Qualitative Observation of Labeled Cells

Quantitative determination of the absolute distance from the surface to a labeled cell membrane at a cell/substrate contact region can be based on the variation of F(d) with 0.(1O6) This effort is challenging because corrections have to be made for 0-dependent reflection and transmission through four stratified layers (glass, culture medium, membrane, and cytoplasm), all with different refractive indices. For 3T3 cells, Lanni et a//1065 derived a plasma membrane/substrate spacing of 49 nm for focal contacts and 69 nm for close contacts elsewhere. They were also able to calculate an approximate refractive index for the cytoplasm of 1.358 to 1.374. 

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 

One TIRF study found that some membrane proteins behave just oppositely to AChR they avoid the cell/substrate contact regions/ 1 When endothelial cells are grown on a bare glass surface or are brought into suspension, a specific membrane protein marked with antibodies appears all over the cell surface, as evidenced by epi-illumination and TIRF. However, when the cells are grown on (or returned to) a surface coated with their own extracellular matrix material, the protein disappears from the basal (cell/substrate-contacting) side of the cells. 

The interaction between immune system cells and their targets often involves a specific and as yet incompletely understood surface reaction. This 

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