Label-free real-time imaging of mitochondrial matrix volume changes and permeability transition in living cells

Along with the membrane potential and respiration, mitochondrial matrix volume is a critical parameter that determines mitochondrial function. Mitochondria undergo constant changes in matrix volume and crista dynamics, and in processes that are critical for normal metabolic rates and pathophysiological responses. Changes in matrix volume cannot be easily measured by conventional fluorescence imaging techniques due to the size of the suborganellar structures, which are below resolution. This challenge was successfully resolved in studies of isolated mitochondria with the use of scattered light. Here, we use dark-field imaging, which relies on scattered light contrast, to measure matrix volume dynamics in living cells. We demonstrate that mitochondrial volume changes can be easily detected as changes in intensity of the scattered light following matrix volume modulation with K⁺ ionophores or by onset of the permeability transition. Specifically, we found that stimulation of K⁺ influx leads to an increase in mitochondrial matrix volume, while stimulation of K⁺ efflux leads to matrix shrinkage, and that activation of the permeability transition leads to high-amplitude mitochondrial swelling in wild-type but not in cells lacking subunit c of ATP synthase. These results directly demonstrate the dynamic nature of mitochondrial matrix volume and its link to physiological and pathological ion transport.