Glass evanescent wave cell

Another complication in the quantitation of TIRF on cells is the effect of the membrane thickness itself on the profile of the evanescent wave. Reichert and Truskey<105) have calculated that, in theory, the thickness of the membrane should have a negligible effect on the fluorescence and that a simplified theory of three stratified layers (glass/water/cytoplasm) should be adequate. The theory approximates for simplicity that scattering plays a negligible role and that fluorescence intensity versus angle of observation and fluorescence lifetime are not functions of distance to the interface z. Experiments that... 

Evanescent wave microscopy or total internal reflection microscopy (TIRM) has been employed in the fields of biology and chemistry since the 1970s. The TIRM technique has long been used in cell biology studies and more recently cell-substrate contacts, vesicle fusion, and single-molecule observation. Here, cells on a microscope cover glass are illuminated by an... 

As illustrated in Fig. 12a, the objective-type TIRFM can be used for this purpose, because this type of microscopes provides a free space above the glass surface and thus it has no limitation on the thickness of samples. The surface of the cell close to the glass surface is locally illuminated by the evanescent wave, lighting up biological events on the cell surface at the single molecule level. In some type of cells, it is possible to observe the other (apical) surface by adjusting the incident light carefully [18]. 

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