IMAGING IN 5 DIMENSIONS - TIME-DEPENDENT MEMBRANE-POTENTIALS IN INDIVIDUAL MITOCHONDRIA

Because of its importance in the chemiosmotic theory, mitochondrial me mbrane potential has been the object of many investigations. Significa ntly, however, quantitative data on how energy transduction might be r egulated or perturbed by the physiological state of the cell has only been gathered via indirect studies on isolated mitochondrial suspensio ns; quantitative studies on individual mitochondria in situ have not b een possible because of their small size, their intrinsic motility, an d the absence of appropriate analytical reagents. In this article, we combine techniques for rapid, high resolution, quantitative three-dime nsional imaging microscopy and mathematical modeling to determine accu rate distributions of a potentiometric fluorescent probe between the c ytosol and individual mitochondria inside a living cell. Analysis of t his distribution via the Nernst equation permits assignment of potenti als to each of the imaged mitochondrial membranes. The mitochondrial m embrane potentials are distributed over a narrow range centered at -15 0 mV within the neurites of differentiated neuroblastoma cells. We fin d that the membrane potential of a single mitochondrion is generally r emarkably stable over times of 40-80 s, but significant fluctuations c an occasionally be seen. The motility of individual mitochondria is no t directly correlated to membrane potential, but mitochondria do becom e immobile after prolonged treatment with respiratory inhibitors or un couplers. Thus, three spatial dimensions, a key physiological paramete r, and their changes over time are all quantitated for objects at the resolution limit of light microscopy. The methods described may be rea dily extended to permit investigations of how mitochondrial function i s integrated with other processes in the intact cell.