Characterization of planar supported membranes and adjacent proteins at dielectric interfaces by fluorescence microscopy: Experiments and theories.

摘要

Total internal reflection with fluorescence correlation spectroscopy (TIR-FCS) has been developed as a technique to measure the dynamics in solution near planar dielectric interfaces. The theoretical form of the autocorrelation function describing combined surface reaction and diffusion through the evanescent wave was derived, and its application to experimental regimes was set forth. The theory was then applied to a system in which a fluorescently labeled antibody diffused adjacent to a model membrane containing no specific binding sites supported on fused silica. The diffusion of the protein was monitored ≈1000 A from the membrane surface and with varying membrane compositions and buffer conditions. The results were compared to the concentration and diffusive characteristics far from the membrane as measured by FCS with a focused beam and to the diffusion coefficient of the protein in the absence of the membrane measured by dynamic light scattering.; One future application of TIR-FCS is to perform the experiments on high refractive index substrates to achieve very shallow evanescent fields (∼200 A). In preparation for these experiments, a protocol for depositing supported bilayers onto TiO2 and SrTiO3 single-crystal substrates was developed. High-quality bilayers were formed using Langmuir-Blodgett/Langmuir-Schaeffer methods or by using the vesicle adsorption and fusion method with 30 mol % cholesterol in the vesicle mixture. Bilayers on these substrates were less intense and more difficult to bleach, which made it necessary to alter the typical experimental setup to create a smaller, more focused observation area. This instrumental alteration facilitated the need for a new theoretical model due to the nature of the inhomogeneous diffusion exhibited by the samples: recovery due to the faster component was observed from outside the observation area in addition to recovery from across the striped pattern. A theoretical model describing the fluorescence pattern photobleaching recovery through an area defined by a finite Gaussian beam intersected by a ruling was developed and experimentally verified. Preliminary measurements of protein diffusion at varying distances from the membrane were performed, and the nature of fluorescence emission and collection near dielectric interfaces was considered.