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Measurement of mitochondrial membrane potential using fluorescent rhodamine derivatives
1Department of Cellular and Molecular Physiology, College of Medicine, The Pennsylvania State University, Hershey, Pennsylvania 17033 USA. rscaduto@psghs.edu
Biophysical Journal
|January 6, 1999
Summary
Rhodamine dyes (R123, TMRM, TMRE) can monitor mitochondrial membrane potential (DeltaPsi) in isolated mitochondria using ratio fluorescence. However, these dyes bind to membranes and affect respiration, limiting their use in intact tissues.
Area of Science:
- Mitochondrial physiology
- Biophysical chemistry
- Fluorescence spectroscopy
Background:
- Mitochondrial membrane potential (DeltaPsi) is crucial for cellular energy production.
- Accurate measurement of DeltaPsi is essential for understanding mitochondrial function.
- Fluorescent lipophilic cations are commonly used probes for DeltaPsi.
Purpose of the Study:
- To evaluate rhodamine 123 (R123), tetramethylrhodamine methyl ester (TMRM), and tetramethylrhodamine ethyl ester (TMRE) as fluorescent probes for mitochondrial DeltaPsi.
- To develop a ratio fluorescence method for dynamic DeltaPsi monitoring in isolated mitochondria.
- To assess the applicability of these probes in intact tissues.
Main Methods:
- Utilized R123, TMRM, and TMRE as fluorescent probes.
- Employed a ratio fluorescence approach for DeltaPsi measurement.
- Determined binding characteristics and effects on mitochondrial respiration.
- Applied the method to isolated rat heart mitochondria and perfused hearts.
Main Results:
- All three dyes exhibited spectral shifts and fluorescence quenching upon mitochondrial accumulation, proportional to DeltaPsi.
- Dye binding exceeded Nernst equation predictions and was temperature-dependent (TMRE > R123 > TMRM).
- Dyes suppressed mitochondrial respiratory control, with TMRE > R123 > TMRM; TMRM showed minimal effect at low concentrations.
- The ratio fluorescence method successfully measured DeltaPsi in isolated mitochondria.
- Spectral shifts in intact perfused hearts occurred in the cytosol, not exclusively in mitochondria, precluding DeltaPsi monitoring.
Conclusions:
- Rhodamine dyes and ratio fluorescence offer a viable method for measuring DeltaPsi in isolated mitochondria.
- Dye binding and respiratory inhibition must be considered for accurate DeltaPsi quantification.
- The spectral properties of these probes in intact tissues limit their application for mitochondrial DeltaPsi monitoring in vivo.