Physics

Enzyme

Rhodamine 123

Membrane

Antibiotics

Inner mitochondrial membrane

Biology

Electrochemical gradient

Biophysics

Multiple drug resistance

Membrane potential

Chemistry

Rhodamine

Oxidative phosphorylation

Quantum mechanics

Mitochondrion

Cytosol

Biochemistry

Inner membrane

Fluorescence

Journal of the American Chemical Society

Mitochondria are the site of aerobic respiration, producing ATP via oxidative phosphorylation as protons flow down their electrochemical gradient through ATP synthase. This negative membrane potential across the inner mitochondrial membrane (ΔΨm) represents a fundamental biophysical parameter central to cellular life. Traditional, electrode-based methods for recording membrane potential are impossible to implement on mitochondria within intact cells. Fluorescent ΔΨm indicators based on cationic, lipophilic dyes are a common alternative, but these indicators are complicated by concentration-dependent artifacts and the requirement to maintain dye in the extracellular solution to visualize reversible ΔΨm dynamics. Here, we report the first example of a fluorescent ΔΨm reporter that does not rely on ΔΨm-dependent accumulation. We redirected the localization of a photoinduced electron transfer (PeT)-based indicator, Rhodamine Voltage Reporter (RhoVR), to mitochondria by masking the carboxylate of RhoVR 1 as an acetoxymethyl (AM) ester. Once within mitochondria, esterases remove the AM ester, trapping RhoVR inside of the mitochondrial matrix, where it can incorporate within the inner membrane and reversibly report on changes in ΔΨm. We show that this Small molecule, Permeable, Internally Redistributing for Inner membrane Targeting Rhodamine Voltage Reporter, or SPIRIT RhoVR, localizes to mitochondria across a number of different cell lines and responds reversibly to changes in ΔΨm induced by exceptionally low concentrations of the uncoupler FCCP without the need for exogenous pools of dye (unlike traditional, accumulation-based rhodamine esters). SPIRIT RhoVR is compatible with multi-color imaging, enabling simultaneous, real-time observation of cytosolic Ca2+, plasma membrane potential, and reversible ΔΨm dynamics.

Imaging Reversible Mitochondrial Membrane Potential Dynamics with a Masked Rhodamine Voltage Reporter