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Biophys J
2008 Oct 01;958:4068-76. doi: 10.1529/biophysj.108.135079.
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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
.
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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.
FIGURE 1. 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.
FIGURE 2. Distribution of polarization-resolved fluorescence intensities and corresponding anisotropy values of DASPMI-stained XTH2 cells. The very distinct high anisotropy at high-intensity regions (inserts) inside several individual mitochondria is apparent. Such submitochondrial zones of higher membrane potential are known to exist from previous studies in XTH2 cells. The plot of mean anisotropy values and corresponding pixel fluorescence intensities (± 50 units) were fitted to an exponential function of the form \documentclass[10pt]{article}
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\begin{equation*}y=y_{0}+Ae^{({\mathrm{x}}/{\mathrm{t}})}.\end{equation*}\end{document}
FIGURE 3. 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 \documentclass[10pt]{article}
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\begin{document}
\begin{equation*}y=y_{0}+Ae^{({\mathrm{x/t}})}.\end{equation*}\end{document} 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.
FIGURE 4. Location and orientation of DASPMI in the mitochondrial inner membrane. DASPMI preferentially locates its positively charged methyl pyridinium ring in the aqueous interface facing the outer mitochondrial membrane, and its hydrophobic moiety along the hydrocarbon chain. As for orientation, it aligns itself along the local electric field for a maximum electrochromic response. Under high-membrane-potential conditions, the delocalization of π-electrons leads to a less polar structure, imposing steric constraints on the single bonds neighboring the olefinic double bond.
FIGURE 5. Emission wavelength-dependent anisotropy of DASPMI. In response to high-membrane-potential conditions, electrons are withdrawn from the electron-rich aniline moiety, leading to a less polar DASPMI structure. Consequently, an increase in rigidity of the single bonds (i.e., double bond-like character) neighboring the olefinic double bond leads to an increase in anisotropy. The dependence of anisotropy on emission wavelength indicates the gradual change in molecular configuration and hence the transition in dipole moment from the initially excited Franck-Condon level.
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