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

Abstract

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).

Rational Optimization and ImagingIn Vivoof a Genetically Encoded Optical Voltage Reporter

“…Our experimental measurements of ⌬F/F also necessarily include contributions from GFP not localized to the plasma membrane. The application of total internal reflection fluorescence microscopy in future studies could allow selective measurement of plasma membrane fluorescence (Thompson et al, 1981), enabling direct experimental measurement of relative quantum yield Q. Despite these qualifications, our model produced predictions that allowed us to optimize hVOS and image fast electrical activity in the fly olfactory system.…”

Fluorescence Fluctuation Spectroscopy in Subdiffraction Focal Volumes

“…Diffraction has, indeed, imposed a maximum molecular concentration that can be handled by far-field optical FFS. All efforts to reduce the detection volume any further implied the use of evanescent fields and mechanical constraints such as near-field optical tips [7], interfaces providing total internal reflection [8,9], and waveguide structures [10,11]. However, mechanical confinement hampers noninvasive intracellular investigations, and the interaction with the probe is likely to affect the dynamics to be probed.…”

Near-field optical probes provide subdiffraction-limited excitation areas for fluorescence correlation spectroscopy on membranes

“…1, where G D (τ) is the diffusional component of the correlation function, erf(ξ) denotes the error function, and τ' D = s 2 /4D where s is the half-length of a side of the square for the excitation profile [19]. Equation 1 is a special case of an autocorrelation function previously derived by Thompson et al [29]. The fit of the experimental FCS-NSOM data to eq.…”