Total internal reflectance fluorescence models

W. M. Reichert, J. T. Suci, J. T. Ives, and J. D. Andrade, Evanescent detection of adsorbed protein concentration-distance profiles Fit of simple models to variable-angle total internal reflection fluorescence data, Appl. Spectrosc. 41, 503-507 (1987). 

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). 

K. H. Pearce, R. G. Hiskey, and N. L. Thompson, Binding kinetics of fluorescently labeled bovine prothrombin fragment 1 at planar model membranes measured by total internal reflection fluorescence microscopy, Biophys. J. 59, 622a (1991). 

SPR imaging was also employed to look at the same cholera toxin-GMl interaction on a supported bilayer by Philips et al. in array experiments [74]. Another investigative technique applied to the model ganglioside-cholera toxin system was total internal reflection fluorescence microscopy applied to spatially addressable supported bilayer membranes [75]. The recognition by HIV-1 surface glycoprotein gpl20 of glycosphingolipids partitioned within an SBM has also been looked at by total internal reflection fluorescence [76]. 

FIGURE 10.7. Total internal reflection fluorescence microscopy of the micro-two-phase system of dode-cane/water. (a) Continuous photons with an average of 11 molecules in the observation area, (b) photon burst with an average of 0.02 molecules in the observation area and (c) extension of the photon burst upon the addition of DMPC. A model for the photon burst upon the observation using single molecule diffusion (d). 

Figure 14 illustrates an example of a structure that can provide enhanced fluorescence capture. It consists of a truncated cone, on top of which the fluorescent species is deposited. The cone angle, a, is chosen in order to cause total internal reflection of the emitted fluorescence (the angular distribution of which is calculated from the model) and is therefore dependent on the refractive indices of the cone material and the environment. The emitted fluorescence is reflected onto a detector positioned directly beneath the cone. 

L. E. Morrison and G. Weber, Biological membrane modeling with a liquid/liquid interface. Probing mobility and environment with total internal reflection excited fluorescence, Biophys. J. 52, 367-379 (1987). 

Total internal reflection intrinsic fluorescence (TIRIF) spectroscopy and molecular graphics have been applied to study the adsorption behavior of two lysozymes on a set of three model surfaces. A recently devised TIRIF quantitation scheme was used to determine adsorption isotherms of both hen egg-white lysozyme (HEWL) and human milk lysozyme on the three model surfaces. This preliminary study suggests that the adsorption properties of the two lysozymes are significantly different, and that further comparative studies of the two lysozymes might prove to be beneficial in understanding how protein structure might influence adsorption properties. Molecular graphics was used to rationalize the adsorption results from TIRIF in terms of the proteins surface hydrophobic/hydrophillic character. 

This equation is valid also for the case of total internal reflection provided that 6i exceeds the critical angle. It relates the EW amplitude to the surface coverage, 6, defined in terms of Ng. In the framework of the Langmuir model of adsorption the quantity is related to the surface temperature by Eqs (2.146), (2.144) and (2.145). Therefore, measuring the EW fluorescence spectra from the gas as a function of surface temperature allows one to obtain information on the adsorbate. 

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