Total internal reflection fluorescence microscopy: application to substrate-supported planar membranes

Abstract

The use of total internal reflection illumination in fluorescence microscopy (TIRFM) is reviewed with emphasis on application to fluorescent macromolecules that specifically and reversibly bind to planar model membranes supported on glass or quartz substrates. Several methods for characterizing macromolecular motion and organization are discussed: the measurement of equilibrium binding curves to obtain values for equilibrium binding constants; the measurement of fluorescence photobleaching recovery curves to obtain values of kinetic rate constants and surface diffusion coefficients; and the measurement of fluorescence intensities as a function of the evanescent field polarization to characterize orientational order. Applications to cell-substrate contact regions are summarized and future directions of TIRFM are outlined.

Total internal reflection spectroscopy for studying soft matter

“…TIRF provides two means to measure kinetics of adsorption even when the system is at equilibrium. 100 The first is to use correlation spectroscopy. Here, only a very small number of molecules (∼100) are in the field-of-view at any one time, 131 by having a small surface coverage, a small sampling area, or by only fluorescently labelling a small fraction of the available adsorbates.…”

Quantitative single-molecule study of TGF-β/Smad signaling

“…TIRFM is the most widely used microscope for SMIT, which can greatly reduce intracellular background signals and effectively improve the SNR of images on cell membrane [23]. It is known that when the illumination laser travels through an optically denser medium to an optically thinner medium and the incident angle is greater than the critical angle, total internal reflection will appear to generate an evanescent field, of which the excitation intensity exponentially decays with distance, resulting in a thin-layer illumination about 100-160 nm at the interface ( Fig.…”

Directin vivomeasurement of targeted binding in a human tumor xenograft

“…This approach has been used extensively to detect binding in cell membranes (7,8), cytoplasm (9,10), and various in vitro preparations (11)(12)(13)(14)(15)(16). We previously applied FRAP in vivo to measure the interstitial diffusion and convection of albumin within a tumor tissue preparation (17), and we report here the application of FRAP to measure binding in vivo.…”