Time Lapse Measurement of Mitochondrial Membrane Potential in Absolute Millivolts in Single Intact Cells

Abstract

Assaying mitochondrial membrane potential (ΔΨM ) in absolute millivolts in intact cells had been previously limited mostly to radioisotope distribution methods. Here we introduce a purely fluorescence based ΔΨM assay to calculate time courses of ΔΨM in absolute millivolts in monolayer cell cultures. We built a biophysical model‐based method to calibrate single‐cell fluorescence of a bis‐oxonol‐type plasma membrane potential (ΔΨP ) indicator and the ΔΨM –probe TMRM to potentials. The ΔΨP ‐dependent distribution of the probes is modeled by Eyring rate theory, which we have verified using fluorescence imaging combined with voltage clamp. ΔΨM is determined in millivolts by deconvoluting TMRM fluorescence in time taking in account the slow, ΔΨP ‐dependent redistribution and its Nernstian behavior. The resting ΔΨM is calculated from a complete step‐depolarization of ΔΨM. The calibration accounts for volume ratios, high and low affinity bindings, activity coefficients, background fluorescence and optical dilution. All of the calibration parameters are back‐calculated from fluorescence intensities or measured by confocal microscopic assays (validated by electron microscopy), allowing comparisons of potentials in cells with different properties. We show that ΔΨM in cultured rat cortical neurons is regulated between −120 and −154 mV as a concerted effect of increased ATP turnover and Ca2+‐dependent metabolic activation.