Abstract
Permeant cationic fluorescent probes are widely employed to monitor mitochondrial transmembrane potential and its changes. The application of such potential-dependent probes in conjunction with both fluorescence microscopy and fluorescence spectroscopy allows the monitoring of mitochondrial membrane potential in individual living cells as well as in large population of cells. These approaches to the analysis of membrane potential is of extremely high value to obtain insights into both the basic energy metabolism and its dysfunction in pathologic cells. However, the use of fluorescent molecules to probe biological phenomena must follow the awareness of some principles of fluorescence emission, quenching, and quantum yield since it is a very sensitive tool, but because of this extremely high sensitivity it is also strongly affected by the environment. In addition, the instruments used to monitor fluorescence and its changes in biological systems have also to be employed with cautions due to technical limits that may affect the signals. We have therefore undertaken to review the most currently used analytical methods, providing a summary of practical tips that should precede data acquisition and subsequent analysis. Furthermore, we discuss the application and feasibility of various techniques and discuss their respective strength and weakness.
Similar content being viewed by others
References
Baracca A, Barogi S, Carelli V, Lenaz G, Solaini G (2000) Catalytic activities of mitochondrial ATP synthase in patients with mitochondrial DNA T8993G mutation in the ATPase 6 gene encoding subunit a. J Biol Chem 275:4177–4182
Baracca A, Sgarbi G, Solaini G, Lenaz G (2003) Rhodamine 123 as a probe of mitochondrial membrane potential: evaluation of proton flux through F0 during ATP synthesis. Biochim Biophys Acta 1606:137–146
Bernardi P, Scorrano L, Colonna R, Petronilli V, Di Lisa F (1999) Mitochondria and cell death: mechanistic aspects and methodological issues. Eur J Biochem 264:687–701
Chen LB (1988) Mitochondrial membrane potential in living cells. Annu Rev Cell Biol 4:155–181
Cooper CE, Bruce D, Nicholls P (1990) Use of oxonol V as a probe of membrane potential in proteoliposomes containing cytochrome oxidase in the submitochondrial orientation. Biochemistry 29:3859–3865
Cossarizza A, Baccarani-Contri M, Kalashnikova G, Franceschi C (1993) A new method for the cytofluorimetric analysis of mitochondrial membrane potential using the J-aggregate forming lipophilic cation 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolcarbocyanine iodide (JC-1). Biochem Biophys Res Commun 97:40–45
Dubot A, Godinot C, Dumur V, Sablonniere B, Stojkovic T, Cuisset JM, Vojtiskova A, Pecina P, Jesina P, Houstek J (2004) GUG is an efficient initiation codon to translate the human mitochondrial ATP6 gene. Biochem Biophys Res Commun 313:687–693
Dykens JA, Stout AK (2001) Assessment of mitochondrial membrane potential in situ using single potentiometric dyes and a novel fluorescence resonance energy transfer technique. Methods Cell Biol. 65:285–309
Ehrenberg B, Montana V, Wei MD, Wuskell JP, Loew LM, (1988) Membrane potential can be determined in individual cells from the nernstian distribution of cationic dyes. Biophys J 53:785–794
Emaus RK, Grunwald R, Lemasters JJ (1986) Rhodamine 123 as a probe of transmembrane potential in isolated rat liver mitochondria: spectral and metabolic properties. Biochim Biophys Acta 850:436–448
Farkas DL, Wei MD, Febbroriello P, Carson JH, Loew LM (1989) Simultaneous imaging of cell and mitochondrial membrane potentials. Biophys J 56:1053–1069
Jackson JB, Nicholls DG (1986) Methods for the determination of membrane potential in bioenergetic systems. Methods Enzymol 127:557–577
Jayaraman S (2005) Flow cytometric determination of mitochondrial membrane potential changes during apoptosis of T lymphocytic and pancreatic beta cell lines: comparison of tetramethylrhodamineethylester (TMRE), chloromethyl-X-rosamine (H2-CMX-Ros) and MitoTracker Red 580 (MTR580). J Immunol Methods 306:68–79
Johnson LV, Walsh ML, Bockus BJ, Chen LB (1981) Monitoring of relative mitochondrial membrane potential in living cells by fluorescence microscopy. J Cell Biol 88:526–535
Johnson LV, Walsh ML, Chen LB (1980) Localization of mitochondria in living cells with rhodamine 123. Proc Natl Acad Sci USA 77:990–994
Juan G, Cavazzoni M, Saez GT, O’Connor JA (1994) A fast kinetic method for assessing mitochondrial membrane potential in isolated hepatocytes with rhodamine 123 and flow cytometry. Cytometry 15:335–342
Kalenak A, McKenzie RJ, Conover TE (1991) Response of the electrochromic dye, merocyanine 540, to membrane potential in rat liver mitochondria. J Membr Biol 123:23–31
Kinnally KW, Tedeschi H, Maloff BL (1978) Use of dyes to estimate the electrical potential of the mitochondrial membrane. Biochemistry 17:3419–3428
Kuznetsov AV, Troppmair J, Sucher R, Hermann M, Saks V, Margreiter R (2006) Mitochondrial subpopulations and heterogeneity revealed by confocal imaging: possible physiological role? Biochim Biophys Acta 1757:686–691
Labajova A, Vojtiskova A, Krivakova P, Kofranek J, Drahota Z, Houstek J (2006) Evaluation of mitochondrial membrane potential using a computerized device with a tetraphenylphosphonium-selective electrode. Anal Biochem 353:37–42
Lakowicz JR (1983) In Principle of fluorescence spectroscopy. Plenum Press, New York
Le SB, Holmuhamedov EL, Narayanan VL, Sausville EA, Kaufmann SH (2006) Adaphostin and other anticancer drugs quench the fluorescence of mitochondrial potential probes. Cell Death Differ 13:151–159
Lecoeur H, Langonne A, Baux L, Rebouillat D, Rustin P, Prevost MC, Brenner C, Edelman L, Jacotot E (2004) Real-time flow cytometry analysis of permeability transition in isolated mitochondria. Exp Cell Res 294:106–117
Lemasters JJ, Chacon E, Ohata H, Harper IS, Nieminen AL, Tesfai SA, Herman B (1995) Measurement of electrical potential, pH, and free calcium ion concentration in mitochondria of living cells by laser scanning confocal microscopy. Methods Enzymol 260:428–444
Mitchell P, Moyle J (1969) Estimation of the membrane potential and pH differences across the cristae membrane of rat liver mitochondria. Eur J Biochem 7:471–484
Montana V, Farkas DL, Loew LM (1989) Dual-wavelength ratiometric measurements of membrane potential. Biochemistry USA 28:4536–4539
Nakayama S, Sakuyama T, Mitaku S, Ohta Y (2002) Fluorescence imaging of metabolic responses in single mitochondria. Biochem Biophys Res Commun 290:23–28
Nicholls DG (2005) Commentary on: ‘old and new data, new issues: the mitochondrial Deltapsi’ by H. Tedeschi. Biochim Biophys Acta 1710:63–65
Nicholls DG (2006) Simultaneous monitoring of ionophore- and inhibitor-mediated plasma and mitochondrial membrane potential changes in cultured neurons. J Biol Chem 281:14864–14874
Nicholls DG, Ward MW (2000) Mitochondrial membrane potential and neuronal glutamate excitotoxicity: mortality and millivolts. Trends Neurosci 23:166–174
Nicholls DG, Ferguson SJ (2002) Bioenergetics 3. Academic press, London
O’Reilly CM, Fogarty KE, Drummond RM, Tuft RA, Walsh JV Jr (2003) Quantitative analysis of spontaneous mitochondrial depolarizations. Biophys J 85:3350–3359
Plasek J, Vojtıskova A, Houstek J (2005) Flow-cytometric monitoring of mitochondrial depolarisation: from fluorescence intensities to millivolts. J Photochem Photobiol – Biol 78:99–108
Reers M, Smith TW, Chen LB (1991) J-aggregate formation of a carbocyanine as a quantitative fluorescent indicator of membrane potential. Biochemistry 30:4480–4486
Rottenberg H (1984) Membrane potential and surface potential in mitochondria: uptake and binding of lipophilic cations. J Membr Biol 81:127–138
Rottenberg H, Wu S (1998) Quantitative assay by flow cytometry of the mitochondrial membrane potential in intact cells. Biochim Biophys Acta 404:393–404
Sack MN (2006) Exploring mitochondria in the intact ischemic heart: advancing technologies to image intracellular function. Circulation 114:1452–1454
Schatten G, Pawley JB (1988) Advances in optical, confocal, and electron microscopic imaging for biomedical researchers. Science 239(4841 Pt 2):G164, G48
Sgarbi G, Baracca A, Lenaz G, Carelli V, Valentino L, Solaini G (2006) Inefficient coupling between proton transport and ATP synthesis may be the pathogenic mechanism for NARP and Leigh syndrome resulting from the T8993G mutation in mtDNA. Biochem J 395:493–500
Smith JC (1990) Potential-sensitive molecular probes in membranes of bioenergetic relevance, Biochim. Biophys Acta 1016:1–28
Toescu EC, Verkhratsky A (2000) Assessment of mitochondrial polarization status in living cells based on analysis of the spatial heterogeneity of rhodamine 123 fluorescence staining. Eur J Physiol 440:941–947
Ubl JJ, Chatton JY, Chen S, Stucki JW (1996) A critical evaluation of in situ measurement of mitochondrial electrical potentials in single hepatocytes. Biochim Biophys Acta 1276:124–132
Vergun O, Reynolds IJ (2004) Fluctuations in mitochondrial membrane potential in single isolated brain mitochondria: modulation by adenine nucleotides and Ca2+. Biophys J 87:3585–3593
Zanotti A, Azzone GF (1980) Safranine as membrane potential probe in rat liver mitochondria. Arch Biochem Biophys 201:255–265
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Solaini, G., Sgarbi, G., Lenaz, G. et al. Evaluating Mitochondrial Membrane Potential in Cells. Biosci Rep 27, 11–21 (2007). https://doi.org/10.1007/s10540-007-9033-4
Published:
Issue Date:
DOI: https://doi.org/10.1007/s10540-007-9033-4