser irradiation. Cells were loaded with TMRE and imaged with a moderate laser intensity (30 µW at objective). Rapid mitochondrial depolarisation events appeared as loss of fluorescence between frames (typically 2–4 s). (A) A TMRE-loaded pancreatic acinar cell. Please note that the image is a median section of a cell where the apical region would project out of the figure and the basal pole behind the figure. The locations of mitochondria that showed clear depolarisation events are mapped in (Aii). The grey areas denote TMRE-stained mitochondria that did not flicker. G, the position of the granular region, which is bounded by the perigranular mitochondria (PG). The subplasmalemmal (SPM) mitochondria can been seen around the outside of the cell. (Bi–Di) Portions of a HeLa cell (Bi), HUVEC cells (Ci) and ventricular cardiomyocyte (Di), following loading with TMRE. (Bii–Dii) The positions of electrically discrete mitochondria are depicted in the HeLa cell (Bii) and HUVEC cell (Cii). The maps of individual electrically isolated mitochondria were constructed by monitoring the locations of individual depolarisation events over time. The magenta, cyan and yellow colouration is used to indicate the positions of the electrically isolated mitochondria, and does not indicate any relationships between the organelles. (Dii) A line-scan plot derived from the region between the arrowheads in (Di), illustrating the asynchronous flickering of the majority of mitochondria. A few mitochondria do appear to depolarise synchronously (Dii, arrowheads). Scale bars, 5 µm.

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