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.     

Fluorescence recovery after photobleaching: direct measurement of diffusion anisotropy

Biomechanics and Modeling in Mechanobiology - Fluorescence recovery after photobleaching (FRAP) is a widely used technique for studying diffusion in biological tissues. Most of the existing...     

Fluorescence recovery after photobleaching as a probe of diffusion in starch systems

The diffusion coefficients of dextran probes of various molecular weights in starch solutions over a wide concentration range were carried out using fluorescent recovery after photobleaching (FRAP), combined with a confocal microscope and tracer probe diffusion. The technique is simple to implement and can be carried out using increasingly common microscopy apparatus, giving access to a wide variety of new structural and kinetic information. The data can be rationalized in terms of the effects of probe molecular weight and on matrix starch concentration and structure. This provides a new tool to investigate the behavior of systems where starch is an ingredient that contributes to the processing and textural properties of food.     

Fluorescence recovery after photobleaching of suspensions of vacuoles

In this work we derive theoretical expressions for the FRAP measured on a liquid suspension of vacuoles labelled by a fluorescent probe bound to the surface membrane of these vacuoles. The bleaching laser beam creates an inhomogeneity in the surface concentration of the probe molecules. We consider the case in which the randomization of these probe molecules on the vacuole surface occurs much faster than the fluorescent recovery due to the vacuole diffusion. For a given value of the bleaching parameter K, we found that the bleaching fraction of the fluorescent molecules and the fluorescence recovery rate are decreasing functions of the square ratio of the vacuole to the laser beam radius of the FRAP instrument.     

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.     

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.     

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.     

Fluorescence photobleaching with spatial Fourier analysis: measurement of diffusion in light-scattering media.

A new method for the measurement of diffusion in thick samples is introduced, based upon the spatial Fourier analysis of Tsay and Jacobson (Biophys. J. 60: 360-368, 1991) for the video image analysis of fluorescence recovery after photobleaching (FRAP). In this approach, the diffusion coefficient is calculated from the decay of Fourier transform coefficients in successive fluorescence images. Previously, the application of FRAP in thick samples has been confounded by the optical effects of out-of-focus light and scattering and absorption by the sample. The theory of image formation is invoked to show that the decay rate is the same for both the observed fluorescence intensity and the true concentration distribution in the tissue. The method was tested in a series of macromolecular diffusion measurements in aqueous solution, in agarose gel, and in simulated tissue consisting of tumor cells (45% v/v) and blood cells (5% v/v) in an agarose gel. For a range of fluorescently labeled proteins (MW = 14 to 600 kD) and dextrans (MW = 4.4 to 147.8 kD), the diffusion coefficients in aqueous solution were comparable to previously published values. A comparison of the spatial Fourier analysis with a conventional direct photometric method revealed that even for the weakly scattering agarose sample, the conventional method gives a result that is inaccurate and dependent on sample thickness whereas the diffusion coefficient calculated by the spatial Fourier method agreed with published values and was independent of sample thickness. The diffusion coefficient of albumin in the simulated tissue samples, as determined by the spatial Fourier analysis, varied slightly with sample thickness.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Determining diffusion coefficients in inhomogeneous tissues using fluorescence recovery after photobleaching

Diffusion plays an important role in the transport of nutrients and signaling molecules in cartilaginous tissues. Diffusion coefficients can be measured by fluorescence recovery after photobleaching (FRAP). Available methods to analyze FRAP data, however, assume homogeneity in the environment of the bleached area and neglect geometrical restrictions to diffusion. Hence, diffusion coefficients in inhomogeneous materials, such as most biological tissues, cannot be assessed accurately. In this study, a new method for analyzing data from FRAP measurements has been developed, which is applicable to inhomogeneous tissues. It is based on a fitting procedure of the intensity recovery after photobleaching with a two-dimensional finite element analysis, which includes Fick's law for diffusion. The finite element analysis can account for distinctive diffusivity in predefined zones, which allows determining diffusion coefficients in inhomogeneous samples. The method is validated theoretically and experimentally in both homogeneous and inhomogeneous tissues and subsequently applied to the proliferation zone of the growth plate. Finally, the importance of accounting for inhomogeneities, for appropriate assessment of diffusivity in inhomogeneous tissues, is illustrated.     

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.     

Fluorescence photobleaching in DNA sequencing

Advances in DNA-sequencing technology suggest many possibilities using either conventional or unconventional means. Several approaches offer conceivable improvements in running time by one hundred fold or more. A fluorescence photobleaching scheme in combination with ultra-thin gel slab or capillary electrophoresis which proposes to shorten the run time and to increase the resolution of multi-fragment DNA analysis is reviewed. The advantages and some of the yet to be resolved obstacles are discussed.     

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.