Total internal reflection fluorescence microscopy evanescent fields

Fig. 15.1. Schematic representation of amyloid fibrils revealed by total internal reflection fluorescence microscopy, (a) The penetration depth of the evanescent field formed by the total internal reflection of laser light is 150nm for a laser light at 455 nm, so only amyloid fibrils lying parallel to the slide glass surface were observed. (b) Schematic diagram of a prism-type TIRFM system on an inverted microscope. ISIT image-intensifier-coupled silicone intensified target camera, CCD charge-coupled device camera...

Wellman AD, Sepaniak MJ (2007) Multiplexed, waveguide approach to magnetically assisted transport evanescent field fluoroassays. Anal Chem 79 6622-6628 Loete F, Vuillemin B, Oltra R et al (2006) Application of total internal reflection fluorescence microscopy for studying pH changes in an occluded electrochemical cell development of a waveguide sensor. Electrochem Commun 8 1016—1020... 

Total internal reflection fluorescence microscopy (TIRFM) is a promising alternative approach to low background fluorescence imaging [68], For excitation of molecules on a surface or within a thin slice of the sample, an evanescent optical field is used traveling along the interface between a medium with a high refractive index n, (typically a quartz glass prism) and... 

Total internal reflection fluorescence microscopy (TIRFM), or simply TIRF, is the application of fluorescence imaging to evanescent-wave microscopy. The material of interest is tagged with fluorescent material that emits light at wavelengths greater than the excitation photons from the evanescent field, which additionally improves the detectability of the evanescent-wave microscopy technique. 

Nanofluidic systems are also ideally suitable for certain single-molecule detection techniques such as total internal reflection fluorescence (TIRF) microscopy. TIRF utilizes evanescent waves, which are generated by total internal reflection of a laser beam, to excite the fluorescence signal, and since evanescent waves decay exponentially, the molecule of interest must locate to the close proximity of the interface of the glass and liquid. Nanofluidic systems confine molecules of interest in nanochannels, which is well in the evanescent field of TIRF microscopy. 

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. 

At the edge between 6a and 6c, the laser beam is incident above critical angle at the glass/water interface at where the beam is totally internally reflected, generating evanescent field in the water. Thus, by shifting the position of the one mirror, the type of illumination can be switched between epi-fluorescence microscopy and objective-type TIRFM. 

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