Figure 1. Experimental setup and fluorescence images obtained with varying distance between fluorochrome and reflector.

Setup (a) was used to investigate fluorescence in air with dry objectives of low numerical aperture and long working distance (e.g. 5 × N.A. 0.15). The fluorochrome, e.g. Atto 532 dye, was deposited as a thin layer on the convex side of a planoconvex lens, which was balanced on an aluminised first-surface reflector. In certain experiments the broad-band reflector was replaced with a plate coated to reflect only the excitation wavelengths or only the emission wavelengths of the dye. For studies at high numerical aperture, setup (b) was used, allowing the fluorochrome to be brought within the limited working distance of the high-N.A. lens and for normal immersion oil and a correctly specified coverslip to be used. The dye in this case was the sodium salt of fluorescein deposited in a thin layer on the underside of the coverslip and the convex surface of a solid silicon planoconvex lens was used as the reflector, since it behaves as such in visible wavelengths. (c) Fluorescence image from the setup in (a), showing concentric fringes from multiple planes of excitation at the standing-wave antinodes. Scale bar = 250 μm. (d) Fluorescence fringes from the setup in (b), consistent with standing-wave excitation in spite of the presence of rays at a wide range of angles of incidence. Scale bar = 50 μm.