Fluorescence microscopy was developed around the turn of the 20th century. The earliest instruments used UV light for excitation; later systems could employ excitation at blue and longer wavelengths, but the requirement for relatively high power at relatively short wavelengths made it necessary to use arc lamps, rather than filament lamps, as light sources. Fluorescence microscopy, in principle, allows visualization of bright objects against a dark background. Earlier systems, however, were likely to fall short of achieving this goal because they were essen tially transmitted-light microscopes with colored glass filters in both the excitation path, that is, between the light source and the condenser, and the emission path, that is, between the objective and the eyepiece. The combination of stray light transmission through both excitation and emission filters and fluorescence excited in the emission filter often resulted in the background being too bright to permit observation of weakly fluorescent material.
An extremely important application of fluorescence microscopy developed during the 1940s was the fluorescent antibody technique introduced by Coons et al. (1941). Other workers had demonstrated that azo dye-conjugated antisera to bacteria retained their reactivity with the organisms and would agglutinate them to form faintly colored precipitates; however, the absorption of the dye-conjugated sera was not strong enough to permit visual detection of bacterial antigens in tissue preparations.
Albert Coons surmised that it might be easier to detect small concentrations of antibody labeled with fluorescent material against a dark background using fluorescence microscopy. He and his coworkers labelled anti-pneumococcal antibodies with anthracene and could detect both isolated organisms and, more importantly, antibody bound to antigen in tissue specimens, by the UV-excited blue fluorescence of this label, as long as tissue autofluorescence and background were not excessive.
In 1950, Coons and Kaplan reported that fluorescein gave better results as an antibody label than did anthracene, because the blue-excited yellow-green fluorescence of fluorescein was easier to discriminate from autofluorescence. Thereafter, fluorescein became and has remained the most widely used immunofluo-rescent label.
A significant advance in fluorescence microscopy, epiillumination, was made in 1967 by Ploem (1967), who substituted dichroic mirrors for the half-silvered mirror normally used in an incident light microscope, and added excitation and emission filters to the optical path. Even when color glass filters were still used for excitation and emission wavelength selection, this configuration greatly reduced both stray light transmission and filter fluorescence, yielding much lower backgrounds. Within a short time, it had been reported that, when an epiillumi-nated apparatus was employed, measurements of nuclei stained by a fluorescent Feulgen procedure using acriflavine yielded results equivalent to those obtained by the standard absorption method (Bohm and Sprenger 1968).
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