Fig. 2. Imaging of TMRE-loaded astrocytes revealed transient fluctuations in _m. (A-D) Imaging of TMRE-loaded cells revealed brief, spontaneous and reversible depolarisations of individual mitochondria. The fluorescence signal over individual mitochondria is plotted as a function of time. Note that the signal over individual organelles frequently returned to baseline, suggesting complete mitochondrial repolarisation following a depolarisation (A) and single organelles could depolarise and repolarise repeatedly (B,C), further suggesting reversible depolarisations of _m. The traces in C and D were obtained from two mitochondria in a single cell, demonstrating that the flickers were independent in time and did not reflect a global loss of _m. The histogram in E, shows the distribution of the relative amplitudes of individual transient changes in signal. FCCP was applied at the end of each imaging sequence, and the amplitudes are expressed as a percentage of the response to FCCP. In some cells the global fluorescence was essentially stable, so that individual depolarisations were easily discernible (F), and were much smaller in amplitude than the response to FCCP. In other cells, the flickers rapidly summated to produce a global rise in whole-cell fluorescence that overwhelmed the signal produced by individual mitochondria (G), in which case application of FCCP induced no further change in signal, suggesting that mitochondrial depolarisation was complete.

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