Clinical Chemistry, Vol 16, 643-650, Copyright © 1970 by the American Association for Clinical Chemistry

The Microscope, Spectra, and Automated Analysis

¹ Department of Engineering Biophysics, University of Alabama in Birmingham, Birmingham, Ala.

Fluorescence spectroscopy (FS) and optical rotatory dispersion (ORD) have been added to the capabilities of the microscope. With them, as with conventional spectroscopy, a single component can be detected, even when in a mixture. This ability is enhanced by use of certain fluorescent dyes or "molecular probes" such as acridine orange (AO). Molecular complexes between dye and substrate may have characteristic physical-optical and physical-chemical properties. The microscope used for FS and ORD studies can produce fluorescence- and ORD-spectra from selected portions of unfixed cells and tissues and from extremely small amorphous samples. Characteristic spectra can be obtained from as little as 10^-17 moles of sample. It is possible to obtain cytochemical data that closely resembles, both spectroscopically and numerically, data obtained from solution studies on in vitro model systems, and consequently it is possible to predict quantitatively the results of interaction of cells with dye. Previous work in cytochemistry cannot produce such correlations, nor is it useful for quantitative prediction. Data so obtained with the microscope and corrected for instrumental parameters, can be used in design of automated analytical systems capable of detecting and quantitating particular biopolymers even in an intact unfixed cell. When a particular biopolymer can serve (qualitatively or quantitatively) to characterize a cell type, automated identification, counting, and separation can readily be devised from predetermined optical properties. The fluorescence and ORD spectra of the microscopic samples determine the complexity of the automated systems. Not only can relatively simple instruments sometimes serve, but automation of an analysis that cannot be definitive can be avoided.