Total internal reflection fluorescence TIRF microscopy

Total internal reflection fluorescence (TIRF) microscopy, fluorescence in situ hybridization (FISH), fluorescence recovery after photobleaching (FRAP), fluorescence lifetime imaging microscopy (FLIM). 

W. M. Reichert and G. A. Truskey, Total internal reflection fluorescence (TIRF) microscopy. I. Modeling cell contact region fluorescence, J. Cell Sci. 96, 219-230 (1990). 

Key words Breast cancer cell migration, invasion, invadopodia. Total Internal Reflection Fluorescence (TIRF) microscopy. Interference Reflection Microscopy (IRM)... 

Total Internal Reflection Fluorescence (TIRF) Microscopy... 

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. 

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. 

Klenerman and coworkers have utilized potential-controlled delivery of water-soluble analytes from nanopipettes in a system which is entirely aqueous. Pulsed voltage-driven delivery of ions was performed from nanopipettes at close proximity to a surface, and the localized concentration profiles were measured immediately with total internal reflectance fluorescence (TIRF) microscopy (Figure 11.14B). Their studies found that the total concentration of analyte delivered was dependent on the size of the nanopipette, applied potential difference, height above the surface during delivery, and initial analyte concentration within the pipette. This localized delivery system was then applied to cellular studies in which the sodium-sensitive E. coli flagellar motor was dosed with Na+ ions. The cellular response of the speed of motor movement was observed and matched well with previously determined dosing profiles under defined delivery conditions. 

Apart from optical microscopy, there are some other optical techniques which are truly surface sensitive and have found widespread use. Examples are ellipsometry (see Section 9.4.1), total internal reflection fluorescence (TIRF) [316], and surface plasmon resonance techniques [348],... 

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. 

On the other hand, optical microscopy, confocal microscopy, ellipsometry, scanning electron microscopy (SEM), scanning tunneling microscopy (STM), atomic force microscopy (AFM) and total internal reflection fluorescence (TIRF) are the main microscopic methods for imaging the surface structure. There are many good books and reviews on spectroscopic and chemical surface analysis methods and microscopy of surfaces description of the principles and application details of these advanced instrumental methods is beyond the scope of this book. 

A variety of other techniques has been used to examine the structure of proteins at surfaces, including electron microscopy (50,51), ellipsometry (52), electrophoretic mobility (53), and total internal reflection fluorescence (TIRF) (54). Several new techniques are being applied at present, including Fourier transform infrared spectroscopy (FTIR) and TIRF (see next section),... 

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. 

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. 

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