File:Stage-Specific-Changes-in-the-Water-Na+-Cl--and-K+-Contents-of-Organelles-during-Apoptosis-pone.0148727.s004.ogv

English: Simultaneous imaging of mitochondrial potential and of nuclear modifications studied by time-lapse confocal imaging after the induction of apoptosis by 500 ng/mL AMD. HeLa cells stably expressing H2B-GFP were stained with TMRE to study mitochondrial polarization. Simultaneous time-lapse confocal imaging of TMRE and H2B-GFP was performed by two-photon excitation, every 5 minutes for 7 h and 15 minutes after the induction of apoptosis by the addition of 500 ng/mL AMD. Z-stacks of 85 optical sections were processed to obtain 3D images (surface rendering for chromatin and volume rendering for mitochondrial polarization), which were assembled to produce movies demonstrating the successive steps of 3D chromatin reorganization and changes in mitochondrial potential (based on the green fluorescence of H2B-GFP and red fluorescence of TMRE respectively). In the field of view shown in this movie, we observed 35 cells containing H2B-GFP. Twelve cells entering apoptosis during the period of 7 hours and 15 minutes were numbered 1 to 12. In these cells, depolarization of mitochondria was initiated at different times as demonstrated by the observation of TMRE red signal reorganization. Mitochondrial polarization disappeared after: three hours and 45 minutes (cell # 1), four hours and 35 minutes (cell # 2), five hours and 10 minutes (cells # 3 and 4), six hours (cells # 5 and 6), six hours and 5 minutes (cell # 7), six hours and 15 minutes (cells # 8 and 9), six hours and 20 minutes (cell # 10) and six hours and 30 minutes (cells # 11 and 12).