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.     

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.     

Lateral diffusion of rhodopsin in photoreceptor cells measured by fluorescence photobleaching and recovery.

Frog rod outer segments were labeled with the sulfhydryl-reactive label iodoacetamido tetramethylrhodamine. The bulk of the label reacted with the major disk membrane protein, rhodopsin. Fluorescence photobleaching and recovery (FPR) experiments on labeled rods showed that the labeled proteins diffused rapidly in the disk membranes. In these FPR experiments we observed both the recovery of fluorescence in the bleached spot and the loss of fluorescence from nearby, unbleached regions of the photoreceptor. These and previous experiments show that the redistribution of the fluorescent labeled proteins after bleaching was due to diffusion. The diffusion constant, D, was (3.0 +/- 10(-9) cm2 s-1 if estimated from the rate of recovery of fluorescence in the bleached spot, and (5.3 +/- 2.4) x 10(-9) cm2 s-1 if estimated from the rate of depletion of fluorescence from nearby regions. The temperature coefficient, Q10, for diffusion was 1.7 +/- 0.5 over the range 10 degrees--29 degrees C. These values obtained by FPR are in good agreement with those previously obtained by photobleaching rhodopsin in fresh, unlabeled rods. This agreement indicates that the labeling and bleaching procedures required by the FPR method did not significantly alter the diffusion rate of rhodopsin. Moreover, the magnitude of the diffusion constant for rhodopsin is that to be expected for an object of its diameter diffusing in a bilayer with the viscosity of the disk membrane. In contrast to the case of rhodopsin, FPR methods applied to other membrane proteins have yielded much smaller diffusion constants. The present results help indicate that these smaller diffusion constants are not artifacts of the method but may instead be due to interactions the diffusing proteins have with other components of the membrane in addition to the viscous drag imposed by the lipid bilayer.     

Depolarized fluorescence photobleaching recovery

The effects of the fact that the laser sources typically used in fluorescence photobleaching recovery (FPR) experiments in the most commonly employed in-line microscope imaging geometries, are highly linearly polarized, are examined in some detail. The implications of the results, in particular for the interpretation of FPR data in complex cell membrane systems in terms of laterally mobile and immobile sub-populations of the labelled molecular species of concern, are discussed. Methods of experimentally eliminating the potentially major rotational diffusion-based artifacts, different from those appropriate to three-dimensional (solution or suspension) systems which require other than in-line geometries, are delineated.Abbreviations FPR fluorescence photobleaching recovery - FRAP fluorescence recovery after photobleaching - 2- and 3-D two- and three-dimensional     

在荧光漂白恢复测量中荧光恢复起始时间的研究

在荧光漂白恢复测量中,漂白过程结束的准确时间相对于漂白控制信号后沿会有一定的延迟时间,不易确定.光电倍增管快门开启又要推迟一段时间.因此,荧光恢复起始时间,即漂白结束的瞬间不能准确给出,开始阶段数据收集不全.本文分析了荧光恢复起始时间不准确所造成的误差,测量了起始时间.并尽量减少此误差,使恢复初期收集到的数据更完全,荧光恢复过程的时间座标更准确.     

Polarized photoacoustic, absorption and fluorescence spectra of chloroplasts and thylakoids oriented in polyvinyl alcohol films

The polarized photoacoustic, absorption and fluorescence spectra of chloroplasts and thylakoids in unstretched and stretched polyvinyl alcohol films were measured. The intensity ratios of fluorescence bands at 674 nm, 700 nm, 730 nm and 750 nm, and the polarized fluorescence excitation spectra are strongly dependent on light polarization and film stretching. In stretched films, thylakoids exhibit predominantly 674 nm emission. The ratio of photoacoustic signal to absorption is different for light polarized parallel and perpendicular to film stretching. This difference is large in the region of chlorophyll a and carotenoids absorption in which the fluorescence excitation spectra are also strongly dependent on light polarization and film stretching. The observed spectral changes are explained by reorientation of pigment molecules influencing the yield of excitation transfer between different pigments.     

Fusion of native Sendai virions with human erythrocytes : Quantitation by fluorescence photobleaching recovery

We have recently developed a method to quantitate the fusion of reconstituted viral envelopes with cells by fluorescence photobleaching recovery (FPR) (Aroeti, B & Henis, Y I, Biochemistry 25 (1986) 4588). The method is based on the incorporation of non quenching concentrations of the fluorescent lipid probe N-(7-nitrobenz-2-oxa-1, 3-diazol-4-yl)phosphatidylethanolamine during the reconstitution of the viral envelopes (the latter probe does not incorporate efficiently into the membrane of native virions). In the present work, we employed the fluorescent dye octadecyl rhodamine B chloride (R18), which can be incorporated directly into the membrane of native enveloped virions, to extend the FPR method to study fusion between native Sendai virions and intact human erythrocytes. The R18 fluorescence was found to be quenched in the viral envelope at the concentration range required for the FPR experiments, possibly due to preferential insertion of the probe into specific domains in the viral membrane. We therefore developed a correction (presented in the Appendix) which takes into account the lower quantum yield of the probe molecules in the membranes of unfused virions in the calculation of the fraction of fused virions from the FPR experiments. The results demonstrate that the method does indeed measure virus-cell fusion, and that the contribution of exchange to the measurements is not significant. The applicability of the method was further verified by the similarity of the results to those obtained independently by fluorescence dequenching measurements, and its ability to measure the distribution of virus-cell fusion within the cell population was demonstrated. These results suggest that the use of R18 can enlarge the scope of the FPR experiments to study the fusion of native virions with cells.     

Fluorescence correlation spectroscopy and photobleaching recovery of multiple binding reactions. II. FPR and FCS measurements at low and high DNA concentrations

The preceding paper develops the theory for the interpretation of fluorescence photobleaching recovery (FPR) measurements of multiple binding of a ligand to a multivalent substrate molecule. Based on a reasonable assumption about the mechanism of the photobleaching process, this analysis shows that the observed behavior of a multivalent system should be practically identical to that of a univalent binding system. This is in contrast to the expected and observed behavior of fluorescence correlation spectroscopy (FCS) measurments. Experimental FPR measurements of multivalent binding of ethidium bromide to DNA confirm these conclusions. The FCS and FPR measurements also reveal an apparently enhanced diffusion of ethidium at high DNA concentration. This enhancement might result from direct transfer of ethidium among DNA molecules.     

The polarization of fluorescence of DNA stains depends on the incorporation density of the dye molecules.

BACKGROUND: The fluorescence induced by polarized light sources, such as the lasers that are used in flow cytometry, is often polarized and anisotropic. In addition, most optical detector systems are sensitive to the direction of polarization. These two factors influence the accuracy of fluorescence intensity measurements. The intensity of two light sources can be compared only if all details of the direction and degree of polarization are known. In a previous study, we observed that fluorescence polarization might be modified by dye-dye interactions. This report further investigates the role of dye density in fluorescence polarization anisotropy. METHODS: We measured the polarization distribution of samples stained with commonly used DNA dyes. To determine the role of fluorophore proximity, we compared the monomeric and a dimeric form of the DNA dyes ethidium bromide (EB), thiazole orange (TO), and oxazole yellow (YO). RESULTS: In all dyes sampled, fluorescence polarization is less at high dye concentrations than at low concentrations. The monomeric dyes exhibit a higher degree of polarization than the dimeric dyes of the same species. CONCLUSIONS: The polarization of fluorescence from DNA dyes is related to the density of incorporation into the DNA helix. Energy transfer between molecules that are in close proximity loosens the linkage between the excitation and emission dipoles, thereby reducing the degree of polarization of the emission.     

Fluorescence Recovery after Merging a Droplet to Measure the Two-dimensional Diffusion of a Phospholipid Monolayer

We introduce a new method to measure the lateral diffusivity of a surfactant monolayer at the fluid-fluid interface, called fluorescence recovery after merging (FRAM). FRAM adopts the same principles as the fluorescence recovery after photobleaching (FRAP) technique, especially for measuring fluorescence recovery after bleaching a specific area, but FRAM uses a drop coalescence instead of photobleaching dye molecules to induce a chemical potential gradient of dye molecules. Our technique has several advantages over FRAP: it only requires a fluorescence microscope rather than a confocal microscope equipped with high power lasers; it is essentially free from the selection of fluorescence dyes; and it has far more freedom to define the measured diffusion area. Furthermore, FRAM potentially provides a route for studying the mixing or inter-diffusion of two different surfactants, when the monolayers at a surface of droplet and at a flat air/water interface are prepared with different species, independently.     

Fluorescence correlation spectroscopy and photobleaching recovery of multiple binding reactions. I. Theory and FCS measurements

Fluorescence correlation spectroscopy (FCS) and fluorescence photobleaching recovery (FPR) are two methods that may be used to measure diffusion and chemical reaction kinetics in small, labile systems such as biological cells. These methods are here applied to systems in which a fluorescent ligand can bind to a polyvalent substrate molecule in a multistep reaction sequence. The analytical theory for both FCS and FPR is extended to allow analysis of these kinds of systems. Experimental measurements of the binding of ethidium bromide to DNA by FCS confirm the theoretical analysis. (FPR measurements on the same system are reported in the accompanying paper.) The analysis shows that FCS and FPR perceive multivalent binding reactions differently. This difference results from the selective effect of the photobleaching process in the chemical reaction system. The development and results we report could have useful applications to a wide range of biopolymeric binding and assembly process.     

Linear dichroism and polarized fluorescence of dye-complexed DNA fibers

Summary A simple method to obtain well orientated DNA fibers for studying the ordered binding of dyes and fluorochromes by linear dichroism and polarized fluorescence is described. The metachromatic dye toluidine blue and the intercalating fluorochromes ethidium bromide and acridine orange showed a perpendicular alignement to DNA; the minor groove binding fluorochromes 33258 Hoechst and DAPI appeared parallel. Thus, DNA fibers represent a suitable cytochemical test substrate for studying the orientation of bound dyes by polarization methods.     

Polarized fluorescence depletion reports orientation distribution and rotational dynamics of muscle cross-bridges

The method of polarized fluorescence depletion (PFD) has been applied to enhance the resolution of orientational distributions and dynamics obtained from fluorescence polarization (FP) experiments on ordered systems, particularly in muscle fibers. Previous FP data from single fluorescent probes were limited to the 2(nd)- and 4(th)-rank order parameters, and , of the probe angular distribution (beta) relative to the fiber axis and , a coefficient describing the extent of rapid probe motions. We applied intense 12-micros polarized photoselection pulses to transiently populate the triplet state of rhodamine probes and measured the polarization of the ground-state depletion using a weak interrogation beam. PFD provides dynamic information describing the extent of motions on the time scale between the fluorescence lifetime (e.g., 4 ns) and the duration of the photoselection pulse and it potentially supplies information about the probe angular distribution corresponding to order parameters above rank 4. Gizzard myosin regulatory light chain (RLC) was labeled with the 6-isomer of iodoacetamidotetramethylrhodamine and exchanged into rabbit psoas muscle fibers. In active contraction, dynamic motions of the RLC on the PFD time scale were intermediate between those observed in relaxation and rigor. The results indicate that previously observed disorder of the light chain region in contraction can be ascribed principally to dynamic motions on the microsecond time scale.     

Protein rotational motion in solution measured by polarized fluorescence depletion.

A microscope-based system is described for directly measuring protein rotational motion in viscous environments such as cell membranes by polarized fluorescence depletion (PFD). Proteins labeled with fluorophores having a high quantum yield for triplet formation, such as eosin isothiocyanate (EITC), are examined anaerobically in a fluorescence microscope. An acousto-optic modulator generates a several-microsecond pulse of linearly polarized light which produces an orientationally-asymmetric depletion of ground state fluorescence in the sample. When the sample is then probed with light polarized parallel to the excitation pulse, fluorescence recovers over 0-1,000 microseconds as the sum of two exponentials. One exponential corresponds to triplet decay and the other to the rotational relaxation. An exciting pulse perpendicular to the probe beam is then applied. Fluorescence recovery following this pulse is the difference of the same two exponentials. Equations for fluorescence recovery kinetics to be expected in various experimentally significant cases are derived. Least-squares analysis using these equations then permits the triplet lifetime and rotational correlation time to be determined directly from PFD data. Instrumentation for PFD measurements is discussed that permits photobleaching recovery measurements of lateral diffusion coefficients using the same microscope system. With this apparatus, both rotational and translational diffusion coefficients (Dr, Dt) were measured for EITC-labeled bovine serum albumin in glycerol solutions. Values obtained for Dr and Dt are discussed in light of both the PFD models and the experimental system.     

Measuring surface dynamics of biomolecules by total internal reflection fluorescence with photobleaching recovery or correlation spectroscopy.

The theoretical basis of a new technique for measuring equilibrium adsorption/desorption kinetics and surface diffusion of fluorescent-labeled solute molecules at solid surfaces has been developed. The technique combines total internal reflection fluorescence (TIR) with either fluorescence photobleaching recovery (FPR) or fluorescence correlation spectroscopy (FCS). A laser beam totally internally reflects at a solid/liquid interface; the shallow evanescent field in the liquid excites the fluorescence of surface adsorbed molecules. In TIR/FPR, adsorbed molecules are bleaching by a flash of the focused laser beam; subsequent fluorescence recovery is monitored as bleached molecules exchange with unbleached ones from the solution or surrounding nonilluminated regions of the surface. In TIR/FCS, spontaneous fluorescence fluctuations due to individual molecules entering and leaving a well-defined portion of the evanescent field are autocorrelated. Under appropriate experimental conditions, the rate constants and surface diffusion coefficient can be readily obtained from the TIR/FPR and TIR/FCS curves. In general, the shape of the theoretical TIR/FPR and TIR/FCS curves depends in a complex manner upon the bulk and surface diffusion coefficients, the size of the iluminated or observed region, and the adsorption/desorption/kinetic rate constants. The theory can be applied both to specific binding between immobilized receptors and soluble ligands, and to nonspecific adsorption processes. A discussion of experimental considerations and the application of this technique to the adsorption of serum proteins on quartz may be found in the accompanying paper (Burghardt and Axelrod. 1981. Biophys. J. 33:455).     

Polarization of fluorescently labeled myosin subfragment-1 fully or partially decorating muscle fibers and myofibrils.

Fluorescently labeled myosin heads (S1) were added to muscle fibers and myofibrils at various concentrations. The orientation of the absorption dipole of the dye with respect to the axis of F-actin was calculated from polarization of fluorescence which was measured by a novel method from video images of muscle. In this method light emitted from muscle was split by a birefringent crystal into two nonoverlapping images: the first image was created with light polarized in the direction parallel to muscle axis, and the second image was created with light polarized in the direction perpendicular to muscle axis. Images were recorded by high-sensitivity video camera and polarization was calculated from the relative intensity of both images. The method allows measurement of the fluorescence polarization from single myofibril irrigated with low concentrations of S1 labeled with dye. Orientation was also measured by fluorescence-detected linear dichroism. The orientation was different when muscle was irrigated with high concentration of S1 (molar ratio S1:actin in the I bands equal to 1) then when it was irrigated with low concentration of S1 (molar ratio S1:actin in the I bands equal to 0.32). The results support our earlier proposal that S1 could form two different rigor complexes with F-actin depending on the molar ratio of S1:actin.     

Effects of second order photobleaching on recovered diffusion parameters from fluorescence photobleaching recovery

In the original theoretical development of fluorescence photobleaching recovery with circular or Gaussian laser intensity profiles (Axelrod et al., 1976, Biophys. J.) the bleaching process is assumed to obey first order kinetics in the fluorescent probe. While this is reasonable in most cases where oxygen participates in the photolysis reaction, some processes may obey second order kinetics in the fluorophore concentration due to dimerization. Accordingly, we present here an analysis of the fluorescence recovery when the photobleaching process is taken to be second order in the probe. Analytical solutions for small bleaching levels indicate that the fluorescence recovery curve is very similar to that measured following a bleaching process first order in the probe. Numerical solutions for moderate bleaching levels show that the recovery is qualitatively similar, but quantitatively different. Because the shape of the recovery curve provides no evidence as to the order of photobleaching, we recommend continued use of the previous theoretical analysis. However, it must be borne in mind that the diffusion coefficient is increasingly underestimated as the extent of photobleaching is increased. The true diffusion coefficient is obtained in the limit of small levels of photobleaching. Estimates of the fractional recovery are not affected by this approach.     

Fringe pattern photobleaching, a new method for the measurement of transport coefficients of biological macromolecules.

The conventional method of studying mass transport in membranes by spot photobleaching and then following the recovery of fluorescence has disadvantages. Among them, the need for a high density of fluorescent molecules, the measurement of the beam profile, and a knowledge of the photobleaching processes are of a crucial importance. The application of a planar fringe pattern of light both for the bleaching and the monitoring of the fluorescent molecules solves these three major difficulties. Brownian diffusion coefficients and flow velocities can be measured independently and are averaged over the whole fringe pattern volume. These transport coefficients are explored over the wide range of experimentally accessible distances (from an interfringe spacing 0.5-50 micron). The quantification of the mobile and immobile components is further simplified by scanning the fringe pattern and detecting only a modulated fluorescence recovery signal. The fringe pattern photobleaching method is particularly adapted to the measurements of diffusion coefficients and flow velocity of membrane components, as well as of cytoplasmic proteins. The theoretical results and the test experiments with fluorescent bovine serum albumin are described.     

Speed of intersubunit communication in proteins.

To determine the speed of communication between protein subunits, time-resolved absorption spectra were measured following partial photodissociation of the carbon monoxide complex of hemoglobin. The experiments were carried out using linearly polarized, 10-ns laser pulses, with the polarization of the excitation pulse both parallel and perpendicular to the polarization of the probe pulse. The substantial contribution to the observed spectra from photoselection effects was eliminated by isotropically averaging the polarized spectra, allowing a detailed comparison of the kinetics as a function of the degree of photolysis. These results show that prior to 1 microsecond both geminate ligand rebinding and conformational relaxation are independent of the number of ligands dissociated from the hemoglobin tetramer, as expected for a two-state allosteric model. After this time the kinetics depend on the ligation state of the tetramer. The conformational relaxation at 10 microseconds can be interpreted in terms of the two-state allosteric model as arising from the R to T quaternary conformational change of both unliganded and singly liganded molecules. These results suggest that communication between subunits requires about 1 microsecond and that the mechanism of the communication which occurs after this time is via the R to T conformational change. The optical anisotropy provides a novel means of accurately determining the extinction coefficients of the transient photoproduct. The decay in the optical anisotropy, moreover, provides an accurate determination of the rotational correlation time of 36 +/- 3 ns.     

Fluorescence recovery spectroscopy as a probe of slow rotational motions.

Pump-and-probe techniques can be used to follow the slow rotational motions of fluorescent labels bound to macromolecules in solution. A strong pulse of polarized light initially anisotropically depletes the ground-state population. A continuous low-intensity beam of variable polarization then probes the anisotropic ground-state distribution. Using an additional emission polarizer, the generated fluorescence can be recorded as it rises towards its prepump value. A general theory of fluorescence recovery spectroscopy (FRS) is presented that allows for irreversible depletion processes like photobleaching as well as slowly reversible processes like triplet formation. In either case, rotational motions modulate recovery through cosine-squared laws for dipolar absorption and emission processes. Certain pump, probe, and emission polarization directions eliminate the directional dependence of either dipole and simplify the resulting expressions. Two anisotropy functions can then be constructed to independently monitor the rotations of either dipole. These functions are identical in form to the anisotropy used in fluorescence depolarization measurements and all rotational models developed there apply here with minor modifications. Several setups are discussed that achieve the necessary polarization alignments. These include right-angle detection equipment that is commonly available in laboratories using fluorescence methods.