QUANTITATIVE FLUORESCENCE MICROSCOPY: FLUORESCENT ANTIBODY AUTOMATIC SCANNING TECHNIQUES

Abstract

The application of microscanning techniques to the detection, classification and counting of fluorescence-tagged microorganisms presents unique and somewhat more severe optical and photometric problems than those encountered in absorption type microscanning. Low fluorescence emission, autofluorescence of optics, filters and specimen mounting media and the close proximity of the excitation and emission wavelengths of typical fluorochromes impose severe limitations on sensitivity and maximum scanning rates. By the use of bright field illumination, liquid dye immersion filters, and careful matching of light source and filter spectral characteristics to those of the fluorochromes, fluorescence output was increased 10-100 times with negligible increase in background level. The effectiveness of a number of light sources filters and phototubes in producing maximal signals from fluorescein isothiocyanate-tagged microorganisms in an experimental fluorescence microscanner are compared. Proper selection produced increases in sensitivity of more than 10 times the initial sensitivity obtained with standard fluorescence microscopes and illuminators. Image plane, object plane and flying spot scanning techniques are compared as to their suitability for fluorescence microscanning. Practical scanner designs are illustrated. A consideration of the advantages of laser excitation of fluorochromes indicates that in addition to greatly simplifying filtering problems, a 10² increase in irradiance can be expected. The possibility of differential diagnosis based upon two color fluorescence discrimination is explored. Overlapping emission spectra presents the major difficulties in such a system. An experimental two color fluorescence microscanner and the optical and electronic techniques employed for the discrimination of overlapping color spectra is described.