Fig. 2. FRAP analysis of mitochondrial lumenal continuity. (Ai-iv) The DsRed1 fluorescence in a HeLa cell before and 10 s, 20 min and 60 min after photobleaching a small area of mitochondria in the perinuclear aggregation (denoted by white box). (Av) At 60 min post-photobleach, 1 µmol 1^-1 TMRE was added to the cells for 5 min. The mitochondria that failed to recover DsRed1 fluorescence in Av were stained with TMRE, indicating that their functional integrity was not compromised during laser irradiation. Note that the laser intensity was reduced by 33% in Av compared to Ai-iv, to prevent reactive oxygen species (ROS) production and permeability transition pore (PTP) opening. See text for details. (Bi-v) The DsRed1 fluorescence in a cortical astrocyte before and 10 s, 20 min and 60 min after photobleaching the area denoted by the white box in Bi. (Bii–v) The region of the cell bounded by the broken line in A on an expanded scale. Scale bars, 5 µm.

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