Inner membrane dynamics in mitochondria

Combining the use of cells with sparse cristae marked with IMP-EGFP and short pulsed sub-saturating fluorescence excitation (non-saturation fluorescence microscopy/NSFM) revealed inhomogeneous fluorescence distribution along mitochondria in living cells. Also the matrix located TMRE was distributed non-uniformly and at least in part filling the gaps between the IMP-EGFP fluorescence: fluorescence intensities are modulated in space and time in part in an antidromic manner. The spatial modulations can be interpreted to represent cristae/matrix distributions. The temporal fluctuations of fluorescence vary within 0.3-3 s. Because most peak positions of IMP fluorescence remain stationary up to at least several minutes, temporal intensity modulations may result from varying emissions related to the degree of excitation and/or represent wobbling of cristae, i.e. lateral movements, bending or size changes. Modulations by noise and non-saturated excitation have been reduced by 3 steps of deconvolution followed by averaging 4 images. This allowed a final temporal resolution of 150 ms. Disappearance of cristae or formation of new ones takes place within a few seconds, but these are rare events. Thus position of cristae seems to be rather stable, but they regularly disassemble close to fission sites. Treatment with oligomycin strongly reduces “wobbling” activity.