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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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Pseudo color time-integrated QA image and corresponding decay kinetics (Table 1) in various ROIs of a DASPMI-stained XTH2 cell. The variable mitochondrial membrane potential can be inferred from its intensity distribution. Fluorescence lifetime analysis was performed over various ROIs, including single mitochondria or several organelles. Fluorescence from ROI 3 is primarily from the nucleus, with a minor contribution from mitochondria only. Bar, 5 μm.
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fig1: Pseudo color time-integrated QA image and corresponding decay kinetics (Table 1) in various ROIs of a DASPMI-stained XTH2 cell. The variable mitochondrial membrane potential can be inferred from its intensity distribution. Fluorescence lifetime analysis was performed over various ROIs, including single mitochondria or several organelles. Fluorescence from ROI 3 is primarily from the nucleus, with a minor contribution from mitochondria only. Bar, 5 μm.

Mentions: Spatially resolved fluorescence lifetime imaging is the method of choice to relate the complex photophysics of DASPMI to its interaction with mitochondria at different physiological states, i.e., to elucidate how DASPMI fluorescence monitors mitochondrial membrane potential. Using a QA detector, fluorescence decay values can be derived even for single mitochondria within living cells (Fig. 1 and Table 1). In Fig. 1, fluorescence intensities have been color-coded and fluorescence decay has been calculated for ROIs containing one mitochondrion or only a very few organelles. Fluorescence intensity is highest in mitochondria in the perinuclear regions and decreases toward the cell periphery, a behavior often observed in larger cells. A whole cell is shown in Fig. 1; the ROIs with the higher numbers lie in the cell periphery. In the extreme periphery, single mitochondria can no longer be identified because of motion-related blur (this QA picture was taken while the cells were alive and mitochondria were moving within the cytoplasm during the photon collection time).


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

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

Pseudo color time-integrated QA image and corresponding decay kinetics (Table 1) in various ROIs of a DASPMI-stained XTH2 cell. The variable mitochondrial membrane potential can be inferred from its intensity distribution. Fluorescence lifetime analysis was performed over various ROIs, including single mitochondria or several organelles. Fluorescence from ROI 3 is primarily from the nucleus, with a minor contribution from mitochondria only. Bar, 5 μm.
© Copyright Policy
Related In: Results  -  Collection

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

fig1: Pseudo color time-integrated QA image and corresponding decay kinetics (Table 1) in various ROIs of a DASPMI-stained XTH2 cell. The variable mitochondrial membrane potential can be inferred from its intensity distribution. Fluorescence lifetime analysis was performed over various ROIs, including single mitochondria or several organelles. Fluorescence from ROI 3 is primarily from the nucleus, with a minor contribution from mitochondria only. Bar, 5 μm.
Mentions: Spatially resolved fluorescence lifetime imaging is the method of choice to relate the complex photophysics of DASPMI to its interaction with mitochondria at different physiological states, i.e., to elucidate how DASPMI fluorescence monitors mitochondrial membrane potential. Using a QA detector, fluorescence decay values can be derived even for single mitochondria within living cells (Fig. 1 and Table 1). In Fig. 1, fluorescence intensities have been color-coded and fluorescence decay has been calculated for ROIs containing one mitochondrion or only a very few organelles. Fluorescence intensity is highest in mitochondria in the perinuclear regions and decreases toward the cell periphery, a behavior often observed in larger cells. A whole cell is shown in Fig. 1; the ROIs with the higher numbers lie in the cell periphery. In the extreme periphery, single mitochondria can no longer be identified because of motion-related blur (this QA picture was taken while the cells were alive and mitochondria were moving within the cytoplasm during the photon collection time).

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