Variable angle total internal reflection fluorescence (TIRF) microscopy of fluorescently labelled lipid films and cells at the glass-liquid interface
Total internal reflection fluorescence (TIRF) microscopy has become the technique of choice for imaging the regions of cells in closest apposition to the substrate surface. For a given wavelength of incident light totally reflected at the glass/solution interface, the depth to which the evanescent wave illuminates the adherent cells decreases in a very defined manner with increasing incident angles of total reflection. Using this relationship, collecting a series of TIRF microscopy images at increasing angles of total internal reflection produces a means of profiling cell/substrate contact regions to a depth of a few tenths of a micron. The system to be discussed here consists of an inverted microscope equipped with a parallel plate flow cell coupled to a hemicylindrical prism to facilitate uniform rotation through the desired illumination angles. The 488 nm excitation radiation is delivered at precise angles by a fiber optic cable and fluorescent images are collected using a thermoelectrically cooled CCD camera. Samples examined consist of Langmuir-Blodgett (L-B) lipid films and bovine aortic endothelial cells (BAEC) labelled with the lipophilic carbocyanine dye dil-C18. The angular dependence of image intensity collected from the L-B films show excellent correspondence of angularly dependence predicted by theory. Angularly dependent cell images, although more complex, also show image intensities that decay with increasing angles of incidence. Attempts to estimate the magnitude of the cell/substrate separations in the focal adhesion sites produced values consistent to those reported in the literature.