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How DASPMI reveals mitochondrial membrane potential: fluorescence decay kinetics and steady-state anisotropy in living cells.

Ramadass R, Bereiter-Hahn J - Biophys. J. (2008)

Bottom Line: Considerable shortening of the short lifetime component (tau(1)) under a high-membrane-potential condition, such as in the presence of ATP and/or substrate, was similar to quenching and a dramatic decrease of lifetime in polar solvents.Inhibiting respiration by cyanide resulted in a notable increase in the mean lifetime and a decrease in mitochondrial fluorescence.Accordingly, determination of anisotropy in DASPMI-stained mitochondria in living cells revealed a dependence of anisotropy on the membrane potential.

View Article: PubMed Central - PubMed

Affiliation: Cluster of Excellence Macromolecular Complexes, Institute of Cell Biology and Neuroscience, Biocenter, Johann Wolfgang Goethe University, Germany. ramadass@bio.uni-frankfurt.de

ABSTRACT
Spectroscopic responses of the potentiometric probe 2-(4-(dimethylamino)styryl)-1-methylpyridinium iodide (DASPMI) were investigated in living cells by means of a time- and space-correlated single photon counting technique. Spatially resolved fluorescence decays from single mitochondria or only a very few organelles of XTH2 cells exhibited three-exponential decay kinetics. Based on DASPMI photophysics in a variety of solvents, these lifetimes were attributed to the fluorescence from the locally excited state, intramolecular charge transfer state, and twisted intramolecular charge transfer state. A considerable variation in lifetimes among mitochondria of different morphologies and within single cells was evident, corresponding to high physiological variations within single cells. Considerable shortening of the short lifetime component (tau(1)) under a high-membrane-potential condition, such as in the presence of ATP and/or substrate, was similar to quenching and a dramatic decrease of lifetime in polar solvents. Under these conditions tau(2) and tau(3) increased with decreasing contribution. Inhibiting respiration by cyanide resulted in a notable increase in the mean lifetime and a decrease in mitochondrial fluorescence. Increased DASPMI fluorescence under conditions that elevate the mitochondrial membrane potential has been attributed to uptake according to Nernst distributions, delocalization of pi-electrons, quenching processes of the methyl pyridinium moiety, and restricted torsional dynamics at the mitochondrial inner membrane. Accordingly, determination of anisotropy in DASPMI-stained mitochondria in living cells revealed a dependence of anisotropy on the membrane potential. The direct influence of the local electric field on the transition dipole moment of the probe and its torsional dynamics monitor changes in mitochondrial energy status within living cells.

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Mean values of anisotropy and SDs at different intensities (± 50 units) in senescent CEFs. Mean pixel fluorescence intensities (± 50 units) from the summation of horizontally and vertically polarized steady-state intensity images of senescent CEFs have been plotted against the mean anisotropy. The plot was fitted to an exponential function of the form  The consistent increase of anisotropy with fluorescence intensity and the loss of membrane potential apparent from low values of anisotropy in senescent CEFs are evident.
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fig3: Mean values of anisotropy and SDs at different intensities (± 50 units) in senescent CEFs. Mean pixel fluorescence intensities (± 50 units) from the summation of horizontally and vertically polarized steady-state intensity images of senescent CEFs have been plotted against the mean anisotropy. The plot was fitted to an exponential function of the form The consistent increase of anisotropy with fluorescence intensity and the loss of membrane potential apparent from low values of anisotropy in senescent CEFs are evident.

Mentions: Because of the severe loss of dye molecules from the mitochondria in the presence of uncouplers, another physiological condition has been used to demonstrate differences of mitochondrial membrane potential by anisotropy. Decreased mitochondrial membrane potential is a widely found property in senescent cells. Therefore, DASPMI fluorescence anisotropy has been compared in young and old CEFs. The anisotropy in mitochondria of young cells (passage 6) was found to be uniform and higher, in comparison to the old (passage 28) CEF cells. This correlates to the reduced uptake of DASPMI in old CEF cells. Mean values of anisotropy and SDs at different total pixel fluorescence intensities (± 50 units) in young and old cells are shown in Fig. 3. The initial, nearly linear increase of anisotropy in response to mitochondrial membrane potential (as visualized by increased fluorescence intensity) in old cells subsequently coincides at 5000 au intensity with young cells. The higher membrane potential in young CEFs, as apparent from its higher intensity values, exhibits a sharp increase in anisotropy thereafter. The mean anisotropy values and corresponding fluorescence intensities from young and old CEF cells were exponentially correlated (Fig. 3). Hence, membrane-potential-dependent anisotropy in DASPMI has the advantage of giving comparable measures of membrane potential from different cell types stained with different concentrations due to their independence from absolute intensity.


How DASPMI reveals mitochondrial membrane potential: fluorescence decay kinetics and steady-state anisotropy in living cells.

Ramadass R, Bereiter-Hahn J - Biophys. J. (2008)

Mean values of anisotropy and SDs at different intensities (± 50 units) in senescent CEFs. Mean pixel fluorescence intensities (± 50 units) from the summation of horizontally and vertically polarized steady-state intensity images of senescent CEFs have been plotted against the mean anisotropy. The plot was fitted to an exponential function of the form  The consistent increase of anisotropy with fluorescence intensity and the loss of membrane potential apparent from low values of anisotropy in senescent CEFs are evident.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2553107&req=5

fig3: Mean values of anisotropy and SDs at different intensities (± 50 units) in senescent CEFs. Mean pixel fluorescence intensities (± 50 units) from the summation of horizontally and vertically polarized steady-state intensity images of senescent CEFs have been plotted against the mean anisotropy. The plot was fitted to an exponential function of the form The consistent increase of anisotropy with fluorescence intensity and the loss of membrane potential apparent from low values of anisotropy in senescent CEFs are evident.
Mentions: Because of the severe loss of dye molecules from the mitochondria in the presence of uncouplers, another physiological condition has been used to demonstrate differences of mitochondrial membrane potential by anisotropy. Decreased mitochondrial membrane potential is a widely found property in senescent cells. Therefore, DASPMI fluorescence anisotropy has been compared in young and old CEFs. The anisotropy in mitochondria of young cells (passage 6) was found to be uniform and higher, in comparison to the old (passage 28) CEF cells. This correlates to the reduced uptake of DASPMI in old CEF cells. Mean values of anisotropy and SDs at different total pixel fluorescence intensities (± 50 units) in young and old cells are shown in Fig. 3. The initial, nearly linear increase of anisotropy in response to mitochondrial membrane potential (as visualized by increased fluorescence intensity) in old cells subsequently coincides at 5000 au intensity with young cells. The higher membrane potential in young CEFs, as apparent from its higher intensity values, exhibits a sharp increase in anisotropy thereafter. The mean anisotropy values and corresponding fluorescence intensities from young and old CEF cells were exponentially correlated (Fig. 3). Hence, membrane-potential-dependent anisotropy in DASPMI has the advantage of giving comparable measures of membrane potential from different cell types stained with different concentrations due to their independence from absolute intensity.

Bottom Line: Considerable shortening of the short lifetime component (tau(1)) under a high-membrane-potential condition, such as in the presence of ATP and/or substrate, was similar to quenching and a dramatic decrease of lifetime in polar solvents.Inhibiting respiration by cyanide resulted in a notable increase in the mean lifetime and a decrease in mitochondrial fluorescence.Accordingly, determination of anisotropy in DASPMI-stained mitochondria in living cells revealed a dependence of anisotropy on the membrane potential.

View Article: PubMed Central - PubMed

Affiliation: Cluster of Excellence Macromolecular Complexes, Institute of Cell Biology and Neuroscience, Biocenter, Johann Wolfgang Goethe University, Germany. ramadass@bio.uni-frankfurt.de

ABSTRACT
Spectroscopic responses of the potentiometric probe 2-(4-(dimethylamino)styryl)-1-methylpyridinium iodide (DASPMI) were investigated in living cells by means of a time- and space-correlated single photon counting technique. Spatially resolved fluorescence decays from single mitochondria or only a very few organelles of XTH2 cells exhibited three-exponential decay kinetics. Based on DASPMI photophysics in a variety of solvents, these lifetimes were attributed to the fluorescence from the locally excited state, intramolecular charge transfer state, and twisted intramolecular charge transfer state. A considerable variation in lifetimes among mitochondria of different morphologies and within single cells was evident, corresponding to high physiological variations within single cells. Considerable shortening of the short lifetime component (tau(1)) under a high-membrane-potential condition, such as in the presence of ATP and/or substrate, was similar to quenching and a dramatic decrease of lifetime in polar solvents. Under these conditions tau(2) and tau(3) increased with decreasing contribution. Inhibiting respiration by cyanide resulted in a notable increase in the mean lifetime and a decrease in mitochondrial fluorescence. Increased DASPMI fluorescence under conditions that elevate the mitochondrial membrane potential has been attributed to uptake according to Nernst distributions, delocalization of pi-electrons, quenching processes of the methyl pyridinium moiety, and restricted torsional dynamics at the mitochondrial inner membrane. Accordingly, determination of anisotropy in DASPMI-stained mitochondria in living cells revealed a dependence of anisotropy on the membrane potential. The direct influence of the local electric field on the transition dipole moment of the probe and its torsional dynamics monitor changes in mitochondrial energy status within living cells.

Show MeSH
Related in: MedlinePlus