Selective imaging of surface fluorescence with very high aperture microscope objectives

Three approaches to selective surface fluorescence detection are described. All three of them depend on the use of extremely high numerical aperture (NA) objectives now commercially available (1.45 NA from Zeiss and Olympus and 1.65 NA from Olympus). The first two approaches are elaborations of "prismless" total internal reflection fluorescence (TIRF), one approach with a laser illumination and the second with arc lamp illumination. The new higher NA objectives are much more suitable for TIRF work on biological cells in culture than are 1.4 NA objectives previously described for prismless TIRF. The third approach is not TIRF at all. It uses the high aperture objective to selectively gather the emission of fluorophores located close enough to the substrate for their near-field energy to be captured by the substrate. Schematic diagrams, experimental demonstrations, and practical suggestions for all these techniques are provided.