Total Internal Reflection Fluorescence TIRF

Recently, a formalism has been developed to determine the second and the fourth order parameters of films using polarized total internal reflection fluorescence (TIRF) [71]. Similarly to IR-ATR spectroscopy (Section 4), the experiment makes use of p- and s-polarized excitation, but the fluorescence emission (analyzed either in p- or s-direction) is detected normal to the substrate. Two approaches are developed based on the measurements of two intensity ratios. In the first one, the S angle has to be known experimentally or theoretically, and the order parameters (P2) and (P4) can be determined. In the second one, the order parameter (R ) is obtained by another technique, for instance IR-ATR spectroscopy, which allows deducing the order parameter (P4) and (cos2<5). 

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

S. A. Rockhold, R. D. Quinn, R. A. VanWegenen, J. D. Andrade, and M. Reichert, Total internal reflection fluorescence (TIRF) as a quantitative probe of protein adsorption,... 

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). 

Techniques which allow one to monitor the boundary layer as a function of time, such as total internal reflection fluorescence (TIRF) spectroscopy 4 43), permit a quantitative evaluation of interfacial mass transport processes using, for example, fluorescently-tagged macromolecules which do not adsorb, such as fluorescein-labeled dextran 40 ... 

The tendency for an adsorbed protein to undergo conformational change is protein-and interface-specific, as well as time-dependent. Total internal reflection fluorescence (TIRF) studies on IgG adsorption and desorption on hydrophobic and hydrophilic surfaces as a function of residence time show clearly both the time-dependence, as well as the surface-dependence, of desorption 92 ... 

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],... 

Nucleic acid biosensors based on optical modes of detection represent another common approach for generating analytical signals based on nucleic acid hybridization. The methods discussed herein are based on methods that are suitable for the study of materials on surfaces. There are a number of different optical methods that have been described, with the most common being attenuated total reflectance (ATR), total internal reflection fluorescence (TIRF) and surface plasmon resonance (SPR) [15]. All of these methods work... 

FIGURE 2.17 Results of total internal reflection fluorescence (TIRF) measurements in which the discrete and processive stepping action of myosin can be seen clearly. SOURCE Reprinted with permission from Yildiz, A., J.N. Forkey, S.A. McKinney, T. Ha, Y.E. Goldman, and P.R. Selvin..2003. Myosin V walks hand-over-hand Single fluorophore imaging with 1.5-nm localization. Science 300 2061-2065. Copyright 2003 AAAS. 

Tschmelak, J., M. Kumpf, N. Kappel, et al. 2006. Total internal reflectance fluorescence (TIRF) biosensor for environmental monitoring of testosterone with commercially available immunochemistry Antibody characterization, assay development and real sample measurements. Talanta 69 343-350. 

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. 

In general, a vast number of optical transduction techniques can be used for biosensor development. These may employ linear optical phenomenon (e.g. adsorption, fluorescence, phosphorescence, and polarization) or nonlinear phenomena (e.g. second harmonic generation). The choice of a particular optical method depends on the analyte and the sensitivity needed. Total internal reflection fluorescence (TIRF) has been used with planar and fibre-optic wave-guides as signal transducers in a number of biosensors. 

The three-dimensional network of actin stress fiber, which is an association of actin filaments, provides mechanical support for the cell, determines the cell shape, and enables cell movement. Thus the shape change in the cell due to the laser tsunami can be examined by observing the laser-induced dynamics of fibers. The actin stress fiber was visualized by binding it with enhanced green fluorescence protein (EGFP), and monitored by total internal reflection fluorescence (TIRF) imaging [34]. 

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),... 

Current understanding of protein adsorption has been synthesized from investigations employing a number of techniques. One of these techniques, total internal reflection fluorescence (TIRF), has emerged as a versatile tool for studying proteins at surfaces. The investigation of protein adsorption at solid-liquid interfaces in this laboratory using TIRF is reviewed. Several reviews of the TIRF technique have appeared recently (13-15). 

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

Total Internal Reflection Fluorescence (TIRF) and Surface Enhanced Fluorescence... 

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