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Abstract
The spectral properties of a novel membrane potential sensitive probe (JC-1) were
characterized in aqueous buffers and in isolated cardiac mitochondria. JC-1 is a carbocyanine
with a delocalized positive charge. It formed under favorable conditions a concentration-dependent
fluorescent nematic phase consisting of J-aggregates. When excited at 490 nm, the
monomers exhibited an emission maximum at 527 nm and J-aggregates at 590 nm. Increasing
concentrations of JC-1 above a certain concentration caused a linear rise in the J-aggregate
fluorescence, while the monomer fluorescence remained constant. The membrane potential
of energized mitochondria (negative inside) promoted a directional uptake of JC-1
into the matrix, also with subsequent formation of J-aggregates. The J-aggregate fluorescence
was sensitive to transient membrane potential changes induced by ADP and to metabolic
inhibitors of oxidative phosphorylation. The J-aggregate fluorescence was found to
be pH independent within the physiological pH range of 7.15-8.0 and could be linearly
calibrated with valinomycin-induced K+ diffusion potentials. The advantage of JC-1
over rhodamines and other carbocyanines is that its color altered reversibly from
green to red with increasing membrane potentials. This can be exploited for imaging
live mitochondria on the stage of a microscope.