Calcium-induced alterations in mitochondrial morphology quantified in situ with optical scatter imaging

Optical scatter imaging (OSI), a technique we developed recently, was used to measure the ratio of wide-to-narrow angle scatter (OSIR) within endothelial cells subjected to calcium overload (1.6 mM) after permeabilization by ionomycin. Within a few minutes of calcium overload, the mitochondria, which started as elongated organelles, rounded up into spherically shaped particles. This change in morphology was accompanied by a statistically significant 14% increase in OSIR in the cells' cytoplasm. Mitochondrial rounding and OSIR increase were suppressed by cyclosporin A (25 μM), implying that the observed geometrical and scattering changes were directly attributable to the mitochondrial permeability transition. The angular scattering properties of a long mitochondrion rounding up were approximated by numerical simulations of light scatter from an ellipsoid rounding up into a sphere. The simulations predicted a relative increase in OSIR comparable to that measured experimentally for the case where the shape transition takes place with little or no volume increase. The simulations also suggested that mitochondrial refractive index changes could not account for the OSIR changes observed. Our data show that changes in OSIR correlate with mitochondrial morphology change in situ. OSI provides a new tool for subcellular imaging and complements other microscopy methods, such as fluorescence.