Total internal reflection fluorescence microscopy method

Mashanov GI, Tacon D, Knight AE et al (2003) Visualizing single molecules inside living cells using total internal reflection fluorescence microscopy. Methods 29 142-152... 

Oreopoulos, J. and Yip, C. M. 2008. Combined scanning probe and total internal reflection fluorescence microscopy. Methods 46,2-10. 

D. Axelrod, Total internal reflection fluorescence microscopy, in Fluorescence Microscopy of Living Cells in Culture B (D. L. Taylor and Y.-L. Wang, eds.), Methods in Cell Biology, Vol. 30, pp. 245-270, Academic Press, San Diego, California (1989). 

Axelrod D. Total internal reflection fluorescence microscopy in cell biology. Methods Enzymol. 2003 361 1-33. 

One of the simplest methods to study adsorption at the oil water interface is to measure the variation of interfacial tension as a function of concentration. If the polymer used for adsorption is monodisperse, then the Gibbs equation (51) may be used to estimate the surface excess. However, if the polymer is polydis-perse, this method will give erroneous values of the surface excess because the larger molecules will tend to adsorb preferentially, and the equation is imable to account for this adsorption behavior. As a result, most of the data available in the literature report the change in the interfacial tension as a function of concentration without attempting to convert it into an adsorbed amoimt. Apart from interfacial tension measurements, other techniques such as total internal reflection fluorescence microscopy (52) and scintillation measurements from radiolabeled polymers (53) have also been used to measure the adsorption at the liquid-liquid interface. 

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. 

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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