Microspectrophotometric studies of Romanowsky stained blood cells. I. Subtraction analysis of a standardized procedure.

This paper describes a microspectrophotometric study of blood smears stained by a simple, standardized Romanowsky technique, using only the dyes azure B and cosin. Absorbance spectra are presented for twenty-two classes of cellular object, and for the two dyes in solution, together with tabulations of spectral maxima, and suitable wavelengths for use in automated image processing. The colours of objects stained with azure B/eosin are discussed in terms of absorbance spectra. By a spectral subtraction technique, it is shown that the differential colouration of various cell structures may be explained satisfactorily in terms of the varying proportions of only four dye components. These are the monomers and dimers of azure B and eosin. Polymerization was found to occur both in solution and on binding to biopolymers. A similar analysis of a conventional Romanowsky stain would present much greater difficulties, due to the greater number of dye components, which, however, contribute little to the colours observed.