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 a
nd 1.65 NA from Olympus). The first two approaches are elaborations of "pri
smless" total internal reflection fluorescence (TIRF), one approach with a
laser illumination and the second with are lamp illumination. The new highe
r 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 objectiv
e to selectively gather the emission of fluorophores located close enough t
o the substrate for their near-field energy to be captured by the substrate
. Schematic diagrams, experimental demonstrations, and practical suggestion
s for all these techniques are provided. (C) 2001 Society of Photo-Optical
instrumentation Engineers.