Monitoring of mitochondrial continuity. (A–D) mCherry FLIP in wild-type cells expressing Tom20-GFP (mitochondrial marker; without photobleach) and matrix-mCherry (photobleach target). The line graphs are the intensity profiles along the lines in the respective images. (A) Loss of matrix-mCherry serves as a continuity marker; a physically separate mitochondrion, indicated by an arrow, retained mCherry signals while a photobleached mitochondrion, indicated by a double arrow, lost the fluorescence. (B) An example of a fusion event. The photobleached mitochondrion in the mother (indicated by a double arrow) lost mCherry fluorescence while a physically separate mitochondrion in the bud (indicated by an arrow) retained the fluorescence, until these two mitochondria fused with each other, leading to equilibration of the mCherry fluorescence between the structures. The fusion event took place between 475 and 513 s. (C) An example of a fission event. A continuous mitochondrion was photobleached and lost mCherry fluorescence from the entire structure until it underwent fission forming three separate mitochondria. One mitochondrion at the bleaching area (indicated by a double arrow) further lost mCherry fluorescence whereas two separated mitochondria (indicated by an arrow and arrowhead) retained the fluorescence after fission. The fission event took place between 209 and 285 s. (D) An example of a continuous mitochondrion between the mother and bud throughout the imaging period. The mCherry fluorescence was lost from the entire structure. Photobleach was applied in the mCherry channel as indicated by white circles. Images are a sum projection of five z-stacks taken at 0.5-μm intervals. Scale bar: 3 µm.