Detection of mitochondrial defects by laser fluorimetry

The mitochondrial function in skeletal muscle biopsies of three patients with chronic progressive external ophthalmoplegia, having deletions of the mitochondrial DNA, was studied by laser-excited fluorescence measurements of NAD(P)H and flavoproteins in saponin-skinned fibers. We detected substantially elevated steady state redox states of the mitochondrial NAD-system in the muscle fibers of these patients. Moreover, the respiratory chain-linked autofluorescence changes in the muscle fibers of these patients were larger in comparison to controls indicating substantial alterations of the mitochondrial content. These results are in line with the presence of elevated numbers of partially respiratory chain inhibited mitochondria in the skeletal muscle of chronic progressive external ophthalmoplegia patients. (Mol Cell Biochem 174: 97–100, 1997)     

Spectral properties of fluorescent flavoproteins of isolated rat liver mitochondria

The different flavoproteins contributing to flavin fluorescence of isolated rat liver mitochondria have distinct excitation and emission spectra. The NAD-linked flavin component was identified as alpha-lipoamide dehydrogenase, while the non-NAD-linked component was found to be electron transfer flavoprotein. The differences in excitation and emission properties of the mitochondrial flavoproteins permit selective recording of their redox state changes in isolated mitochondria.     

DNA-binding properties of Mblk-1, a putative transcription factor from the honeybee

The membrane potentials, rates of NAD(P)H formation, and rates of flavoprotein reduction have been measured for single mitochondria isolated from porcine hearts. These metabolic responses were elicited by the addition of malate and measured using fluorescence microscopy. For the measurements of mitochondrial membrane potential, mitochondria were stained with tetramethylrhodamine ethyl ester, and the membrane potentials of single mitochondria were determined. Individual mitochondria maintained the membrane potential at around -80 mV before addition of malate. Upon the addition of malate, each mitochondrion was rapidly polarized to around -100 approximately -140 mV and underwent repeated cycles of polarization and depolarization, which were probably caused by openings and closings of permeability transition pores. NAD(P)(+) and flavoprotein were reduced immediately after addition of malate and then slowly became reoxidized. Thus, single mitochondria can undergo rapid and repetitive changes in membrane potential, but not in the redox state of NAD(P)H and flavoprotein.     

Contribution of different enzymes to flavoprotein fluorescence of isolated rat liver mitochondria

The fluorescence signal of flavoproteins of rat liver mitochondria was investigated to determine the respective contributions of the various flavoenzymes. About 50% of the overall signal were found to be NAD-linked and caused by alpha-lipoamide dehydrogenase flavin (Em7.4 = -283 mV). Roughly 25% were due to a flavoprotein reducible in a non-NAD-linked reaction. This fluorescent flavoenzyme (Em7.4 = -52 mV) has been tentatively identified as a flavoprotein of the fatty-acid-oxidizing system, most probably the electron transfer flavoprotein. The remaining 25% of the signal are accounted for by flavoenzymes which are reducible by dithionite only. These flavoenzymes were not involved in the flavoprotein fluorescence alterations accompanying changes in electron flow through the respiratory chain. Contributions of other mitochondrial flavoproteins such as succinate dehydrogenase, NADH dehydrogenase, alpha-glycerophosphate dehydrogenase, proline dehydrogenase, and choline oxidase, to the overall flavin fluorescence signal of isolated rat liver mitochondria can be neglected.     

Two-photon fluorescence spectroscopy and microscopy of NAD(P)H and flavoprotein

Two-photon (2P) ratiometric redox fluorometry and microscopy of pyridine nucleotide (NAD(P)H) and flavoprotein (FP) fluorescence, at 800-nm excitation, has been demonstrated as a function of mitochondrial metabolic states in isolated adult dog cardiomyocytes. We have measured the 2P-excitation spectra of NAD(P)H, flavin adenine dinucleotide (FAD), and lipoamide dehydrogenase (LipDH) over the wavelength range of 720-1000 nm. The 2P-excitation action cross sections (sigma2P) increase rapidly at wavelengths below 800 nm, and the maximum sigma2P of LipDH is approximately 5 and 12 times larger than those of FAD and NAD(P)H, respectively. Only FAD and LipDH can be efficiently excited at wavelengths above 800 nm with a broad 2P-excitation band around 900 nm. Two autofluorescence spectral regions (i.e., approximately 410-490 nm and approximately 510-650 nm) of isolated cardiomyocytes were imaged using 2P-laser scanning microscopy. At 750-nm excitation, fluorescence of both regions is dominated by NAD(P)H emission, as indicated by fluorescence intensity changes induced by mitochondrial inhibitor NaCN and mitochondria uncoupler carbonyl cyanide p-(trifluoromethoxy) phenyl hydrazone (FCCP). In contrast, 2P-FP fluorescence dominates at 900-nm excitation, which is in agreement with the sigma2P measurements. Finally, 2P-autofluorescence emission spectra of single cardiac cells have been obtained, with results suggesting potential for substantial improvement of the proposed 2P-ratiometric technique.     

The respiratory chain of plant mitochondria: x. Oxidation-reduction potentials of the flavoproteins of skunk cabbage mitochondria

The oxidation-reduction potentials of the flavoproteins of skunk cabbage (Symplocarpus foetidus) mitochondria have been measured under anaerobic conditions by means of a combined spectrophotometric or fluorimetric-potentiometric method. Five components were resolved whose oxidation-reduction reactions corresponded to two-electron changes, as expected for flavoproteins. The midpoint potentials at pH 7.2 are as follows, listed in order of increasingly negative potential: +170 millivolts, +110 millivolts, +20 millivolts, −70 millivolts, and −155 millivolts. The most negative component was highly fluorescent; the other components could only be identified by their characteristic absorbance changes. In addition to these components, which are mitochondrial, variable amounts of a very highly fluorescent flavoprotein with a midpoint potential of −215 millivolts was found. This component appears to be extra-mitochondrial. The same midpoint potential values at pH 7.2 were obtained with mitochondria in the uncoupled state as in mitochondria energized with ATP in the absence of phosphate.     

Effects of hypoxia on relationships between cytosolic and mitochondrial NAD(P)H redox and superoxide generation in coronary arterial smooth muscle

Since controversy exists on how hypoxia influences vascular reactive oxygen species (ROS) generation, and our previous work provided evidence that it relaxes endothelium-denuded bovine coronary arteries (BCA) in a ROS-independent manner by promoting cytosolic NADPH oxidation, we examined how hypoxia alters relationships between cytosolic and mitochondrial NAD(P)H redox and superoxide generation in BCA. Methods were developed to image and interpret the effects of hypoxia on NAD(P)H redox based on its autofluorescence in the cytosolic, mitochondrial, and nuclear regions of smooth muscle cells isolated from BCA. Aspects of anaerobic glycolysis and cytosolic NADH redox in BCA were assessed from measurements of lactate and pyruvate. Imaging changes in mitosox and dehydroethidium fluorescence were used to detect changes in mitochondrial and cytosolic-nuclear superoxide, respectively. Hypoxia appeared to increase mitochondrial and decrease cytosolic-nuclear superoxide under conditions associated with increased cytosolic NADH (lactate/pyruvate), mitochondrial NAD(P)H, and hyperpolarization of mitochondria detected by tetramethylrhodamine methyl-ester perchlorate fluorescence. Rotenone appeared to increase mitochondrial NAD(P)H and superoxide, suggesting hypoxia could increase superoxide generation by complex I. However, hypoxia decreased mitochondrial superoxide in the presence of contraction to 30 mM KCl, associated with decreased mitochondrial NAD(P)H. Thus, while hypoxia augments NAD(P)H redox associated with increased mitochondrial superoxide, contraction with KCl reverses these effects of hypoxia on mitochondrial superoxide, suggesting mitochondrial ROS increases do not mediate hypoxic relaxation in BCA. Since hypoxia lowers pyruvate, and pyruvate inhibits hypoxia-elicited relaxation and NADPH oxidation in BCA, mitochondrial control of pyruvate metabolism associated with cytosolic NADPH redox regulation could contribute to sensing hypoxia.     

Quantitative NAD(P)H/flavoprotein autofluorescence imaging reveals metabolic mechanisms of pancreatic islet pyruvate response

Glucose-stimulated insulin secretion is a multistep process dependent on beta-cell metabolic flux. Our previous studies on intact pancreatic islets used two-photon NAD(P)H imaging as a quantitative measure of the combined redox signal from NADH and NADPH (referred to as NAD(P)H). These studies showed that pyruvate, a non-secretagogue, enters beta-cells and causes a transient rise in NAD(P)H. To further characterize the metabolic fate of pyruvate, we have now developed one-photon flavoprotein microscopy as a simultaneous assay of lipoamide dehydrogenase (LipDH) autofluorescence. This flavoprotein is in direct equilibrium with mitochondrial NADH. Hence, a comparison of LipDH and NAD(P)H autofluorescence provides a method to distinguish the production of NADH, NADPH, or both. Using this method, the glucose dose response is consistent with an increase in both NADH and NADPH. In contrast, the transient rise in NAD(P)H observed with pyruvate stimulation is not accompanied by a significant change in LipDH, which indicates that pyruvate raises cellular NADPH without raising NADH. In comparison, methyl pyruvate stimulated a robust NADH and NADPH response. These data provide new evidence that exogenous pyruvate does not induce a significant rise in mitochondrial NADH. This inability likely results in its failure to produce the ATP necessary for stimulated secretion of insulin. Overall, these data are consistent with either a restricted pyruvate dehydrogenase-dependent metabolism or a buffering of the NADH response by other metabolic mechanisms.     

The Respiratory Chain of Plant Mitochondria: VI. Flavoprotein Components of the Respiratory Chain of Mung Bean Mitochondria

Redox changes of the flavoproteins of mung bean (Phaseolus aureus) mitochondria were measured by differential absorbance at 468 to 493 nanometers and by fluorescence emission above 500 nanometers excited at 436 nanometers. Four flavoproteins are distinguishable by the ratio of their fluorescence to absorbance changes, and by their requirement, or lack of it, for energy-linked reverse electron transport for reduction by succinate. Two flavoproteins are reduced by succinate in fully depleted mitochondria which lack the capacity for reverse electron transport. These are designated Fp_ha and Fp_hf and have fluorescence to absorbance ratios of 0 and 1.4, respectively. The two flavoproteins have the same half-time for oxidation, but Fp_hf is reduced more slowly than Fp_ha by substrate in the presence of cyanide. One flavoprotein with a fluorescence to absorbance ratio of 0 is not reduced by succinate in anaerobic, fully depleted mitochondria, but is rapidly reduced on subsequent addition of malate; it is designated Fp_m. The fourth distinguishable flavoprotein component is reducible by succinate in an energy-linked reaction, even in partially depleted mitochondria. This component has a fluorescence to absorbance ratio of 3.8 and is designated Fp_1f. In addition to these four flavoproteins reducible by substrates, there is a highly fluorescent flavin-containing component in or associated with these mitochondria, which is rapidly reduced by dithionite.     

Transcranial flavoprotein fluorescence imaging of mouse cortical activity and plasticity

Endogenous fluorescence signals derived from mitochondria reflect activity-dependent changes in brain metabolism and may be exploited in functional brain imaging. Endogenous flavoprotein fluorescence imaging in mice is especially important because many genetically manipulated strains of mice are available and the transparent skull of mice allows transcranial fluorescence imaging of cortical activities. In the primary sensory areas of mice, cortical activities and experience-dependent plasticity have been investigated using transcranial fluorescence imaging. Furthermore, differential imaging, based on stimulus specificity of cortical areas, distinguished activities in higher visual areas around the primary visual cortex from those in primary visual cortex. The combination of transcranial fluorescence imaging with the suppression of cortical activities using photobleaching of flavoproteins is expected to aid in elucidating the roles of sensory cortices including higher areas in mice.     

Nicotinamide adenine dinucleotide fluorescence spectroscopy and imaging of isolated cardiac myocytes.

Nicotinamide adenine dinucleotide (NADH) plays a critical role in oxidative phosphorylation as the primary source of reducing equivalents to the respiratory chain. Using a modified fluorescence microscope, we have obtained spectra and images of the blue autofluorescence from single rat cardiac myocytes. The optical setup permitted rapid acquisition of fluorescence emission spectra (390-595 nm) or intensified digital video images of individual myocytes. The spectra showed a broad fluorescence centered at 447 +/- 0.2 nm, consistent with mitochondrial NADH. Addition of cyanide resulted in a 100 +/- 10% increase in fluorescence, while the uncoupler FCCP resulted in a 82 +/- 4% decrease. These two transitions were consistent with mitochondrial NADH and implied that the myocytes were 44 +/- 6% reduced under the resting control conditions. Intracellular fluorescent structures were observed that correlated with the distribution of a mitochondrial selective fluorescent probe (DASPMI), the mitochondrial distribution seen in published electron micrographs, and a metabolic digital subtraction image of the cyanide fluorescence transition. These data are consistent with the notion that the blue autofluorescence of rat cardiac myocytes originates from mitochondrial NADH.     

Oxidation-reduction midpoint potentials of mitochondrial flavoproteins and their intramitochondrial localization

Spectrophotometric and fluorimetric substrate couple titrations and potentiometric spectrophotometric titrations were used to determine the oxidation-reduction potentials of components showing absorbance or fluorescence at the wavelengths attributable to the flavoproteins of mitochondria fractionated using digitonin together with sonication. A pure mitoplast fraction devoid of cytochrome b₅ contamination could be obtained using 230 µg digitonin/mg of mitochondrial protein. The digitonin-soluble fraction contained a species havingE _m 7 .4=–123 mV and probably represents the outer membrane flavoproteins. The inner membrane-matrix fraction, treated with ultrasound, provided evidence of a flavoprotein species with low redox potential (E _m 7 .4=–302 mV) in the matrix fraction. The –302 mV component is probably lipoamide dehydrogenase. A high redox potential species withE _m 7 .4=+19 mV in titrations with the succinate fumarate couple was located in the inner membrane vesicles and is probably identical with succinate dehydrogenase. The electron-transferring flavoprotein (ETF) was isolated from bovine heart mitochondria and itsE _m 7 .4=–74 mV determined. The component in the matrix fraction with an apparentE _m 7 .4=–56 mV probably represents ETF, and that in the inner membrane fraction with an apparentE _m 7 .4=–43 mV the NADH dehydrogenase flavoprotein. A component in an apparently low concentration withE _m 7 .4=+30 mV was detected in the inner membrane fraction. This probably represents the ETF-dehydrogenase flavoprotein. The origin of the flavoprotein fluorescence of mitochondria and intact tissues is discussed.     

Skeletal muscle NAD(P)H two-photon fluorescence microscopy in vivo: topology and optical inner filters

Two-photon excitation fluorescence microscopy (TPEFM) permits the investigation of the topology of intercellular events within living animals. TPEFM was used to monitor the distribution of mitochondrial reduced nicotinamide adenine dinucleotide (NAD(P)H) in murine skeletal muscle in vivo. NAD(P)H fluorescence emission was monitored (~460 nm) using 710–720 nm excitation. High-resolution TPEFM images were collected up to a depth of 150 μm from the surface of the tibialis anterior muscle. The NAD(P)H fluorescence images revealed subcellular structures consistent with subsarcolemmal, perivascular, intersarcomeric, and paranuclear mitochondria. In vivo fiber typing between IIB and IIA/D fibers was possible using the distribution and content of mitochondria from the NAD(P)H fluorescence signal. The intersarcomeric mitochondria concentrated at the Z-line in the IIB fiber types resulting in a periodic pattern with a spacing of one sarcomere (2.34 ± 0.17 μm). The primary inner filter effects were nearly equivalent to water, however, the secondary inner filter effects were highly significant and dynamically affected the observed emission frequency and amplitude of the NAD(P)H fluorescence signal. These data demonstrate the feasibility, and highlight the complexity, of using NAD(P)H TPEFM in skeletal muscle to characterize the topology and metabolic function of mitochondria within the living mouse.     

Do single mitochondria contain zones with different membrane potential?

Observations of Lan Bo Chen’s group using a mitochondria-selective fluorochrome 5,5’,6,6’- tetrachloro- 1,1’,3,3’- tetraethylbenzimidazolocarbocyanine iodide (JC-1) indicate that mitochondria in situ may have zones of different electrochemical potential along their length. This was indicated by the formation of J-aggregates of this dye at distinct sites along a single mitochondrion. Also, intensity variations along single mitochondria were found with diamino-styryl-pyridinium methiodide (DASPMI), another fluorochrome that selectively stains mitochondria depending on their electrochemical potential. DASPMI exchanges easily with the cytoplasm and changes its quantum yield when bound to mitochondrial membranes. Therefore, fluorescence intensity is primarily controlled by the membrane environment rather than by mass accumulation. Two possible explanations of intramitochondrial fluorescence intensity variations have to be discussed: variations in the amount of mitochondrial inner membrane per unit of projection area (or voxel), and differences in the electrochemical gradient. This problem has been approached by comparing fluoro-micrographs of mitochondria in endothelial cells stained with either JC-1 or DASPMI with electron micrographs of the same mitochondria after fixation with glutardialdehyde and osmium tetroxide and ultrathin sectioning. JC-1 red fluorescence (revealing J-aggregate formation) as well as high-intensity staining with DASPMI correlate roughly with the local thickness of mitochondria; no differences in the crista organization are revealed for those areas or mitochondria exhibiting red JC-1 fluorescence and those with green fluorescence. The distance between red fluorescing areas in a single mitochondrion seem to be caused by competition for dye molecules placed in between centres of JC-1 aggregation. Isolated mitochondria are of uniform small size and spherical shape; therefore, no differences in shape interfere with JC-1 staining. Thus JC-1 may be an appropriate indicator of membrane potential in isolated mitochondria. In living cells mitochondria often are large and elongated, and thus the situation is not straightforward to interpret. However, evidence is provided that there are submitochondrial zones, which differ in membrane potential from one adjacent area to another, because DASPMI staining of intramitochondrial zones reveals differences in fluorescence intensity and preferred photodamage of these areas. In some cases separation of the zones of higher membrane potential by cristae traversing the whole diameter of a mitochondrion has been observed. Local photobleaching of stained mitochondria results in a loss of fluorescence along the total length of a mitochondrion. However, this type of bleaching develops over tens of seconds, not in the very short time range (e.g. ms) expected from the discharge of all the membranes if they were electrically coupled.     

Regulation of brain mitochondrial H2O2 production by membrane potential and NAD(P)H redox state

Mitochondrial production of reactive oxygen species (ROS) at Complex I of the electron transport chain is implicated in the etiology of neural cell death in acute and chronic neurodegenerative disorders. However, little is known regarding the regulation of mitochondrial ROS production by NADH-linked respiratory substrates under physiologically realistic conditions in the absence of respiratory chain inhibitors. This study used Amplex Red fluorescence measurements of H2O2 to test the hypothesis that ROS production by isolated brain mitochondria is regulated by membrane potential (DeltaPsi) and NAD(P)H redox state. DeltaPsi was monitored by following the medium concentration of the lipophilic cation tetraphenylphosphonium with a selective electrode. NAD(P)H autofluorescence was used to monitor NAD(P)H redox state. While the rate of H2O2 production was closely related to DeltaPsi and the level of NAD(P)H reduction at high values of DeltaPsi, 30% of the maximal rate of H2O2 formation was still observed in the presence of uncoupler (p-trifluoromethoxycarbonylcyanide phenylhydrazone) concentrations that provided for maximum depolarization of DeltaPsi and oxidation of NAD(P)H. Our findings indicate that ROS production by mitochondria oxidizing physiological NADH-dependent substrates is regulated by DeltaPsi and by the NAD(P)H redox state over ranges consistent with those that exist at different levels of cellular energy demand.     

Changes in mitochondrial redox state, membrane potential and calcium precede mitochondrial dysfunction in doxorubicin-induced cell death

Mitochondria play central roles in cell life as a source of energy and in cell death by inducing apoptosis. Many important functions of mitochondria change in cancer, and these organelles can be a target of chemotherapy. The widely used anticancer drug doxorubicin (DOX) causes cell death, inhibition of cell cycle/proliferation and mitochondrial impairment. However, the mechanism of such impairment is not completely understood. In our study we used confocal and two-photon fluorescence imaging together with enzymatic and respirometric analysis to study short- and long-term effects of doxorubicin on mitochondria in various human carcinoma cells. We show that short-term (< 30 min) effects include i) rapid changes in mitochondrial redox potentials towards a more oxidized state (flavoproteins and NADH), ii) mitochondrial depolarization, iii) elevated matrix calcium levels, and iv) mitochondrial ROS production, demonstrating a complex pattern of mitochondrial alterations. Significant inhibition of mitochondrial endogenous and uncoupled respiration, ATP depletion and changes in the activities of marker enzymes were observed after 48 h of DOX treatment (long-term effects) associated with cell cycle arrest and death.     

Fluorimetry of mitochondria in cells vitally stained with DASPMI or rhodamine 6 GO

The fluorescent dyes DASPMI and rhodamine 6 GO specifically stain mitochondria in living cells. Dye concentrations from 10^?8 to 5 × 10^?6 mole l^?1 can be used. Excitation and emission spectra, and quantum efficiency of DASPMI depend on solvent characteristics. Spectra of mitochondria in living cells correspond to those in phospholipids (excitation around 470 nm, emission 560–570 nm). Fluorescence intensity of DASPMI is a measure for the energization of mitochondria, as revealed by in vitro studies. In living cells uptake of the dye is strongly influenced by inhibitors of oxidative phosphorylation (i.e. by oligomycin, FCCP). Distribution of fluorescence intensity indicates an intracellular gradient in energy load of endothelial cells. Single mitochondria exhibit oscillations in fluorescence. Mitochondria loaded with DASPMI release the dye suddenly into the cytoplasm on treatment with FCCP. Cyanide and antimycin however, do not diminish fluorescence in vivo under optimal nutritional conditions, while they do so in mitochondrial suspension, indicating different mitochondrial behaviour in vivo and in suspension.     

Genetically encoded fluorescent sensors for intracellular NADH detection

We have developed genetically encoded fluorescent sensors for reduced nicotinamide adenine dinucleotide (NADH), which manifest a large change in fluorescence upon NADH binding. We demonstrate the utility of these sensors in mammalian cells by monitoring the dynamic changes in NADH levels in subcellular organelles as affected by NADH transport, glucose metabolism, electron transport chain function, and redox environment, and we demonstrate the temporal separation of changes in mitochondrial and cytosolic NADH levels with perturbation. These results support the view that cytosolic NADH is sensitive to environmental changes, while mitochondria have a strong tendency to maintain physiological NADH homeostasis. These sensors provide a very good alternative to existing techniques that measure endogenous fluorescence of intracellular NAD(P)H and, owing to their superior sensitivity and specificity, allow for the selective monitoring of total cellular and compartmental responses of this essential cofactor.     

A comparative analysis of the action of oxidative metabolic inhibitors on the luminescence parameters of normal and tumorous cells]

Comparative investigation of different mitochondrial oxidative metabolism inhibitors action on NAD(P)H and flavoproteins fluorescence intensity of minimal transformed 3T3 NIH mouse fibroblasts and rat HTC hepatoma cells was made. Principle differences were shown between these cells in oxidized flavoproteins fluorescence intensity changes under the action of used inhibitors. It is suggested that the unusual HTC hepatoma cells flavin fluorescence intensity increase is connected with the oxidation of unidentified flavin-containing component functionally attached to mitochondrial respiratory chain.     

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

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 (τ₁) 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 τ₂ and τ₃ 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 π-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.