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PubMed2006

In vivo resolution of oligomers with fluorescence photobleaching recovery histograms.

Simple independent enzyme-catalyzed reactions distributed homogeneously throughout an aqueous environment cannot adequately explain the regulation of metabolic and other cellular processes in vivo. Such an unstructured system results in unacceptably slow substrate turnover rates and consumes inordinate amounts of cellular energy. Current approaches to resolving compartmentalization in living cells requires the partitioning of the molecular species in question such that its localization can be resolved with fluorescence microscopy. Standard imaging approaches will not resolve localization of protein activity for proteins that are ubiquitously distributed, but whose function requires a change in state of the protein. The small heat shock protein sHSP27 exists as both dimers and large multimers and is distributed homogeneously throughout the cytoplasm. A fusion of the green fluorescent protein variant S65T and sHSP27 is used to assess the ability of diffusion rate histograms to resolve compartmentalization of the 2 dominant oligomeric species of sHSP27. Diffusion rates were measured by multiphoton fluorescence photobleaching recovery. Under physiologic conditions, diffusion rate histograms resolved at least 2 diffusive transport rates within a living cell potentially corresponding to the large and small oligomers of sHSP27. Given that oligomerization is often a means of regulation, compartmentalization of different oligomer species could provide a means for efficient regulation and localization of sHsp27 activity.

Animals↗
PubMed2001

On the Use of Pattern Fluorescence Photobleaching Recovery with Modulation Detection to Obtain Colloidal Size Distributions.

Fluorescence photobleaching recovery, long a staple tool of complex fluid experimentation, is discussed as a method for size characterization of colloidal particles. Specific comparisons are made to dynamic light scattering, which is often used for sizing particles despite some important limitations. The capabilities of fluorescence photobleaching recovery for size determination are demonstrated for aggregating core-shell silica-polypeptide composite particles suspended in polar organic solvents. Copyright 2001 Academic Press.

Journal Article↗
PubMed1983

Theoretical analysis of fluorescence photobleaching recovery experiments.

We derive an exact closed formula for the fluorescence recovery curve measured in fluorescence photobleaching recovery experiments employing uniform circular laser beams. In contrast to the expression used currently, this result is very simple and free of mathematical drawbacks, thus facilitating the quantitative analysis of experimental data.

Mathematics↗
PubMed1993

Measurement of restricted rotational diffusion of fluorescent lipids in supported planar phospholipid monolayers using angle-dependent polarized fluorescence photobleaching recovery.

A theory describing the shapes of polarized fluorescence photobleaching recovery (PFPR) curves for a population of fluorophores undergoing restricted rotational diffusion in two-dimensional systems such as planar membranes has been developed. In this model, restricted rotational diffusion of the fluorophores is described by using reflective boundary conditions, in which the fluorophores are assumed to diffuse freely but only within an angular space of width 2 omega. The magnitude and apparent rate of the PFPR postbleach fluorescence curves are a function of both omega and the angle between the bleaching and observation beam polarizations psi. It is shown that estimates of the degree of rotational restriction omega may be obtained from changes in the psi-dependent postbleach fluorescence intensities. Using angle-dependent PFPR, slow rotational reorientations of the fluorescent lipid analogue 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine in distearoylphosphatidylcholine Langmuir-Blodgett monolayers deposited on octadecyltrichlorosilane-treated fused quartz were measured. As theoretically predicted for a rotationally restricted fluorophore population, both the initial F psi (0) and final F psi (infinity) postbleach fluorescence intensities varied as a function of psi, and no measurable change in the postbleach fluorescence intensities was observed for psi = 45 degrees. Using the theory for restricted rotational motion, the psi-dependent variations of the final fluorescence intensities F psi (infinity) obtained at two bleaching intensities gave an average apparent omega approximately 52 degrees. However, to adequately fit the F psi (0) data, inclusion of the theoretical effects of rapid (faster than the duration of the photobleaching pulse) fluorophore dynamics was also required. Best fits of the F psi (0) and F psi (infinity) data were obtained when the fluorophores were assumed to rapidly wobble within a cone of semiangle delta approximately 30 degrees-50 degrees while slowly rotating within an angular space defined by semiangle omega approximately 35 degrees-60 degrees. Subsequent analysis of the time- and psi-dependent changes in the post-bleach fluorescence curves F psi (t) gave apparent diffusion coefficients ranging from D approximately 10(-3) s-1 to 4 x 10(-2) s-1.

Carbocyanines↗
PubMed1988

Class I major histocompatibility complex proteins diffuse isotropically on immune interferon-activated endothelial cells despite anisotropic cell shape and cytoskeletal organization: application of fluorescence photobleaching recovery with an elliptical beam.

Interferon gamma induces striking phenotypic alterations in confluent cultures of human vascular endothelial cells (HEC), including cell shape change from polygonal to elongated and cytoskeletal actin rearrangement from dense peripheral bands to longitudinal bundles of stress fibers. Since many transmembrane proteins, including class I major histocompatibility complex (MHC) proteins, interact with cytoskeletal actin, an interferon-gamma-induced anisotropic arrangement of stress fibers might cause anisotropic lateral diffusion of HEC class I MHC proteins. To test this hypothesis, we adapted the fluorescence photobleaching recovery technique to allow measurement of anisotropic diffusion of fluorescently labeled molecules on two-dimensional surfaces. A highly eccentric elliptical Gaussian laser beam was used to photobleach the sample and to monitor fluorescence recovery. In this technique, named "line fluorescence photobleaching recovery," lateral diffusion is measured along that axis of the sample that is perpendicular to the major axis of the elliptical beam. The lateral diffusion coefficient and fractional mobility are obtained by fitting the experimental data to a theoretical recovery curve, the form of which is determined by the solution to a modified version of the diffusion equation in which a tensor is used to describe diffusion in two orthogonal directions. Fluorescein-conjugated murine monoclonal antibodies were used to label class I MHC proteins on interferon-gamma-treated HEC and human dermal fibroblasts. These two cultured human cell types were found to be similar in their elongated shape and anisotropic stress fiber organization. Class I MHC protein lateral mobility was compared to that of fluorescein-labeled phosphatidyl-ethanolamine, a membrane phospholipid probe. Class I MHC proteins diffused anisotropically on human dermal fibroblasts, whereas fluorescein-labeled phosphatidylethanolamine diffused isotropically on this cell type. In contrast, both class I MHC proteins and fluorescein-labeled phosphatidylethanolamine diffused isotropically on interferon-gamma-treated HEC. These data suggest that neither elongated shape nor anisotropic stress fiber arrangement is sufficient to induce anisotropic diffusion of proteins on the HEC plasma membrane.

Algorithms↗
PubMed1994

Deoxygenation affects fluorescence photobleaching recovery measurements of red cell membrane protein lateral mobility.

We have used the fluorescence photobleaching recovery technique to study the dependence on oxygen tension of the lateral mobility of fluorescently labeled band 3, the phospholipid analogue fluorescein phosphatidylethanolamine, and glycophorins in normal red blood cell membranes. Band 3 protein and sialic acid moieties on glycophorins were labeled specifically with eosin maleimide and fluorescein thiosemicarbazide, respectively. The band 3 diffusion rate increased from 1.7 x 10(-11) cm2 s-1 to 6.0 x 10(-11) cm2 s-1 as oxygen tension was decreased from 156 to 2 torr, and a further increase to 17 x 10(-11) cm2 s-1 occurred as oxygen tension was decreased from 2 to 0 torr. The fractional mobility of band 3 decreased from 58 to 32% as oxygen tension was decreased from 156 to 0 torr. The phospholipid diffusion coefficient remained constant as oxygen tension was decreased from 156 to 20 torr, but increased from 2.3 x 10(-9) cm2 s-1 to 7.1 x 10(-9) cm2 s-1 as oxygen tension was decreased from 20 to 0 torr. Neither the diffusion coefficient nor the fractional mobility of glycophorins changed significantly at low oxygen tension. Under non-bleaching excitation conditions, intensities of fluorescence emission were identical for oxygenated and deoxygenated eosin-labeled RBCs. Deoxygenated eosin-labeled RBCs required 160-fold greater laser intensities than did oxygenated RBCs to achieve comparable extents of photobleaching, however. Oxygen seems to act as a facilitator of fluorophore photobleaching and may thereby protect the fluorescently labeled red cell membrane from photodamage. Removal of oxygen may allow excited state fluorophores in close proximity to the plasma membrane to react with neighboring proteins or lipids during photobleaching. This effect has important implications for the ability of the fluorescence photobleaching recovery technique to report accurate lateral mobilities of cell membrane molecules under hypoxic conditions.

Anion Exchange Protein 1, Erythrocyte↗
PubMed1991

Flow rate measurements in isolated perfused kidney tubules by fluorescence photobleaching recovery.

We have developed a new application of the fluorescence photobleaching recovery (FPR) technique for instantaneous measurement of volume flow rates at any axial position along isolated perfused kidney tubules. The method requires fast data acquisition of emitted fluorescence through a photomultiplier (time resolution, 0.5 ms) coupled with differential interference contrast microscopy to measure luminal diameters accurately. While the tubule is perfused in vitro with an impermeant fluorophore (fluorescein sulfonate), a 20-ms bleach pulse reduces the fluorescence in the observation region by 20-25%. Fluorescence recovery is a direct function of perfusate velocity; diffusion plays no significant role in the early phase of recovery. A fluid dynamics approach to data analysis shows that fractional recovery increases linearly with time until t = L/2vm, where L is the length of the observation window and vm is the mean axial velocity. Practically, a linear regression analysis of the early recovery phase allows measurement of vm of up to 0.14 cm/s, i.e., a 40-nl/min flow rate in a 25-microns-diameter tubule. Calibration experiments in small glass tubes perfused at predetermined flow rates demonstrated good accuracy (within 10%) and reproducibility (coefficient of variation, 8.7%). In rat inner medullary collecting ducts microperfused at 4-40 nl/min, the correlation with a standard fluid collection method was excellent (r2 greater than 0.97). The method should also be suitable for the direct measurement of fluid flow rate in kidney tubules or blood vessels microperfused in vivo.

Animals↗
PubMed1985

The use of the fluorescence photobleaching recovery technique to study the self-assembly of tubulin.

Fluorescently labeled microtubule-associated proteins or poly-L-lysine (13,000 MW) were prepared by reaction with fluorescein isothiocyanate. The labeled compounds were used as probes of the assembly of calf brain tubulin using fluorescence photobleaching recovery techniques which allow measurement of the diffusion coefficient and percentage mobility of the fluorescent probe. When unfractionated tubulin (defined as material containing tubulin and microtubule-associated proteins) was polymerized at room temperature or 37 degrees C, either probe could be incorporated into microtubules, since the observed diffusion coefficient (approximately 1.7 X 10(-8) cm2/s) was much slower than that for either probe free in solution. The microtubules formed in the presence of labeled microtubule-associated proteins were free to diffuse while those formed in the presence of labeled polylysine were partially immobilized. Thus the fluorescence photobleaching recovery technique can be used to measure crosslinking of microtubules as well as assembly or interactions with other structures. When unfractionated tubulin was incubated with labeled polylysine in the presence of Ca2+ at room temperature, the observed diffusion coefficient (approximately 5.1 X 10(-8) cm2/s) probably represents the formation of rings of tubulin. The effect of mild and vigorous shearing, of cholchicine, and of different Mg2+ concentrations on the properties of the system were examined.

Animals↗
PubMed1995

Diffusion and binding measurements within oral biofilms using fluorescence photobleaching recovery methods.

Numerous studies have postulated that bacteria which reside in a biofilm differ from planktonic bacteria. These differences are thought to affect biofilm permeability and, indirectly, the susceptibility of biofilm bacteria to antibacterial agents. In this study fluorescence recovery after photobleaching (FRAP) was used to monitor the diffusion and binding characteristics of a set of size fractionated fluorescein isothiocyanate (FTTC)-conjugated dextrans over small areas (ca. 10 micron) in bacterial biofilms. From these measurements it was straightforward to calculate apparent diffusion rates. Initial studies on the concentration dependence of dextran interaction with planktonic bacteria showed that no irreversible interaction was occurring, however, anomalous faster than free solution diffusion rates were obtained. This phenomenon was modelled using novel analytical and numerical methods which incorporate reversible binding with associated fluorescence changes. Apparent diffusion rates measured in biofilms were highly dependent on biofilm preparation. Sucrose starved biofilms produced an apparent slow-down of two- to fivefold depending on dextran molecular mass and location within the biofilm, indicating that diffusion within the biofilm is hindered. Sucrose supplemented biofilms produced apparent diffusion rates close to those in free solution, suggesting less hindered diffusion. Ex vivo plaque showed diffusion and binding similar to the sucrose supplemented biofilms. The FRAP approach provides a fast and convenient method for determining diffusion rates over small areas within bacterial biofilms. This study reinforces the importance of considering the influence of reversible binding and associated fluorescence changes, as these may have a marked effect on the measured apparent diffusion rate.

Bacteria↗
PubMed1983

Simple computer method for evaluation of lateral diffusion coefficients from fluorescence photobleaching recovery kinetics.

A method is presented for the analysis of fluorescence photobleaching recovery curves. Based on the simplified kinetic expression of Yguerabide, J., J.A. Schmidt, and E.E. Yguerabide (1982, Biophys. J., 40:69-75), a linearization procedure is described that permits unequivocal determination of all diffusion parameters. The presence of additional membrane flow or multiple diffusion coefficients can easily be detected by this method, and simple corrections for the presence of these alternative recovery processes can be made by the use of a regular mini-computer. The validity of the method is tested on simulated recovery curves, varying the contribution of flow, multiple diffusion coefficients, and statistical noise due to counting error.

Diffusion↗
PubMed1979

Fluorescence photobleaching recovery measurement of protein absolute diffusion constants.

The technique of fluorescence photobleaching recovery [Axelrod et al., Biophys. J. 16 (1976) 1055] has been applied to the measurement of absolute diffusion constants of a number of fluoiescein isothiocyanate-labeled proteins. Measured diffusion constants agree to within +/- 7% of published values for the underivatized proteins. The method has sufficient sensitivity to reveal the concentration dependence at neutral pH of the diffusion constant of alpha-chymotrypsin. The rapidity with which the labelling and measurements can be performed and the small amount of material required suggest the technique may be useful in rapid characterization of small protein samples. Some developments in optical and electronic systems and in data processing for this technique are discussed.

Journal Article↗
PubMed1988

Polarized fluorescence photobleaching recovery for measuring rotational diffusion in solutions and membranes.

A variation of fluorescence photobleaching recovery (FPR) suitable for measuring the rate of rotational molecular diffusion in solution and cell membranes is presented in theory and experimental practice for epi-illumination microscopy. In this technique, a brief flash of polarized laser light creates an anisotropic distribution of unbleached fluorophores which relaxes by rotational diffusion, leading to a time-dependent postbleach fluorescence. Polarized FPR (PFPR) is applicable to any time scales from seconds to microseconds. However, at fast (microsecond) time scales, a partial recovery independent of molecular orientation tends to obscure rotational effects. The theory here presents a method for overcoming this reversible photobleaching, and includes explicit results for practical geometries, fast wobble of fluorophores, and arbitrary bleaching depth. This variation of a polarized luminescence "pump-and-probe" technique is compared with prior ones and with "pump-only" time-resolved luminescence anisotropy decay methods. The technique is experimentally verified on small latex beads with a variety of diameters, common fluorophore labels, and solvent viscosities. Preliminary measurements on a protein (acetylcholine receptor) in the membrane of nondeoxygenated cells in live culture (rat myotubes) show a difference in rotational diffusion between clustered and nonclustered receptors. In most experiments, signal averaging, high laser power, and automated sample translation must be employed to achieve adequate statistical accuracy.

Animals↗
PubMed1981

Fluorescence photobleaching recovery measurements reveal differences in envelopment of Sindbis and vesicular stomatitis viruses.

Fluorescence photobleaching recovery (FPR) measurements of virus glycoproteins on the surfaces of cells infected with vesicular stomatitis virus (VSV) and Sindbis virus showed that the VSV glycoprotein (G) remained mobile throughout the infectious cycle, whereas Sindbis virus glycoproteins (E1, E2) were partially mobile early after infection and immobile at later times when greater amounts of these proteins were on the cell surface. A highly mobile fraction of Sindbis virus glycoproteins was detected throughout the replication cycle of a temperature-sensitive mutant unable to form virus particles. This immobilization of E1 and E2 was the result of increasing surface glycoprotein concentrations and virus budding. Together with other data, which included the detection of E1 and E2 in particles as soon as these proteins were transported to the cell surface, the FPR results suggest that Sindbis virus assembly initiates on intracellular vesicles, where glycoproteins aggregate and bind nucleocapsids. In contrast, our FPR data on VSV support a model previously suggested by others, in which a small fraction of cell-surface G is immobilized into budding sites formed by interactions with virus matrix and nucleoproteins. FPR measurements also provide direct evidence for strong interactions between E1 and E2, as well as between E1 and PE2, the precursor form of E2.

Animals↗
PubMed1981

Fluorescence photobleaching recovery in solutions of labeled actin.

We have demonstrated that the technique of fluorescence photobleaching recovery (FPR) can be used to examine the state of a single component in complex self-assembling macromolecular systems. Polymerization of actin, initiated by addition of salt or Mg+2 to a low-ionic-strength solution of G-actin, has been observed by sequential measurement of FPR with the aid of fluorescein-labeled actin. Solutions of actin which had been labeled using 5-iodoacetamido fluorescein (5-IAF) showed anomalous recovery of fluorescence above the initial value, which indicates a photoinduced increase in local polymerization. No such anomaly was observed with actin that had been labeled with fluorescein isothiocyanate (FITC). The FPR data are directly interpretable in terms of the fraction of labeled protein that is immobilized in the supramolecular assembly and in terms of the average diffusion coefficient of the mobile fraction. Our data are consistent with the "treadmill" model of actin polymerization, in that they show that actin is present under polymerizing conditions either as a high polymer or as monomer or low oligomer. We believe that the FPR technique can be applied to the study of many types of reconstituted motile or cytoskeletal systems in vitro or in vivo.

Actins↗
PubMed1980

Lateral mobility of band 3 in the human erythrocyte membrane studied by fluorescence photobleaching recovery: evidence for control by cytoskeletal interactions.

Band 3, the major intrinsic protein of the human erythrocyte membrane, was specifically labeled with the covalent fluorescent probe eosin isothiocyanate. The lateral mobility of labeled band 3 in the plane of the membrane under various conditions of ionic strength and temperature was examined by using the fluorescence photobleaching recovery technique. Low temperature (21 degrees C) and high ionic strength (46 mM NaPO(4)) favored immobilization of band 3(10% mobile) as well as slow diffusion of the mobile fraction (diffusion coefficient D = 4 x 10(-11) cm(2)sec(-1)). Increasing temperature (37 degrees C) and decreasing ionic strength (13 mM NaPO(4)) led to an increase in the fraction of mobile band 3(90% mobile) and a reversible increase in the diffusion rate of the mobile fraction (D = 200 x 10(-11) cm(2)sec(-1)). The increase in the fraction of mobile band 3 was markedly dissociated, however, from the increase in the diffusion rate of the mobile fraction. Thus, the fraction of mobile band 3 always increased at higher ionic strength and lower temperature than the ionic strength and temperature at which the diffusion rate increased. This dissociation was manifested kinetically on prolonged incubation of ghosts at constant ionic strength and temperature: the diffusion rate of the mobile fraction increased slowly at first and much more rapidly after the initial lag period, whereas the fraction of mobile band 3 increased almost immediately to 90% and remained maximal for the duration of the experiment. Further, changes in diffusion rate with temperature were promptly and totally reversible, whereas increases in the mobile fraction were only slowly and partially reversible. These effects were shown not to be due to complete dissociation of spectrin, the major protein of the erythrocyte cytoskeleton, from the membrane. This evidence suggests control of band 3 lateral mobility by at least two separate processes. The process that determines the diffusion coefficient of the mobile band 3 is completely reversible, and it probably involves a metastable state of cytoskeleton structure intermediate between tight binding to the membrane and complete dissociation from it.

Cytoskeleton↗
PubMed1986

Fluorescence photobleaching recovery as a method to quantitate viral envelope-cell fusion: application to study fusion of Sendai virus envelopes with cells.

A method to quantitate viral envelope-cell fusion at the single-cell level is presented. The method is based on the incorporation of nonquenching concentrations of a fluorescent lipid probe into the viral envelope; fluorescence photobleaching recovery (FPR) is then applied to measure the lateral mobilization of the probe in the cell membrane following fusion. In adsorbed (unfused) viral envelopes, the probe is constricted to the envelope and is laterally immobile on the micrometer scale of FPR. After fusion, the envelope lipids intermix with the plasma membrane, the probe becomes laterally mobile, and the fraction of fused viral envelopes can be extracted from the fraction of mobile probe molecules. The method has several advantages: (i) It clearly distinguishes fused from internalized envelopes, as probes in the latter are immobile in FPR studies; (ii) focusing the laser beam on specific regions of the cell enables region-specific measurements of the fusion level; (iii) one cell is measured at a time, enabling studies on the distribution of the fusion level within the cell population. The new method was employed to study fusion of reconstituted Sendai virus envelopes (RSVE) containing N-(7-nitro-2,1,3-benzoxadiazol-4-yl)phosphatidylethanolamine with several cell types. Experiments with human erythrocytes demonstrated that the lateral mobilization measured is due to fusion and not the result of exchange processes. The extent of RSVE-erythrocyte fusion determined by FPR was similar to that measured by two other independent methods (fluorescence dequenching and removal of adsorbed RSVE by dithiothreitol).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1998

Exchangeability of actin in cardiac myocytes and fibroblasts as determined by fluorescence photobleaching recovery.

Rhodamine (Rho)-labeled muscle and non-muscle actins were microinjected into cultured embryonic chicken cardiac myocytes and fibroblasts. After incorporation of the fluorescent actin analog into cellular structures, small areas of labeled structures were photobleached with a laser pulse, and fluorescence recovery (FR) was measured to determine the exchangeability of isoactins in these structures. With both Rho-muscle and Rho-non-muscle actins, the FR rate in any part of stress fibers was consistently faster than that observed in any part of myofibrils. Thus, although non-striated (proximal and terminal) portions of nascent myofibrils are similar in appearance and composition to stress fibers, our data clearly revealed differences in actin stability between these two structures. Further, although cardiomyocytes were incapable of discriminating between the incorporation of muscle and non-muscle actin isoforms into myofibrils, FR after photobleaching of Rho-muscle actin was faster than that of Rho-non-muscle actin in immature non-striated portions. This indicates that actin molecules in cardiac myofibrils cannot be readily exchanged by heterotypic non-muscle actin. Fluorescently labeled actin incorporated into non-striated (proximal and terminal) portions of myofibrils and terminal portions of stress fibers was found to be more stable than alpha-actinin. The relative stability of actin could facilitate the formation of nascent Z-bands of myofibrils and the reorganization of stress fibers at these portions.

Actinin↗