Cytoplasmic viscosity near the cell plasma membrane: measurement by evanescent field frequency-domain microfluorimetry.

The purpose of this study was to determine whether the unique physical milieu just beneath the cell plasma membrane influences the rheology of fluid-phase cytoplasm. Cytoplasmic viscosity was evaluated from the picosecond rotation of the small fluorophore 2',7'-bis-(2-carboxyethyl)-5-carboxyfluorescein (BCECF) by parallel-acquisition Fourier transform microfluorimetry (Fushimi and Verkman, 1991). Information about viscosity within < 200 nm of cell plasma membranes was obtained by selective excitation of fluorophores in an evanescent field created by total internal reflection (TIR) of impulse-modulated s-plane-polarized laser illumination (488 nm) at a glass-aqueous interface. Measurements of fluorescence lifetime and time-resolved anisotropy were carried out in solutions containing fluorescein or BCECF at known viscosities, and monolayers of BCECF-labeled Swiss 3T3 fibroblasts and Madin-Darby canine kidney (MDCK) cells. Specific concerns associated with time-resolved fluorescence measurements in the evanescent field were examined theoretically and/or experimentally, including variations in lifetime due to fluorophore proximity to the interface, and the use of the s and p polarized excitation. In fluorescein solutions excited with s-plane polarized light, there was a 5-10% decrease in fluorescein lifetime with TIR compared to trans (subcritical) illumination, but no change in rotational correlation time (approximately 98 ps/cP). Intracellular BCECF had a single lifetime of 3.7 +/- 0.1 ns near the cell plasma membrane. Apparent fluid-phase viscosity near the cell plasma membrane was 1.1 +/- 0.2 cP (fibroblast) and 1.0 +/- 0.2 cP (MDCK), not significantly different from the viscosity measured in bulk cytoplasm far from the plasma membrane. The results establish the methodology for time-resolved microfluorimetric measurement of polarization in the evanescent field and demonstrate that the cell plasma membrane has little effect on the fluid-phase viscosity of adjacent cytoplasm.     

Construction and evaluation of a frequency-domain epifluorescence microscope for lifetime and anisotropy decay measurements in subcellular domains

The measurement of time-resolved fluorescence parameters in living cells provides a powerful approach to study cell structure and dynamics. An epifluorescence microscope was constructed to resolve multi-component fluorescence lifetimes and complex anisotropy decay rapidly in labile biological samples. The excitation source consisted of focused, polarized laser light modulated by an impulse-driven Pockels' cell; parallel acquisition of phase angles and modulation amplitudes at more than 40 frequencies (5-250 MHz) was obtained by multi-harmonic cross-correlation detection. Lifetime decay was measured against standard solutions introduced into the light path proximal to the microscope objective. Anisotropy decay was measured by rotation of a Glan-Thompson polarizer in the emission path. Phase reference light was split from the beam proximal to the microscope. Optical components were selected to avoid depolarization and to optimize fluorescence detection efficiency. The dichoric was replaced by a 1 mm square mirror. Fitting routine statistics were optimized for model discrimination in realistic biological samples. Instrument performance was evaluated using fluorescein in H2O/glycerol and H2O/ethylene glycol mixtures and in Swiss 3T3 fibroblasts in monolayer culture. Objective depolarization effects were evaluated by measurement of anisotropy decay using objectives of different numerical aperture. Lifetime and anisotropy decay measured by microscopy (0.5 micron laser spot) agreed with data obtained by cuvette fluorimetry. New biological applications for time-resolved fluorescence microscopy are discussed.     

Low viscosity in the aqueous domain of cell cytoplasm measured by picosecond polarization microfluorimetry

Information about the rheological characteristics of the aqueous cytoplasm can be provided by analysis of the rotational motion of small polar molecules introduced into the cell. To determine fluid-phase cytoplasmic viscosity in intact cells, a polarization microscope was constructed for measurement of picosecond anisotropy decay of fluorescent probes in the cell cytoplasm. We found that the rotational correlation time (tc) of the probes, 2,7-bis-(2-carboxyethyl)-5-(and-6-)carboxyfluorescein (BCECF), 6-carboxyfluorescein, and 8-hydroxypyrene-1,3,6-trisulfonic acid (HPTS) provided a direct measure of fluid-phase cytoplasmic viscosity that was independent of probe binding. In quiescent Swiss 3T3 fibroblasts, tc values were 20-40% longer than those in water, indicating that the fluid-phase cytoplasm is only 1.2-1.4 times as viscous as water. The activation energy of fluid-phase cytoplasmic viscosity was 4 kcal/mol, which is similar to that of water. Fluid-phase cytoplasmic viscosity was altered by less than 10% upon addition of sucrose to decrease cell volume, cytochalasin B to disrupt cell cytoskeleton, and vasopressin to activate phospholipase C. Nucleoplasmic and peripheral cytoplasmic viscosities were not different. Our results establish a novel method to measure fluid-phase cytoplasmic viscosity, and indicate that fluid-phase cytoplasmic viscosity in fibroblasts is similar to that of free water.     

Subtraction of background fluorescence in multiharmonic frequency-domain fluorimetry.

Background fluorescence is a major concern in time-resolved microfluorimetry studies of biological samples. A general method for subtraction of an arbitrary background signal in measurements of lifetime and anisotropy decay by multiharmonic Fourier transform spectroscopy is presented. Multifrequency phase and modulation values are measured in parallel by transformation of digitized time-domain waveforms into the frequency domain. For subtraction of background, time-domain waveforms are acquired for emission and reference photomultipliers for sample (e.g., cell containing fluorophore) and blank (e.g., unlabeled cell). Time-domain waveforms obtained in a series of measurements (e.g., sample and blank for parallel and perpendicular orientations of an emission polarizer) are time-justified by least-squares fitting of reference channel waveforms or by phase comparison of the first Fourier harmonics of the reference channel. Background is then subtracted directly in the time domain, and the subtracted waveform is Fourier transformed to the frequency domain for analysis of lifetime or anisotropy decay. This approach yielded excellent background correction over a wide range of background intensities and decay profiles. The method was tested in cuvette fluorimetry with fluorescein and acridine orange and in fluorescence microscopy with living MDCK cells loaded with the pH indicator BCECF. Sample lifetimes and rotational parameters could be recovered accurately with greater than 50% of the signal arising from background. These results establish a direct and practical approach to subtraction of background in complex biological and chemical samples studied by frequency-domain fluorimetry.     

Anisotropy decay associated fluorescence spectra and analysis of rotational heterogeneity. 2. 1,6-Diphenyl-1,3,5-hexatriene in lipid bilayers

The application of a new spectroscopic tool [Knutson, J. R., Davenport, L., & Brand, L. (1986) Biochemistry (preceding paper in this issue)] for studying rotational microheterogeneity of probe location in lipid bilayer systems is described. Anisotropy decay associated spectra are derived from experimentally obtained polarized emission components. "Early" difference spectra (IV - IH) contain contributions from both fast and slow rotors, while "late" difference spectra predominantly reflect the emission from slowly rotating fluorophores. Anisotropy decay associated spectra have been used to resolve the emission spectra of 1,6-diphenyl-1,3,5-hexatriene (DPH) imbedded within a known rotationally heterogeneous mixture of two vesicle types (L-alpha-dimyristoyllecithin and L-alpha-dipalmitoyllecithin). At 29 degrees C, diphenylhexatriene within pure dimyristoyllecithin vesicles rotates rapidly, with a small r infinity, while diphenylhexatriene in dipalmitoyllecithin vesicles exhibits a large r infinity. Spectra for diphenylhexatriene imbedded in the two vesicle types show small but significant spectral differences. A spectrum of a mixture of the two vesicle types with DPH lies between these characteristic component spectra. The spectrum extracted for "immobilized" probes in the mixture correctly overlays the dipalmitoyllecithin spectrum. Further studies have shown that diphenylhexatriene exhibits more than one emission anisotropy decay associated spectrum in vesicles of a single lipid type, when that lipid is near its phase transition temperature. Diphenylhexatriene apparently inhabits more than one rotational environment even in these "homogeneous" vesicle preparations.     

Development of a time-resolved fluorometric method for observing hybridization in living cells using fluorescence resonance energy transfer.

We previously showed that a specific kind of mRNA (c-fos) was detected in a living cell under a microscope by introducing two fluorescently labeled oligodeoxynucleotides, each labeled with donor or acceptor, into the cytoplasm, making them hybridize to adjacent locations on c-fos mRNA, and taking images of fluorescence resonance energy transfer (FRET) (A. Tsuji, H. Koshimoto, Y. Sato, M. Hirano. Y. Sei-Iida, S. Kondo, and K. Ishibashi, 2000, Biophys. J. 78:3260-3274). On the formed hybrid, the distance between donor and acceptor becomes close and FRET occurs. To observe small numbers of mRNA in living cells using this method, it is required that FRET fluorescence of hybrid must be distinguished from fluorescence of excess amounts of non-hybridizing probes and from cell autofluorescence. To meet these requirements, we developed a time-resolved method using acceptor fluorescence decays. When a combination of a donor having longer fluorescence lifetime and an acceptor having shorter lifetime is used, the measured fluorescence decays of acceptors under FRET becomes slower than the acceptor fluorescence decay with direct excitation. A combination of Bodipy493/503 and Cy5 was selected as donor and acceptor. When the formed hybrid had a configuration where the target RNA has no single-strand part between the two fluorophores, the acceptor fluorescence of hybrid had a sufficiently longer delay to detect fluorescence of hybrid in the presence of excess amounts of non-hybridizing probes. Spatial separation of 10-12 bases between two fluorophores on the hybrid is also required. The decay is also much slower than cell autofluorescence, and smaller numbers of hybrid were detected with less interference of cell autofluorescence in the cytoplasm of living cells under a time-resolved fluorescence microscope with a time-gated function equipped camera. The present method will be useful when observing induced expressions of mRNA in living cells.     

Picosecond rotation of small polar fluorophores in the cytosol of sea urchin eggs.

A new fluorescence method to measure viscosity in cell cytosol [Fushimi, K., & Verkman, A. S. (1991) J. Cell Biol. 112, 719-725] has been applied to determine fluid-phase viscosity in sea urchin eggs. Freshly harvested eggs from Lytechinus pictus were loaded with the dyes 2,7-bis(2-carboxyethyl)-5-(and-6-)carboxyfluorescein (BCECF), 6-carboxyfluorescein (6CF), fluorescein, or calcein. Fluorescence lifetimes and anisotropy decay were measured in single eggs by multiharmonic, frequency-domain microfluorometry using a 1-2-micron focused laser spot and 25x air objective. In calibration solutions consisting of glycerol in pH 8 buffered sea water, probe lifetime was single exponential and probe rotation was isotropic with a single correlation time which increased linearly with viscosity in the range 1-3.6 cP. In eggs at 22 degrees C, there were single lifetimes (in nanoseconds) of 3.6 (BCECF), 3.4 (6CF), 3.2 (fluorescein), and 3.3 (calcein). Probe rotation in eggs had two components, a fast component (in picoseconds, mean +/- SE, 10-18 eggs) of 568 +/- 39 (BCECF), 311 +/- 21 (6CF), 313 +/- 15 (fluorescein), and 516 +/- 44 (calcein) and a slow component of 10-40 ns. The fractional amplitude of the fast component, corresponding to unbound dye, was 0.72-0.81. Apparent viscosities of fluid-phase cytoplasm (centipoises) given by the four different probes were in good agreement: 2.3 +/- 0.2 (BCECF), 2.1 +/- 0.1 (6CF), 2.5 +/- 0.1 (fluorescein), and 2.3 +/- 0.2 (calcein). The viscosity in cytosol of sea urchin eggs (2.1-2.5 cP) is thus relatively low, yet significantly greater than that of water (1 cP) or cytosol in cultured fibroblasts (1.2-1.4 cP).(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of the core and surface of human plasma lipoproteins. A study based on the use of five fluorophores.

The physical properties of the core and the surface of five classes of human plasma lipoproteins were investigated using five fluorescent probes. The location of the fluorescence probes in the lipoprotein assembly was determined using collisional quenching and resonance energy transfer. The fluorophores monitor different regions of the lipoproteins, as shown by fluorescence quenching. Diphenylhexatriene (DPH) and methyl trans-parinaric acid (MTPA), which are apolar molecules, are localized mainly in the lipoprotein core. Their distribution into the surface is dependent upon the volume ratio of the hydrophobic part of the envelope and the core. The polar fluorophores, trimethylaminodiphenylhexatriene (TMADPH), hydroxycoumarin (HC) and trans-parinaric acid (TPA) are anchored in the glycerol skeleton region of the surface monolayer with the fluorophore group of HC in the headgroup region of the phospholipids. We determined the temperature-dependent steady-state fluorescence anisotropy (r) of these fluorophores in the four major classes of lipoproteins: VLDL, LDL, HDL2, HDL3 and in abnormal HDL from abetalipoproteinemia patients (HDLab). The hydrophobic probes, DPH and MTPA, reported the r values in the lipoproteins in the following order: LDL greater than HDL2 greater than HDL3 much greater than VLDL. This order correlates with the triglyceride-to-cholesterol ester (TG/CE) ratio in the core of lipoproteins. The polar probes HC, TPA and TMADPH reported the r value in a different order: HDL2, HDL3 greater than or equal to LDL much greater than VLDL. This is compatible with the decreasing order of the protein to lipid ratio in the envelope of these lipoproteins. HDLab was investigated by three fluorescent probes: DPH, TMADPH and HC. The anisotropy of DPH in HDLab was larger than that of either HDL2 or HDL3 in normal donors, probably due to the smaller TG/CE ratio in HDLab. The lower r values reported by HC and TMADPH for HDLab are not fully understood and may be related to other factors such as acyl chains composition. The characterization of lipoproteins by fluorescence depolarization using probes of known location in the lipoprotein assembly is very sensitive and may be used to report deviation from the norm.     

Protein rotational dynamics investigated with a dual EPR/optical molecular probe. Spin-labeled eosin.

An acyl spin-label derivative of 5-aminoeosin (5-SLE) was chemically synthesized and employed in studies of rotational dynamics of the free probe and of the probe when bound noncovalently to bovine serum albumin using the spectroscopic techniques of fluorescence anisotropy decay and electron paramagnetic resonance (EPR) and their long-lifetime counterparts phosphorescence anisotropy decay and saturation transfer EPR. Previous work (Beth, A. H., Cobb, C. E., and J. M. Beechem, 1992. Synthesis and characterization of a combined fluorescence, phosphorescence, and electron paramagnetic resonance probe. Society of Photo-Optical Instrumentation Engineers. Time-Resolved Laser Spectroscopy III. 504-512) has shown that the spin-label moiety only slightly altered the fluorescence and phosphorescence lifetimes and quantum yields of 5-SLE when compared with 5-SLE whose nitroxide had been reduced with ascorbate and with the diamagnetic homolog 5-acetyleosin. In the present work, we have utilized time-resolved fluorescence anisotropy decay and linear EPR spectroscopies to observe and quantitate the psec motions of 5-SLE in solution and the nsec motions of the 5-SLE-bovine serum albumin complex. Time-resolved phosphorescence anisotropy decay and saturation transfer EPR studies have been carried out to observe and quantitate the microseconds motions of the 5-SLE-albumin complex in glycerol/buffer solutions of varying viscosity. These latter studies have enabled a rigorous comparison of rotational correlation times obtained from these complementary techniques to be made with a single probe. The studies described demonstrate that it is possible to employ a single molecular probe to carry out the full range of fluorescence, phosphorescence, EPR, and saturation transfer EPR studies. It is anticipated that "dual" molecular probes of this general type will significantly enhance capabilities for extracting dynamics and structural information from macromolecules and their functional assemblies.     

Fluorescence quenching dynamics of tryptophan in proteins. Effect of internal rotation under potential barrier.

In many proteins fluorescence from single tryptophan exhibits a nonexponential decay function. To elucidate the origin of this nonexponential decay, we have examined the fluorescence decay function and time-resolved fluorescence anisotropy of a fluorophore covalently bound to a macromolecule by solving a rotational analogue of the Smoluchowski equation. An angular-dependent quenching constant and potential energy for the fluorophore undergoing internal rotation were introduced into the equation of motion for fluorophore. Results of numerical calculations using the equations thus obtained predict that both the fluorescence decay function and time-resolved anisotropy are dependent on rotational diffusion coefficients of fluorophore and potential energy for the internal rotation. The method was applied to the observed fluorescence decay curve of the single tryptophan in apocytochrome c from horse heart. The calculated decay curves fit the observed ones well.     

Time-dependent rotational rates of excited fluorophores. A linkage between fluorescence depolarization and solvent relaxation

It is generally assumed that the rotational diffusion coefficients of fluorophores are independent of time subsequent to excitation, and that the rotational diffusion coefficients of the ground and the excited states are the same. We now describe a linkage between the extent of solvent relaxation and the rate of fluorescence depolarization. Specifically, if a fluorophore displays time-dependent solvent relaxation it may also show a time-dependent decrease in its rotational rate. A decreased rate of rotation could result from the increased interaction with polar solvent molecules which occurs as a result of solvent relaxation. The decays of anisotropy predicted from our model closely mimic those often observed for fluorophores which are bound to macromolecules. For example, the decays are more complex than a single exponential, and the time-resolved anisotropy can display a limiting value which does not decay to zero. The effect of solvent relaxation upon the rates of rotational diffusion is expected to be most dramatic for solvent-sensitive fluorophores in a viscous environment. These conditions are frequently encountered for fluorophore-macromolecule complexes. Consideration of the linkage between solvent relaxation and rotational diffusion leads to two unusual predictions. First even spherical fluorophores in an isotropic environment could display multi- or nonexponential decays of fluorescence anisotropy. Secondly, for the special case in which the fluorophore dipole moment decreases upon excitation, the theory predicts that the anisotropy decay rate may increase with time subsequent to pulsed excitation. The predictions of this theory are consistent with published data on the effects of red-edge excitation upon the apparent rotational rates of fluorophores in polar solvents.     

Cell membrane fluidity in the intact kidney proximal tubule measured by orientation-independent fluorescence anisotropy imaging.

Membrane fluidity was measured in the isolated perfused proximal tubule from rabbit kidney. The apical and basolateral plasma membranes of tubule cells were stained separately with the fluidity-sensitive fluorophore trimethylammonium-diphenyl-hexatriene (TMA-DPH) by luminal or bath perfusion. Fluorescence anisotropy (r) of TMA-DPH was mapped with spatial resolution using an epifluorescence microscope (excitation 380 nm, emission greater than 410 nm) equipped with rotatable polarizers and a quantitative imaging system. To measure r without the confounding effects of fluorophore orientation, images were recorded with emission polarizer parallel and perpendicular to a continuum of orientations of the excitation polarizer. The theoretical basis of this approach was developed and its limitations were evaluated by mathematical modeling. The tubule inner surface (brush border) was brightly stained when the lumen was perfused with 1 microM TMA-DPH for 5 min; apical membrane r was 0.281 +/- 0.006 (23 degrees C). Staining of the tubule basolateral membrane by addition of TMA-DPH to the bath gave a significantly lower r of 0.242 +/- 0.010 (P less than 0.005); there was no staining of the brush border membrane. To interpret anisotropy images quantitatively, effects of tubule geometry, TMA-DPH lifetime, fluorescence anisotropy decay, and objective-depolarization were evaluated. Steady-state and time-resolved r and lifetimes in the intact tubule, measured by a nanosecond pulsed microscopy method, were compared with results in isolated apical and basolateral membrane vesicles from rabbit proximal tubule measured by cuvette fluorometry; r was 0.281 (apical membrane) and 0.276 (basolateral membrane) (23 degrees C). These results establish a methodology to quantitate membrane fluidity in the intact proximal tubule, and demonstrate a significantly higher fluidity in the basolateral membrane than in the apical membrane.     

Steady-state fluorescence polarization data in membranes. Resolution into physical parameters by an extended Perrin equation for restricted rotation of fluorophores

An extended Perrin equation is derived applicable to the restricted rotation of fluorophores. The equation results in a relation between time-resolved (r infinity) and steady-state fluorescence anisotropy (rs) data. This relation contains a parameter m, which expresses the difference between rotational diffusion in a lipid membrane and that in an isotropic reference oil having the same rs value. The relation is in agreement with rs, r infinity literature data for a variety of artificial and biological membranes labeled with various probes. Cholesterol and fatty acyl unsaturation affect the value of m, but temperature does not. The results indicate that, as far as fluorescence depolarization is concerned, either liposomes of saturated phospholipids without cholesterol or liposomes of unsaturated phospholipids containing cholesterol are good model systems for biological membranes. The accuracy of estimating order parameters or rotational diffusion constants from rs data is discussed. The formalism described here introduces a novel way of applying Arrhenius plots and allows for an unambiguous interpretation of rs data.     

Development of a streak-camera-based time-resolved microscope fluorimeter and its application to studies of membrane fusion in single cells

A time-resolved microscope fluorimeter based on a synchroscan streak camera and a fast pulsed laser system has been developed to measure the fluorescence lifetime decay under the fluorescence microscope. This system allows one to measure the nanosecond fluorescence lifetimes of fluorophores in a small spot (0.8-6.3 microns diameter) in single cultured cells under a fluorescence microscope, while the cells are being viewed under a high-power objective lens. A signal acquisition time between a second and a minute was usually sufficient to obtain fluorescence decay curves with good quality for 10(3)-10(5) fluorophores localized in 1 microns 2 domain. A signal-to-noise ratio better than 30 was obtained for approximately 30,000 fluorescein-labeled band 3 molecules in a 2 microns 2 region in a single human erythrocyte ghost after signal accumulation for 30 s. The measured lifetimes for a variety of fluorescent probes attached to proteins in solution and lipids in liposomes showed a good agreement with those measured in a cuvette under standard conditions by time-correlated single photon counting. With the development of this instrument, microscope fluorimetry has become a practical, straightforward, quantitative technique for investigation of molecular processes in single cells in culture. Time-resolved microscope fluorimetry has been applied to observe fusion of liposomes in vitro and that of endosomes in single cells by monitoring resonance energy transfer. Inspection of individual liposomes and endosomes revealed the extent of fusion for each vesicle. Since the use of time-resolved microscope fluorimetry eliminates the need for subcellular fractionation or the complex correction procedures in steady-state microfluorimetry, it greatly simplifies the assay for endosome fusion in vivo. The results showed that extensive fusion of sequentially formed endosomes takes place all over the cell matrix in cultured cells. This suggests that extensive fusion with incoming endosomes takes place in many endosomal compartments, possibly sorting organelles, or that the early endosomes fuse with the preexisting network of tubular cisternae of the endosomal compartment at many points in the network. It is concluded that time-resolved microscope fluorimetry is a powerful noninvasive technique for studies of in situ biochemistry and biophysics using cells and tissues.     

Time-resolved fluorescence microscopy.

In fluorescence microscopy, the fluorescence emission can be characterised not only by intensity and position, but also by lifetime, polarization and wavelength. Fluorescence lifetime imaging (FLIM) can report on photophysical events that are difficult or impossible to observe by fluorescence intensity imaging, and time-resolved fluorescence anisotropy imaging (TR-FAIM) can measure the rotational mobility of a fluorophore in its environment. We compare different FLIM methods: a chief advantage of wide-field time-gating and phase modulation methods is the speed of acquisition whereas for time-correlated single photon counting (TCSPC) based confocal scanning it is accuracy in the fluorescence decay. FLIM has been used to image interactions between proteins such as receptor oligomerisation and to reveal protein phosphorylation by detecting fluorescence resonance energy transfer (FRET). In addition, FLIM can also probe the local environment of fluorophores, reporting, for example, on the local pH, refractive index, ion or oxygen concentration without the need for ratiometric measurements.     

Time-resolved polarization imaging by pump-probe (stimulated emission) fluorescence microscopy

We report the application of pump-probe fluorescence microscopy in time-resolved polarization imaging. We derived the equations governing the pump-probe stimulated emission process and characterized the pump and probe laser power levels for signal saturation. Our emphasis is to use this novel methodology to image polarization properties of fluorophores across entire cells. As a feasibility study, we imaged a 15-microm orange latex sphere and found that there is depolarization that is possibly due to energy transfer among fluorescent molecules inside the sphere. We also imaged a mouse fibroblast labeled with CellTracker Orange CMTMR (5-(and-6)-(((4-chloromethyl)benzoyl)amino)tetramethyl-rhodamine). We observed that Orange CMTMR complexed with gluthathione rotates fast, indicating the relatively low fluid-phase viscosity of the cytoplasmic microenvironment as seen by Orange CMTMR. The measured rotational correlation time ranged from approximately 30 to approximately 150 ps. This work demonstrates the effectiveness of stimulated emission measurements in acquiring high-resolution, time-resolved polarization information across the entire cell.     

Shape of the fluidity gradient in the plasma membrane of living HeLa cells

The shape of the fluidity gradient of the outer hemi-leaflet of the plasma membrane of living HeLa cells was determined using a series of n-(9-anthroyloxy) fatty acid probes where n = 2, 3, 6, 7, 9, 10, 11, 12, and 16. Fluorescence uptake and steady-state anisotropy values were obtained with a flow cytometer capable of continuous recording over time of vertical and horizontal emission intensities, and of the output of these intensities as calculated anisotropy values. The fluorescence uptake of all of the membrane probes was rapid up to about 15 min. The magnitudes of the uptake of fluorescence were, for the n-(9-anthroyloxy) series, in the order 2 greater than 3 greater than 6 greater than 7 greater than 9 greater than 10 greater than 11 = 12 = 16. Anisotropy values were constant from 5 to 30 min after addition of the various probes, and the magnitudes were in the order 7 greater than 6 greater than 9 = 10 greater than 2 = 3 greater than 11 greater than 12 greater than 16, indicative of the shape of the fluidity gradient. No differences were noted between the values obtained with 12-(9-anthroyloxy) stearic acid and 12- (9-anthroyloxy) oleate. The kinetics of anisotropy exhibited by those probes with the anthroyloxy group in positions deeper than 9, where initially higher values declined until equilibrium was reached, were probably indicative of an energy barrier at the approximate depth sensed by 7 AS.     

Dynamics of biomolecules: assignment of local motions by fluorescence anisotropy decay.

Many biological systems have multiple fluorophores that experience multiple depolarizing motions, requiring multiple lifetimes and correlation times to define the fluorescence intensity and anisotropy decays, respectively. To simplify analyses, an assumption often made is that all fluorophores experience all depolarizing motions. However, this assumption usually is invalid, because each lifetime is not necessarily associated with each correlation time. To help establish the correct associations and recover accurate kinetic parameters, a general kinetic scheme that can examine all possible associations is presented. Using synthetic data sets, the ability of the scheme to discriminate among all nine association models possible for two lifetimes and two correlation times has been evaluated. Correct determination of the association model, and accurate recovery of the decay parameters, required the global analysis of related data sets. This general kinetic scheme was then used for global analyses of liver alcohol dehydrogenase anisotropy data sets. The results indicate that only one of the two tryptophan residues in each subunit is depolarized by process(es) independent of the enzyme's rotations. By applying the proper kinetic scheme and appropriate analysis procedures to time-resolved fluorescence anisotropy data, it is therefore possible to examine the dynamics of specific portions of a macromolecule in solution.     

Physical properties of the fluorescent sterol probe dehydroergosterol

Spectroscopic studies were performed on the fluorescent sterol probes ergosta-5,7,9(11),22-tetraen-3 beta-ol (dehydroergosterol) and cholesta-5,7,9(11)-trien-3 beta-ol (cholestatrienol). In most isotropic solvents, these molecules exhibited a single lifetime near 300 ps. Fluorescence lifetimes in 2-propanol were independent of emission wavelength and independent of excitation wavelength. Excited state behavior of these probes appears relatively simple. In isotropic solvents, dehydroergosterol fluorescence emission underwent at most a small Stokes shift as solvent polarity was modified. Time-resolved anisotropy decays indicated that dehydroergosterol decay was monoexponential, with rotational correlation times dependent on solvent viscosity. When incorporated into L-alpha-dimyristoylphosphatidylcholine liposomes at a concentration of 0.9 mol%, dehydroergosterol fluorescence lifetime decreased at the phase transition of this phospholipid indicating that the sterol probe was detecting physical changes of the bulk phospholipids. Furthermore, total fluorescence decays and anisotropy decays were sensitive to the environment of the sterol. Dehydroergosterol and cholestatrienol are thus useful probes for monitoring sterol behavior in biological systems.