Assessment of mitochondrial polarization status in living cells based on analysis of the spatial heterogeneity of rhodamine 123 fluorescence staining

The mitochondrial membrane potential (Psi (mito)) is an important parameter not only of mitochondrial but also of cellular status. Prolonged mitochond rial depolarization is associated with various forms of neuronal death. Ass essment or mitochondrial depolarization can take advantage of the specific properties or the lipophilic dyes that distribute in a potentiometrically d etermined ratio across membranes. Using conventional imaging, we showed tha t rhodamine 123 accumulated in the mitochondria, generating a highly hetero geneous pattern of spatial distribution of fluorescence across the cell bod y. Collapse of the Psi (mito) following exposure to a protonophore, carbony lcyanide p-chloromethoxyphenylhydrazone (CCCP), released rhodamine 123 from mitochondria into the cytosol. Under acutely changed conditions, this incr eased the overall intensity of the fluorescence signal and significantly de creased the degree of spatial heterogeneity of the signal. If mitochondrial depolarization was sustained chronically, the intensity of the signal decr eased, but the increase in the spatial homogeneity of the fluorescent signa l was maintained. Image analysis showed that the level of spatial heterogen eity of the signal can be assessed by calculating, for each individual neur one, the spread of pixel intensities values around the mean. This spread is defined by the coefficient of variation (CV), which is a measure of the st andard deviation normalized to the average, and was inversely related to mi tochondrial depolarization measured under different conditions. Thus, the d egree of spatial heterogeneity of the rhodamine 123 signal measured from a neurone is a reliable indicator for the assessment of mitochondrial depolar ization and can lte used in experiments to monitor Psi (mito) over shorter or longer periods.