APPLICATIONS OF MICROSPECTROFLUOROMETRY IN QUANTITATION OF IMMUNOFLUORESCENCE ON SINGLE CELLS *

An approach to the quantitation of immunofluorescence was made by developing a model system in which the amount of antigen taking part in an immunofluorescent reaction was varied and the fluorescence intensity was quantitated on a single-cell basis.’ This report concerns the application of quantitative immunofluorescence, based on this model system, to certain biological problems. The model system employed, as previously described,‘ utilized formalinized human red blood cells coated with different amounts of I3’I-labeled human IgM. The cells were then exposed to fluorescein-conjugated antiserum to human IgM, resulting in membrane fluorescence.$ The fluorescence intensity of individual cells was measured with a microspectrofluorometer. As the cells were coated with varying amounts of labeled protein, it was possible to investigate the relationship between the quantity of protein antigen and the intensity of fluorescence. It can be seen (FIGURE 1) that the mean fluorescence values per cell decrease as the amount of coated IgM in the different cell populations, expressed in counts per minute, decreases. Control cells were coated with albumin to measure nonspecific fluorescence of the cells coated with protein as such, and such fluorescence was low. Two microfluorometers were employed in these studies. The first was a Zeiss ultramicrospectrophotometer modified for microfluorometry as described by Caspersson and colleagues’ in which cells were measured by transmitted light. Prism monochromators were used for both excitation at 365 nm and emission at 520 nm. This will be referred to as microfluorometer A. The second microfluorometer employed (referred to as B) was a Zeiss photomicroscope I1 equipped with an RCA IP 28 photomultiplier. Cells were centered for measurement with the use of phase contrast in transmitted light. (The light source was a 12 v 60 w tungsten lamp). For measurement of fluorescence, cells were excited in incident light using an Osram HBO 100w/2 superpressure mercury lamp, a light modulator, a 2.5-mm BG 38 heat filter, and a 2-mm UG 5 or a 3-mm BG 12 filter. A I1 E epicondenser was used, and cells were enclosed by a fixed aperture of appropriate size. An FL reflector allowed passage of light below 460 nm. A 40 x “Neofluar” phase objective (N.A.=0.75) or a 100 X Neofluar oil immersion phase objective (N.A.=1.3) was used. Emitted light passed through a barrier filter