Evanescent excitation and emission in fluorescence microscopy.

Evanescent light-light that does not propagate but instead decays in intensity over a subwavelength distance-appears in both excitation (as in total internal reflection) and emission (as in near-field imaging) forms in fluorescence microscopy. This review describes the physical connection between these two forms as a consequence of geometrical squeezing of wavefronts, and describes newly established or speculative applications and combinations of the two. In particular, each can be used in analogous ways to produce surface-selective images, to examine the thickness and refractive index of films (such as lipid multilayers or protein layers) on solid supports, and to measure the absolute distance of a fluorophore to a surface. In combination, the two forms can further increase selectivity and reduce background scattering in surface images. The polarization properties of each lead to more sensitive and accurate measures of fluorophore orientation and membrane micromorphology. The phase properties of the evanescent excitation lead to a method of creating a submicroscopic area of total internal reflection illumination or enhanced-resolution structured illumination. Analogously, the phase properties of evanescent emission lead to a method of producing a smaller point spread function, in a technique called virtual supercritical angle fluorescence.