Daniel Axelrod

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.

Total Internal Reflection Fluorescence Microscopy in Cell Biology

Key events in cellular trafficking occur at the cell surface, and it is desirable to visualize these events without interference from other regions deeper within. This review describes a microscopy technique based on total internal reflection fluorescence which is well suited for optical sectioning at cell‐substrate regions with an unusually thin region of fluorescence excitation. The technique has many other applications as well, most notably for studying biochemical kinetics and single biomolecule dynamics at surfaces. A brief summary of these applications is provided, followed by presentations of the physical basis for the technique and the various ways to implement total internal reflection fluorescence in a standard fluorescence microscope.

Dynamics of fluorescence marker concentration as a probe of mobility.

We have developed an effective experimental system for the characterization of molecular and structural mobility. It incorporates a modified fluorescence microscope geometry and a variety of analytical techniques to measure effective diffusion coefficients ranging over almost six orders of magnitude, from less than 10(-11) cm2/s to greater than 10(-6) cm2/s. Two principal techniques, fluorescence correlation spectroscopy (FCS) and fluorescence photobleaching recovery (FPR), are employed. In the FPR technique, translational transport rates are measured by monitoring the evolution of a spatial inhomogeneity of fluorescence that is produced photochemically in a microscopic volume by a short burst of intense laser radiation. In contrast, FCS uses laser-induced fluorescence to probe the spontaneous concentration fluctuations in microscopic sample volumes. The kinetics are analyzed by computing time-correlation functions of the stochastic fluctuations of the measured fluorescence intensity. The optical system and digital photocount correlator designed around a dedicated minicomputer are described and discussed. The general power of these techniques is demonstrated with examples from studies conducted on bulk solutions, lipid bilayer membranes, and mammalian cell plasma membranes.

Carbocyanine dye orientation in red cell membrane studied by microscopic fluorescence polarization

The orientation of an amphipathic, long acyl chain fluorescent carbocyanine dye [diI-C18-(3)] in a biological membrane is examined by steady-state fluorescence polarization microscopy on portions of single erythrocyte ghosts. The thermodynamically plausible orientation model most consistent with the experimental data is one in which the diI-C18-(3) conjugated bridge chromophore is parallel to the surface of the cell and the acyl chains are imbedded in the bilayer parallel to the phospholipid acyl chains. Comparison of the predictions of this model with the experimental data yields information on the intramolecular orientations of the dyes transition dipoles and on the dyes rate of rotation in the membrane around an axis normal to the membrane. To interpret the experimental data, formulae are derived to account for the effect of high aperture observation on fluorescence polarization ratios. These formulae are generally applicable to any high aperture polarization studied on microscopic samples, such as portions of single cells.

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).

Total internal reflection fluorescence microscopy in cell biology

Key events in cellular trafficking occur at the cell surface, and it is desirable to visualize these events without interference from other regions deeper within. This review describes a microscopy technique based on total internal reflection fluorescence which is well suited for optical sectioning at cell-substrate regions with an unusually thin region of fluorescence excitation. The technique has many other applications as well, most notably for studying biochemical kinetics and single biomolecule dynamics at surfaces. A brief summary of these applications is provided, followed by presentations of the physical basis for the technique and the various ways to implement total internal reflection fluorescence in a standard fluorescence microscope.

Fluorescent tetramethyl rhodamine derivatives of α-bungarotoxin: Preparation, separation, and characterization

Abstract α-Bungarotoxin is fluorescently labeled with tetramethyl rhodamine isothiocyanate and then fractionated on Sephadex G-25 and CM-Sephadex C-50 columns. The elution profile of the CM-Sephadex C-50 columns exhibits four distinct fluorescent peaks and a peak of unlabeled toxin. All four fluorescent peaks can fluorescently stain mouse diaphragm motor end plates. The most slowly eluting peak, Peak IV, has the highest quantum efficiency. Peak IV, which is identified as monolabeled tetramethyl rhodamine α-bungarotoxin, binds irreversibly to acetylcholine receptors on electroplax fragments and labels the fragments more intensely than Peaks I–III, which are identified as mixtures of multiply labeled tetramethyl rhodamine α-bungarotoxin.

Fluorescence emission at dielectric and metal-film interfaces

It is well known that the classical optical properties of a bare or metal-film-coated dielectric surface significantly the emission pattern of a fluorophore in close proximity to it. Most previous classical calculations of this perturb model the fluorophore as a continuous fixed-amplitude dipole acting as a simple radiator. However, for effect modeling steady-state excitation, a fixed-power dipole is more appropriate. This modification corresponds to normalizing fixed-amplitude dipole intensities by the total dissipated power, which is itself dependent on fluorophore orientation and proximity to the surface. The results for the fixed-power model differ nontrivially from the fixed-amplitude model. Using the fixed-power dipole model, we calculate the observation-angle-dependent intensity as a function of the fluorophore’s orientation and distance from the surface. The surface can have an intermediate layer of arbitrary thickness on it, which is used to model a metal-film-coated dielectric. In addition, general expressions are derived for the emission power as observed through a circular-aperture collection system (such as a microscope objective) located on either side of the interface. These expressions are applied to several common cases of fluorophore spatial and orientational distributions at bare glass–water and metal-film-coated glass-water interfaces. The results suggest practical experimental approaches for measuring the spatial and orientational distribution of fluorophores adsorbed at a surface, utilizing the distance-dependent fluorescence near a metalized surface and optimizing the collection efficiency from a well-defined volume near a quenching surface.

Total internal reflection fluorescent microscopy

This review discusses applications of fluorescence microscopy using totally internally reflected excitation light. When totally internally reflected in a transparent solid at its interface with liquid, the excitation light beam penetrates only a short distance into the liquid. This surface electromagnetic field, called the ‘evanescent wave’, can selectively excite fluorescent molecules in the liquid near the interface. Total internal reflection fluorescence (TIRF) has been used to examine the cell/substrate contact regions of primary cultured rat myotubes with acetylcholine receptors labelled by fluorescent α‐bungarotoxin and human skin fibroblasts labelled with a membrane‐incorporated fluorescent lipid. TIRF examination of cell/substrate contacts dramatically reduces background from cell autofluorescence and debris. TIRF has also been combined with fluorescence photobleaching recovery and correlation spectroscopy to measure the chemical kinetic binding rates and surface diffusion constant of fluorescent labelled serum protein binding (at equilibrium) to a surface.

Total internal reflection/fluorescence photobleaching recovery study of serum albumin adsorption dynamics

The total internal reflection/fluorescence photobleaching recovery (TIR/FPR) technique (Thompson et al. 1981. Biophys. J. 33:435) is used to study adsorbed bovine serum albumin dynamics at a quartz glass/aqueous buffer interface. Adsorbed fluorescent labeled protein is bleached by a brief flash of the evanescent wave of a focused totally internally reflected laser beam. The rates of adsorption/desorption and surface diffusion determine the subsequent fluorescence recovery. The protein surface concentration is low enough to be proportional to the observed fluorescence and high enough to insure that the observed recovery rates arise mainly from adsorbed rather than bulk protein dynamics. The photobleaching recovery curves for rhodamine-labeled bovine serum albumin reveal both an irreversibly bound state and a multiplicity of reversibly bound states. The relative amount of reversible to irreversible adsorption increases with increasing bulk protein concentration. Since the adsorbed protein concentration appears to be too high to pack into a homogeneous surface monolayer, the wide range of desorption rates possibly results from multiple layers of protein on the surface. Comparison of the fluorescence recovery curves obtained with various focused laser beam widths suggests that some of the reversibly bound bovine serum albumin molecules can surface diffuse. Aside from their relevance to the surface chemistry of blood, these results demonstrate the feasibility of the TIR/FPR technique for measuring molecular dynamics on solid surfaces.