Evanescent field excitation of fluorescence by epi-illumination microscopy.

By simple modification of the pattern of fluorescence excitation light in an epi-illumination inverted microscope, one can achieve conditions that produce total internal reflection fluorescence (TIRF) by evanescent wave excitation. Though traditionally requiring a collimated beam traversing through a special prism, TIRF also can be achieved by epi-illumination through the periphery of a 1.4 numerical aperture objective. An opaque disk of appropriate size is placed in the illumination path external to the microscope so as to cast a sharp, real-image shadow at the objective's back focal plane. This shadow allows a hollow cone of epiillumination rays traveling at only super-critical angles to reach the glass/water interface at the sample plane. Three kinds of TIRF illumination patterns can be produced by variations of this scheme: (1) a small spot of illumination of 1.5 microm radius by use of a laser light source, (2) a large region of illumination by use of a laser-illuminated diffusing screen located upbeam from the opaque disk, and (3) a large region of illumination by use of a conventional mercury arc.