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At least 19 recordsLinked to original sources
PubMed1992

Binding kinetics of an anti-dinitrophenyl monoclonal Fab on supported phospholipid monolayers measured by total internal reflection with fluorescence photobleaching recovery.

Fluorescence photobleaching recovery with total internal reflection illumination (TIR-FPR) has been used to measure the dissociation kinetics of a fluorescein-labeled anti-dinitrophenyl monoclonal Fab specifically bound to supported monolayers composed of a mixture of dipalmitoylphosphatidylcholine and dinitrophenyl-conjugated dipalmitoylphosphatidylethanolamine. The fluorescence recovery curves were not monoexponential; when analyzed as a sum of two exponentials, the rates and fractional recoveries were approximately 1 s-1 (approximately 50%) and approximately 0.1 s-1 (approximately 30%). The data did not change as a function of the Fab solution concentration, indicating that the fluorescence recovery curves were not influenced by the rate of diffusion in bulk solution. Also, the recovery curves were independent of the size of the illuminated area, indicating that surface diffusion did not significantly contribute to the rate and shape of the fluorescence recovery. The measured off rates and apparent association constant (1.6 x 10(5) M-1) were analyzed with the theoretical formalism for a proposed mechanism that accounts for the nonmonoexponential kinetics.

Animals↗
PubMed1984

Detection and characterization of actin monomers, oligomers, and filaments in solution by measurement of fluorescence photobleaching recovery.

Fluorescence photobleaching recovery (FPR) was measured to determine the diffusion coefficient of fluorescein-labeled G-actin in low-salt buffer. The result obtained, 7.15 +/- 0.35 X 10(-7) cm2/s, is in good agreement with that computed from the molecular weight, partial specific volume, and sedimentation coefficient, but is higher than previously obtained values. It is demonstrated from theory that at low ionic strength, the electrostatic contribution to the intrinsic viscosity leads to an overestimate of the hydrodynamic eccentricity of G-actin. Data from FPR, sedimentation, and fluorescence polarization experiments all indicate that the true low-salt form of the actin monomer has an axial ratio less than or equal to 3.0. The G-F transformation of actin was also observed by measurement of FPR during the assembly phase, in the steady state, and in the presence of ligands such as cytochalasin and aldolase. Each FPR record in general yields three data: relative proportion of rapidly and slowly diffusing actin, diffusion coefficient for the high-mobility fraction, and a mean diffusion coefficient for the low-mobility fraction. A relation between the mean low-mobility diffusion coefficient and the number-average filament length is derived and applied to the analysis of FPR data. Under typical conditions, the average filament length was much greater than 10 micron in the steady state. Cytochalasin D was found to decrease filament length and total amount of filament proportionally; total filament number was not greatly affected. In all polymerizations of G-actin, the high-mobility material observed in situ was found to be essentially monomeric actin. Relatively stable oligomers of actin were separated by fractionating G-AF-actin by gel filtration in 50 microM MgCl2 at 4 degrees C. On the basis of the diffusion coefficient, we conclude that monomer and dimer constitute the major particle types present under these conditions. Sedimentation of labeled actin polymerized in 1.0 mM MgCl2 yielded a graded supernatant that contained actin oligomers significantly larger than the monomer.

Actins↗
PubMed1999

Measurement of molecular diffusion in solution by multiphoton fluorescence photobleaching recovery.

Multiphoton fluorescence photobleaching recovery (MP-FPR) is a technique for measuring the three-dimensional (3D) mobility of fluorescent molecules with 3D spatial resolution of a few microns. A brief, intense flash of mode-locked laser light pulses excites fluorescent molecules via multiphoton excitation in an ellipsoidal focal volume and photobleaches a fraction. Because multiphoton excitation of fluorophores is intrinsically confined to the high-intensity focal volume of the illuminating beam, the bleached region is restricted to a known, three-dimensionally defined volume. Fluorescence in this focal volume is measured with multiphoton excitation, using the attenuated laser beam to measure fluorescence recovery as fresh unbleached dye diffuses in. The time course of the fluorescence recovery signal after photobleaching can be analyzed to determine the diffusion coefficient of the fluorescent species. The mathematical formulas used to fit MP-FPR recovery curves and the techniques needed to properly utilize them to acquire the diffusion coefficients of fluorescently labeled molecules within cells are presented here. MP-FPR is demonstrated on calcein in RBL-2H3 cells, using an anomalous subdiffusion model, as well as in aqueous solutions of wild-type green fluorescent protein, yielding a diffusion coefficient of 8.7 x 10(-7) cm(2)s(-1) in excellent agreement with the results of other techniques.

Cell Line↗
PubMed1984

Separation of translational and rotational contributions in solution studies using fluorescence photobleaching recovery.

Although fluorescence photobleaching recovery (FPR) experiments are usually interpreted in terms of the translational motions of a fluorescently labeled species, rotational motions can also modulate recovery through the cosine-squared laws for dipolar absorption and emission processes. In a complex interacting system, translational and rotational contributions may both be simultaneously present. We show how these contributions can be separated in solution studies using an FPR setup in which (a) the linear polarization of the low-intensity observation beam and the high-intensity photobleaching pulse can be varied independently, and (b) all emitted fluorescent photons are counted equally. The fluorescence recovery signal obtained with the observation beam polarized at the magic angle, 54.7 degrees, from the bleach polarization direction is independent of label orientation, whereas the anisotropy function formed from a combination of parallel and perpendicular polarizations isolates the orientational recovery. The anisotropy function is identical to that in fluorescence correlation spectroscopy and, for rigid-body rotational diffusion, can be expressed as a sum of five exponential terms.

Mathematics↗
PubMed1976

Mobility measurement by analysis of fluorescence photobleaching recovery kinetics.

Fluorescence photobleaching recovery (FPR) denotes a method for measuring two-dimensional lateral mobility of fluorescent particles, for example, the motion of fluorescently labeled molecules in approximately 10 mum2 regions of a single cell surface. A small spot on the fluorescent surface is photobleached by a brief exposure to an intense focused laser beam, and the subsequent recovery of the fluorescence is monitored by the same, but attenuated, laser beam. Recovery occurs by replenishment of intact fluorophore in the bleached spot by lateral transport from the surrounding surface. We present the theoretical basis and some practical guidelines for simple, rigorous analysis of FPR experiments. Information obtainable from FPR experiments includes: (a) identification of transport process type, i.e. the admixture of random diffusion and uniform directed flow; (b) determination of the absolute mobility coefficient, i.e. the diffusion constant and/or flow velocity; and (c) the fraction of total fluorophore which is mobile. To illustrate the experimental method and to verify the theory for diffusion, we describe some model experiments on aqueous solutions of rhodamine 6G.

Biological Transport↗
PubMed1990

Slow rotational mobilities of antibodies and lipids associated with substrate-supported phospholipid monolayers as measured by polarized fluorescence photobleaching recovery.

Polarized fluorescence photobleaching recovery has been used to monitor slow rotational motions of a fluorescently-labeled anti-dinitrophenyl mouse IgGl monoclonal antibody (ANO2) specifically bound to substrate-supported monolayers composed of a mixture of distearoylphosphatidylcholine (DSPC) and dinitrophenyldioleoylphosphatidylethanolamine (DNP-DOPE). ANO2 antibodies were labeled with a new bifunctional carbocyanine fluorophore that has two amino-reactive groups; steady-state fluorescence anisotropy data confirmed the expected result that the ANO2-conjugated bifunctional probe had less independent flexibility than ANO2-conjugated unifunctional fluorescence labels. Rotational mobilities were also measured for the fluorescent lipid 1,1'-dioctadecyl 3,3,3',3'-tetramethylindocarbocyanine (dil) in DSPC and in mixed DSPC/DNP-DOPE monolayers in the presence and absence of unlabeled ANO2 antibodies. The apparent rotational correlation time and fractional mobility of ANO2 on supported monolayers were approximately 70 and approximately 0.3 s, respectively. These measured parameters of rotational mobility did not depend on the ANO2 surface density or on kinetic factors, but addition of unlabeled polyclonal anti-(mouse IgG) antibodies significantly decreased the apparent mobile fraction. The measured fluorescence recovery curves for dil were consistent with two fluorophore populations with rotational correlation times of approximately 4 and approximately 100 s and a population of immobile fluorescent lipid. No difference in fluorescence recovery and decay curves was measured for dil in DSPC monolayers, DSPC/DNP-DOPE monolayers, and DSPC/DNP-DOPE monolayers treated with unlabeled ANO2 antibodies.

Animals↗
PubMed1981

Antigen-specific mouse lymphocyte stimulation by DNP-conjugated T-independent antigens studied by photobleaching recovery.

Fluorescence photobleaching recovery techniques have allowed us to measure the lateral mobility of T-independent antigens bound to antigen-specific mouse B cells. The in vitro immunogenicity or tolerogenicity of antigens we have examined, DNP-polymerized flagellin (DNP-POL), and DNP-linear dextran (DNP-DEX), depend upon the antigen dose and epitope density. These factors also determine the mobility of antigen bound to B cell surfaces. For DNP-POL bound to DNP-specific cells, the observed diffusion constants D decrease monotonically with increasing antigen dose and epitope density. Values of D range from 10.4 X 10(-11) cm2 sec-1 for DNP0.4-POL at 0.15 microgram/ml to 0.8 X 10(-11) cm2 sec-1 for DNP3.5-POL at 30 microgram/ml. For receptor bound DNP-DEX, D depends strongly on antigen epitope density but not observably on antigen concentration. For epitope densities of 1.2 or less, D is close to the value of 21 X10(-11) cm2 sec-1 observed for single sIg receptors. By an epitope density of 4.8, D has fallen to 2.1 X 10(-11) cm2 sec-1. Peak immunogenicities for DNP-POL and DNP-DEX are observed when antigen-receptor aggregates have mobilities 14-fold and 3-fold lower, respectively, than a single sIg molecule.

Animals↗
PubMed2004

Analysis of binding reactions by fluorescence recovery after photobleaching.

Fluorescence recovery after photobleaching (FRAP) is now widely used to investigate binding interactions in live cells. Although various idealized solutions have been identified for the reaction-diffusion equations that govern FRAP, there has been no comprehensive analysis or systematic approach to serve as a guide for extracting binding information from an arbitrary FRAP curve. Here we present a complete solution to the FRAP reaction-diffusion equations for either single or multiple independent binding interactions, and then relate our solution to the various idealized cases. This yields a coherent approach to extract binding information from FRAP data which we have applied to the question of transcription factor mobility in the nucleus. We show that within the nucleus, the glucocorticoid receptor is transiently bound to a single state, with each molecule binding on average 65 sites per second. This rapid sampling is likely to be important in finding a specific promoter target sequence. Further we show that this predominant binding state is not the nuclear matrix, as some studies have suggested. We illustrate how our analysis provides several self-consistency checks on a FRAP fit. We also define constraints on what can be estimated from FRAP data, show that diffusion should play a key role in many FRAP recoveries, and provide tools to test its contribution. Overall our approach establishes a more general framework to assess the role of diffusion, the number of binding states, and the binding constants underlying a FRAP recovery.

Algorithms↗
PubMed2006

Optimizing detection of tissue anisotropy by fluorescence recovery after photobleaching.

Fluorescence recovery after photobleaching (FRAP) has been widely used to measure fluid flow and diffusion in gels and tissues. It has not been widely used in detection of tissue anisotropy. This may be due to a lack of applicable theory, or due to inherent limitations of the method. We discuss theoretical aspects of the relationship between anisotropy of tissue structure and anisotropy of diffusion coefficients, with special regard to the size of the tracer molecule used. We derive a semi-mechanistic formula relating the fiber volume fraction and ratio of fiber and tracer molecule diameters to the expected anisotropy of the diffusion coefficients. This formula and others are tested on simulated random walks through random simulated and natural media. We determine bounds on the applicability of FRAP for detection of tissue anisotropy, and suggest minimum tracer sizes for detection of anisotropy in tissues of different composition (fiber volume fraction and fiber diameter). We find that it will be easier to detect anisotropy in monodisperse materials than in polydisperse materials. To detect mild anisotropy in a tissue, such as cartilage, which has a low fiber fraction would require a tracer molecule so large that it would be difficult to deliver to the tissue. We conclude that FRAP can be used to detect tissue anisotropy when the tracer molecule is sufficiently large relative to the fiber diameter, volume fraction, and degree of polydispersivity, and when the anisotropy is sufficiently pronounced.

Anisotropy↗
PubMed1994

Saturation effects in polarized fluorescence photobleaching recovery and steady state fluorescence polarization.

The time-resolved anisotropy produced in polarized fluorescence photobleaching recovery experiments has been successfully used to measure rotational correlation times in a variety of biological systems, however the magnitudes of the reported initial anisotropies have been much lower than the theoretically predicted maximum values. This small time-zero anisotropy has been attributed to fluorophore motion, wobble and rotation, during the photobleaching pulse. We demonstrate that inclusion of the possibility of saturation of the fluorophore's transition from its ground state to its excited state during the photobleaching pulse leads to the prediction of reduced time-zero anisotropy. This eliminates the need to rely solely on the assumption of fluorophore motion during the photobleaching pulse as the cause of the reduced initial anisotropy. We present theoretical and experimental results which show that the initial anisotropy decreases as both the bleach pulse intensity is increased and bleach pulse duration is decreased so as to keep the total integrated bleach pulse constant. We also show theoretical and experimental results demonstrating that at high excitation intensity the effects of saturation cause the steady state fluorescence polarization to decrease. We estimate that saturation may occur using common photobleaching conditions.

Biophysical Phenomena↗
PubMed1998

Macromolecular diffusion of biological polymers measured by confocal fluorescence recovery after photobleaching.

Fluorescence recovery after photobleaching with an unmodified confocal laser scanning microscope (confocal FRAP) was used to determine the diffusion properties of network forming biological macromolecules such as aggrecan. The technique was validated using fluorescein isothiocyanate (FITC)-labeled dextrans and proteins (molecular mass 4-2000 kDa) at 25 degrees C and with fluorescent microspheres (207 nm diameter) over a temperature range of 5-50 degrees C. Lateral diffusion coefficients (D) were independent of the focus position, and the degree and extent of bleach. The free diffusion coefficient (Do) of FITC-aggrecan determined by confocal FRAP was 4.25 +/- 0.6 x 10(-8) cm2 s-1, which is compatible with dynamic laser light scattering measurements. It appeared to be independent of concentration below 2.0 mg/ml, but at higher concentrations (2-20 mg/ml) the self-diffusion coefficient followed the function D = Do(e)(-Bc). The concentration at which the self-diffusion coefficient began to fall corresponded to the concentration predicted for domain overlap. Multimolecular aggregates of aggrecan ( approximately 30 monomers) had a much lower free diffusion coefficient (Do = 6.6 +/- 1.0 x 10(-9) cm2 s-1) but showed a decrease in mobility with concentration of a form similar to that of the monomer. The method provides a technique for investigating the macromolecular organization in glycan-rich networks at concentrations close to those found physiologically.

Aggrecans↗
PubMed1994

The intracellular mobility of a viral membrane glycoprotein measured by confocal microscope fluorescence recovery after photobleaching.

Fluorescence recovery after photobleaching (FRAP) has been a powerful tool for characterizing the mobility of cell surface membrane proteins. However, the application of FRAP to the study of intracellular membrane proteins has been hampered by the lack of specific probes and their physical inaccessibility in the cytoplasm. We have measured the mobility of a model transmembrane protein, the temperature-sensitive vesicular stomatitis viral membrane glycoprotein (ts-O45-G), in transit from the endoplasmic reticulum (ER) to the Golgi complex. ts-O45-G accumulates in the ER at nonpermissive temperature (39.5 degrees C) and is transported via the Golgi complex to the surface upon shifting cells to the permissive temperature (31 degrees C). Rhodamine-labeled Fab fragments against a cytoplasmic epitope of ts-O45-G (rh-P5D4-Fabs) were microinjected into cells to visualize the intracellular viral membrane protein and to determine its mobility by FRAP with a confocal microscope. Moreover, we have measured the effects of microinjected antibodies against beta-COP on the mobility of ts-O45-G following release of the temperature block. FRAP was essentially complete when rh-P5D4-Fab-injected cells were bleached either following release of labeled ts-O45-G from the ER or upon its accumulation at 20 degrees C in the trans-Golgi network (TGN). In contrast, recovery was reduced by about one third when infected cells had been injected with antibodies that bind to beta-COP in vivo. The diffusion constant of mobile ts-O45-G under all conditions was approximately 10 x 10(-10) cm2/s. These results validate the feasibility of FRAP for the study of an intracellular transmembrane protein and provide the first evidence that such a protein is highly mobile.

Animals↗
PubMed1994

Theory for measuring bivalent surface binding kinetics using total internal reflection with fluorescence photobleaching recovery.

Total internal reflection with fluorescence photobleaching recovery (TIR-FPR) is a method for experimentally examining coupled diffusion and reaction kinetics at surfaces. In a previous work (Thompson et al. 1981. Biophys. J. 33:435-454), a theoretical basis for interpreting TIR-FPR data was described for monovalent ligands that undergo a reversible reaction with monovalent surface sites in a single step. Here, the theory for TIR-FPR has been extended to two different surface binding mechanisms that involve sequential, bivalent surface attachment. Methods for obtaining the intrinsic surface association and dissociation kinetic rates from measured fluorescence photobleaching recovery curves are described. The new theory should be applicable to the association of bivalent protein ligands such as antibodies with supported planar model membranes.

Animals↗
PubMed1996

Cytoplasmic viscosity near the cell plasma membrane: translational diffusion of a small fluorescent solute measured by total internal reflection-fluorescence photobleaching recovery.

Total internal reflection-fluorescence recovery after photobleaching (TIR-FRAP) was applied to measure solute translational diffusion in the aqueous phase of membrane-adjacent cytoplasm. TIR fluorescence excitation in aqueous solutions and fluorescently labeled cells was produced by laser illumination at a subcritical angle utilizing a quartz prism; microsecond-resolution FRAP was accomplished by acousto-optic modulators and electronic photomultiplier gating. A mathematical model was developed to determine solute diffusion coefficient from the time course of photobleaching recovery, bleach time, bleach intensity, and evanescent field penetration depth; the model included irreversible and reversible photobleaching processes, with triplet state diffusion. The validity and accuracy of TIR-FRAP measurements were first examined in aqueous fluorophore solutions. Diffusion coefficients for fluorescein isothiocyanate-dextrans (10-2000 kDa) determined by TIR-FRAP (recovery t1/2 0.5-2.2 ms) agreed with values measured by conventional spot photobleaching. Model predictions for the dependence of recovery curve shape on solution viscosity, bleach time, and bleach depth were validated experimentally using aqueous fluorescein solutions. To study solute diffusion in cytosol, MDCK epithelial cells were fluorescently labeled with the small solute 2',7'-bis-2-carboxyethyl-5-carboxyfluorescein-acetoxymethyl-ester (BCECF). A reversible photobleaching process (t1/2 approximately 0.5 ms) was identified that involved triplet-state relaxation and could be eliminated by triplet-state quenching with 100% oxygen. TIR-FRAP t1/2 values for irreversible BCECF bleaching, representing BCECF translational diffusion in the evanescent field, were in the range 2.2-4.8 ms (0.2-1 ms bleach times), yielding a BCECF diffusion coefficient 6-10-fold less than that in water. These results establish the theory and the first experimental application of TIR-FRAP to measure aqueous-phase solute diffusion, and indicate slowed translational diffusion of a small solute in membrane-adjacent cytosol.

Animals↗
PubMed2006

Fluorescence photobleaching recovery using total internal reflection interference fringes.

Lateral diffusion measurements on cell membrane molecules, most commonly accomplished through fluorescence photobleaching recovery (FPR or FRAP), provide information on such molecules' size, environment, and participation in intermolecular interactions. However, difficulties arise in FPR measurements of lateral dynamics of materials, such as visible fluorescent protein (VFP) fusion proteins, where fluorescent intracellular species contribute to the fluorescence recovery signal and thus distort measurements intended to reflect surface molecules only. A new method helps eliminate these difficulties. In total internal reflection interference fringe FPR, interfering laser beams enter a 1.65-numercial aperture (NA) Olympus objective at the periphery of the back focal plane where the NA exceeds 1.38. This creates an extended interference pattern totally internally reflected at the coverslip-medium interface which excites fluorescence only from fluorescent molecules located where the cell contacts the coverslip. The large illuminated area interrogates many more membrane receptors than spot methods and hence obtains more diffusion information per measurement while rejecting virtually all interfering intracellular fluorescence. We report successful measurements of membrane dynamics of both VFP-containing and conventionally labeled molecules by this technique and compare them with results of other FPR methods.

Animals↗
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↗
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↗