Total Internal Reflection Fluorescence TIRF Spectroscopy

Total Internal Reflection Fluorescence (TIRF) Spectroscopy 

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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 ... 

SHG) [610], total internal reflection fluorescence (TIRF) spectroscopy [611], UV/Vis spectroscopy, IR spectroscopy, ellipsometry [612], quartz crystal microbalance (QCM) [613], STM [614], and AFM (see Refs. [618-623] for review of in sitn spectroscopies). From those, only the vibrational methods (SHG, SERS, and IR spectroscopy) are able to provide information on both the chemical composition and the strnctnre of the species adsorbed. The IR SEC experiment is simpler and more accessible than SHG and SERS, making IR spectroscopy the dominant tool for stndying electrochemical reactions at metallic electrodes [616, 617, 624-641]. 

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. 

Figure 7 shows a human (milk) Lyso adsorption isotherm recorded on hydrophobic silica slides treated with dimethyldichlorosilane (DDS) [29]. Total internal reflectance fluorescence (TIRF) spectroscopy was used to assess the adsorbed mass of Lyso. The isotherm experiment was carried out at pH 7.4, with protein dissolved in a phosphate-buffered saline (PBS) (0.013 M KH2PO4, 0.054 Af Na2HP04) buffer including 0.1 M NaCl. Tables 8 and 9 show the relevant thermodynamic and regression data determined for this isotherm. 

This approach has common features with the well-known total internal reflection fluorescence (TIRF) spectroscopy that is also a surface-sensitive and surface-specific detection method, but lacks, however, the enormous enhancement of the optical fields that can be obtained at resonant excitation of a surface plasmon wave which is responsible for the substantial sensitivity enhancement in bio-affinity studies. 

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). 

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),... 

Among the other spectroscopies, either in the direct or reflection mode, fluorescence spectroscopy may be mentioned. A promising variant is Total Internal Reflection Fluorescence spectroscopy (TIRF). The decay rate of an excited fluorescing probe is usually Interpreted in terms of the local fluidity and polarity. The technique has been used to estimate the extent of ordering inside adsorbed surfactant layers, but this is not an absolute method because a fluorescent probe has to be inserted, and such probes themselves affect the local fluidity. More rigorous are fluorescence experiments with molecules that possess such a probe as an intrinsic part of their structure, such as tryptophans in... 

The application of total internal reflection fluorescence spectroscopy (TIRF) by this laboratory to the study of protein adsorption at solid-liquid interfaces is reviewed. TIRF has been used to determine adsorption isotherms and adsorption rates from single-and multi-component protein solutions. Initial adsorption rates of BSA can be explained qualitatively by the properties of the adsorbing surface. Most recently, a TIRF study using monoclonal antibodies to probe the conformation of adsorbed sperm whale myoglobin (Mb) elucidated two aspects of the Mb adsorption process 1) Mb adsorbs in a non-random manner. 2) Conformational changes of adsorbed Mb, if they occur, are minor and confined to local regions of the molecule. Fluorescence energy transfer and proteolytic enzyme techniques, when coupled with TIRF, can characterize, respectively, the conformation and orientation of adsorbed Mb. 

Leutenegger M, Ringemann C, Lasser T, Hell SW, Eggeling C (2012) Fluorescence correlation spectroscopy with a total internal reflection fluorescence STED microscope (TIRF-STED-FCS). Opt Express 20(5) 5243-5263... 

The surface plasmon related optical enhancements at resonance are in sharp contrast to the maximum value one might find at the critical angle c for a mere total internal reflection geometry displayed in Figure 3(c). Here, the coherent superposition of the incoming and the outgoing light beam results in an enhancement of a factor of 4 only However, this has been utilized in the past for a surface-specific fluorescence technique, i.e., the total internal reflection fluorescence spectroscopy (TIRF) ... 

FRET fluorescence resonance energy transfer FCS fluorescence correlation spectroscopy TIRF total internal reflection fiuorescence PCFI photon counting histogram ICCD intensified charge coupled device EMCCD eiectron muitipiying charge coupled device CMOS complimentary metal oxide semiconductor AFM atomic force microscope. 

While the SFA provided direct evidence as to the thickness of a polymer layer adhering to a surface, TIRF provides a measure of the surface area concentration, r. TIRF spectroscopy makes use of the total internal reflection of light at the interface between a solid adsorption substrate of relatively high refractive index and a polymer solution of lower refractive index. Ihe total internal reflection, however, generates a standing evanescent wave, a nonclassical penetration of the light into the lower refractive index phase. The evanescent wave may penetrate some 60 to 65 nm into the lower refractive index phase, be absorbed by it, fluoresce, and so on (72,73). 

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