Total internal reflectance fluorescence evanescent wave

To discriminate between surface bound protein molecules and those in bulk solution, total internal reflection fluorescence microscopy (TIRFM)41 55 was employed. TIRFM creates an evanescence wave that decays as a function of distance from the surface as ... 

The use of evanescent waves is very valuable to the study of interfacial properties. Techniques such as total internal reflection fluorescence (TIRF) and attenuated transmitted reflectance (ATR) use the energy of evanescent waves to probe thin regions in the vicinity of an interface to determine surface concentrations of interfacial species. 

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. 

It can be used to detect the variations in optical properties of chemical and biological films placed around the fiber [5]. The laser optical detection method based on evanescent waves is widely used in biosensors. For example, a tapered optical fiber is used to analyze the total internal reflectance fluorescence. As light is propagated down the fiber, an evanescent wave excites fluorescent tracers bound to the fiber surface. Because the evanescent wave decays exponentially with the distance from the fiber surface, the excitation radius rally extends about 100 nm into the buffer medium. A portion of the emission is captured and propagated back through the fiber to the detector. 

Total internal reflection fluorescence (TIRF) microscopy is one of the more complicated techniques for more advanced users of fluorescence microscopy techniques. In order to obtain fluorescent information about small features less than 0.5 pm, this technique uses decaying evanescent waves to probe a focal volume below the diffraction limit of light. 

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]). 

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. 

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. 

When light traversing an optically dense medium approaches an interface with a more optically rare medium at an angle exceeding a critical value, Bent = sin (rerare/ dens), total internal reflection occurs and an evanescent wave of exponentially deca5ung intensity penetrates the rarer medium. This phenomenon is at the heart of certain spectroscopic methods used to probe biomolecules at interfaces (199). In total internal reflection fluorescence (TIRF) spectroscopy (200-202), the evanescent wave excites fluorescent probes attached to the biomolecules, and detection of the emission associated with their decay provides information on the density, composition, and conformation of adsorbed molecules. In fourier transform infrared attenuated total reflection (FTIR-ATIR) spectroscopy (203,204), the evanescent wave excites certain molecular vibrational degrees of freedom, and the detected loss in intensity due to these absorbances can provide quantitative data on density, composition, and conformation. 

A large number of different biochemical systems have been studied by optical measurement on continuous surfaces, using ellipsometry, attenuated total refiection, interference techniques and total internal reflection fluorescence (reference 6 lists 45 different experiments). It is therefore likely that evanescent wave spectroscopy will become a widely applied technique in the future. 

Abel and co-workers [106] at Ciba-Geigy Ltd. have reported an automated optical biosensor system. Their device utilizes 5 -biotinylated-16-mer oligonucleotide probes bound to an optical fiber functionalised with avidin to detect complementary oligonucleotides pre-labeled with fluorescein moieties in a total internal reflection fluorescence (TIRF) evanescent wave motif similar to that of Squirrell. Immobilization of nucleic acid probes onto the optical fiber substrate was achieved by functionalisation of the surface with (3-aminopropyl)triethoxysilane (APTES) or mercaptomethyldimethylethoxysilane (MDS). Onto the short alkylsilane layer was... 

The background problem can be further overcome when using a surface-confined fluorescence excitation and detection scheme at a certain angle of incident light, total internal reflection (TIR) occurs at the interface of a dense (e.g. quartz) and less dense (e.g. water) medium. An evanescent wave is generated which penetrates into the less dense medium and decays exponentially. Optical detection of the binding event is restricted to the penetration depth of the evanescent field and thus to the surface-bound molecules. Fluorescence from unbound molecules in the bulk solution is not detected. In contrast to standard fluorescence scanners, which detect the fluorescence after hybridization, evanescent wave technology allows the measurement of real-time kinetics (www.zeptosens.com, www.affinity-sensors.com). 

The utihzation of fluorescence dyes for analytical measurements enhances the sensitivity for the detection of the molecules of interest. First, Cronick and Little made use of evanescent wave excitation for a fluorescence immunoassay, in 1975. By using totally internally reflected light, they excited the fluorescence of a fluorescein-labeled antibody which has become bound to a hapten-protein conjugate adsorbed on a quartz-plate in an antibody solution [41]. Contrary to the label-free high-refractive-index sensors where the mass of the molecule of interest is... 

Figure 2 Studying membrane fusion with supported bilayers. A supported bilayer is suspended from a quartz substrate (top, gray background) and illuminated by the evanescent wave of a totally internally reflected laser beam (angled cylinders red). A membrane vesicle is observed to approach, hemifuse, and then fully fuse with the supported membrane. Vesicle contents, lipids, or proteins may be labeled fluorescently to monitor this process.

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