A Zeiss scanning microscope photometer with a monochromator was assembled and interfaced with a Digital Equipment Corporation PDP-12 computer to form an automated single-beam microspec-trophotometer for making spatial and particularly spectral scans of Romanowsky-stained blood cells. The spectra, covering 400-700 nm, are stored on tape for subsequent data processing. The limitations on the accuracy and precision of the measurements are discussed. Photometric precision was limited by ±1% peak-to-peak fluctuations associated with the source and with mechanical instability. However, in strong absorption bands digitization error predominated. These random errors were reduced by time-averaging of multiple sampling points, and by averaging data for several cells. Photometric accuracy was limited by stray light, which produced an apparent transmittance of 0.014 for an 8-μm-square opaque object. Distributional error was found to be 6.6% in a neutrophil nucleus. Spectra were obtained for 24 cells each for nucleus and cytoplasm of neutrophils, basophils, eosinophils, lymphocytes and monocytes, and for erythrocytes. Six blood specimens were used. The biological variability in absorbance produced ±17% coefficient of variation for cytoplasm and erythrocytes and ±7% for nuclei. The spectra are interpreted on the basis of metachromatic and nonmetachromatic thiazine absorption peaks, the eosin absorption peak, and unresolved eosin-thiazine peaks. Copyright © 1979 by The Institute of Electrical and Electronics Engineers, Inc.
