Total internal reflection fluorescence combination

The use of an evanescent wave to excite fluorophores selectively near a solid-fluid interface is the basis of the technique total internal reflection fluorescence (TIRF). It can be used to study theadsorption kinetics of fluorophores onto a solid surface, and for the determination of orientational order and dynamics in adsorption layers and Langmuir-Blodgett films. TIRF microscopy (TIRFM) may be combined with FRAP ind FCS measurements to yield information about surface diffusion rates and the formation of surface aggregates. 

A new technique for measuring equilibrium adsorption/desorption kinetics and surface diffusion of fluorescent-labelled solute molecules at surfaces was developed by Thompson et al.74). The technique combines total internal reflection fluorescence with either fluorescence photobleaching recovery or fluorescence correlation spectroscopy with lasers. For example, fluorescent labelled protein was studied in regard to the surface chemistry of blood 75). 

Another exciting technique combines FRAP with total internal reflection fluorescence microscopy. This technique, called total internal reflection interference fringe fluorescence photobleaching... 

Application of total internal reflection fluorescence Dependent on tbe combined method 171... 

Oreopoulos, J., Epand, R. E, Epand, R. M., and Yip, C. M. 2010. Peptide-induced domain formation in supported lipid bilayers Direct evidence by combined atomic force and polarized total internal reflection fluorescence microscopy, Biophys J 98,815-823. 

Nishida, S., Funabashi, Y, and Ikai, A. 2002. Combination of AFM with an objective-type total internal reflection fluorescence microscope (TIRFM) for nanomanipulation of single cells. 

Oreopoulos, J. and Yip, C. M. 2008. Combined scanning probe and total internal reflection fluorescence microscopy. Methods 46,2-10. 

In micro- and nanoscale fluid mechanics, measurements of mass transport and fluid velocity are used to probe fundamental physical phenomena and evaluate the performance of microfluidic devices. Evanescent wave illumination has been combined with several other diagnostic techniques to make such measurements within a few hundred nanometers of fluid—solid interfaces with a resolution as small as several nanometers. Laser Doppler velocimetry has been applied to measure single-point tracer particle velocities in the boundary layer of a fluid within 1 pm of a wall. By seeding fluid with fluorescent dye, total internal reflection fluorescence recovery after photobleaching (FRAP) has been used to measure near-wall diffusion coefficients and velocity (for a summary of early applications, see Zettner and Yoda [2]). 

Conibear, PB and Bi shaw, CR, A comparison of optical geometries for combined flash photolysis and total internal reflection fluorescence microscopy. Journal of Microscopy 200 (2000) 218-229. 

Yordanov S, Best A, Weisshart K, Koynov K (2011) An easy way to enable total internal reflection-fluorescence correlation spectroscopy (TIR-FCS) by combining commercial devices for FCS and TIR microscopy. Rev Sci Instrum 82 036105... 

T. E. Starr and N. L. Thompson, Total internal reflection with fluorescence correlation spectroscopy Combined surface reaction and solution diffusion, Biophys. J. 80, 1575-1584 (2001). 

Here, a laser beam totally internally reflects at a sohd/hquid interface, creating an evanescent field, which penetrates only a fraction of the wavelength into the liquid domain. When using planar phosphoHpid bilayer and fluorescently labeled proteins, this method allows the determination of adsorption/desorption rate constants and surface diffusion constants [171—173]. Figure 6.29 shows a representative TIRF-FPR curve for fluorescein-labeled prothrombin bound to planar membranes. In this experiment the experimental conditions are chosen such that the recovery curve is characterized by the prothrombin desorption rate. It should be mentioned that, similar to other applications of fluorescence microscopy, two and three photon absorption might be combined with FRAP in the near future. 

Wakelin, S. and Bagshaw, C. R. 2003. A prism combination for near isotropic fluorescence excitation by total internal reflection, / Microsc 209,143-148. 

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