Fluorescence membrane potentials

Fluorescence-based methods do not directly measure ionic current but, rather, measure either membrane-potential-dependent or ion-concentration-dependent changes of fluorescence signals (from fluorescent dyes loaded into the cytosol or cell membrane) as a result of ionic flux. Because fluorescence-based methods give robust and homogeneous cell population measurement, these assays are relatively easy to set up and achieve high throughput. 

Mandala M, Serck-Hanssen G, Martino G, Helle KB (1999) The fluorescent cationic dye rhodamine 6G as a probe for membrane potential in bovine aortic endothelial cells. Anal Biochem 274 1-6... 

The method described here can be applied to gain insight about environmental effects on the absorption and fluorescence of other chromophores and environments, including dyes used to probe the membrane potentials. 

Owing to the small changes induced by typical membrane potentials, the voltage sensitivity may be accurately expressed as a sum of independent terms. A fractional change in signal (absorbance or fluorescence) per 100 mV of applied membrane potential change was given by Fromherz et al. [66] as ... 

Grinvald A, Hildesheim R, Farber IC, Anglister L (1982) Improved fluorescent-probes for the measurement of rapid changes in membrane-potential. Biophys J 39(3) 301—308... 

Ross WN, Salzberg BM, Cohen LB et al (1977) Changes in absorption, fluorescence, dichroism and birefringence in stained giant axons optical measurement of membrane potential. J Membr Biol 33 141-183... 

Schummer, U., Schiefer, H. G. and Gerhardt, U. (1979). Mycoplasma membrane-potentials determined by potential-sensitive fluorescent dyes. Curr. Microbiol. 2, 191-194. 

Adkins, C. E., Pillai, G. V., Kerby, J., et al. (2001) alpha4beta3delta GABA(A) receptors characterized by fluorescence resonance energy transfer-derived measurements of membrane potential. J. Biol. Chem. 276, 38934-38939. 

Mitochondria are distinct organelles with two membranes. The outer membrane limits the organelle and the inner membrane is thrown into folds or shelves that project inward and are called cristae mitochondriales. The uptake of most mitochondrion-selective dyes is dependent on the mitochondrial membrane potential. Conventional fluorescent stains for mitochondria, such as rhodamine and tetramethylrosamine, are readily sequestered by functioning mitochondria. They are, however, subsequently washed out of the cells once the mitochondrion s membrane potential is lost. This characteristic limits their use in experiments in which cells must be treated with aldehyde-based fixatives or other agents that affect the energetic state of the mitochondria. To overcome this limitation, the research... 

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... 

Figure 5.3. (a) Example for a compound used in the study of membrane potentials and (b) principle of funcboning of visualizing nerve pulses in a living cell. When a nerve pulse passes, the membrane potential changes, and this induces a change in the fluorescence intensity of the probe. The temporal and spatial profile of these changes can be followed by time-resolved methods. 

L. B. Cohen, B. M. Salzberg, H. V. Davila, W. N. Ross, D. Landowne, A. S. Waggoner, and C. H. Wang, Changes in axon fluorescence during activity Molecular probes of membrane potential, J. Membrane Biol. 19, 1 (1974). 

Szollosi, J., Damjanovich, S., Mulhem, S. A., and Tron, L. (1987) Fluorescence energy transfer and membrane potential measurements monitor dynamic properties of cell membranes a critical review. Prog. Biophys. Mol. Biol. 49, 65-87. 

Mitochondrial function and potential dmg effects such as uncoupling and inhibition of OXPHOS are most directly studied by monitoring oxygen consumption or membrane potential, using polarographic electrodes and fluorescent dyes, respectively, but other techniques are available, as noted below. 

Dykens, J.A. and Stout, A.K. (2001) Fluorescent dyes and assessment of mitochondrial membrane potential in FRET Modes. Fluorescent dyes and assessment of mitochondrial membrane potential in FRET modes. Methods in Cell Biology, 65, 285-309. 

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