Ec. Toescu et A. Verkhratsky, Assessment of mitochondrial polarization status in living cells based on analysis of the spatial heterogeneity of rhodamine 123 fluorescence staining, PFLUG ARCH, 440(6), 2000, pp. 941-947
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.