A dual wavelength, imaging microdensitometer, with specific application to measurements of DNA ploidy and to monoclonal antibody related immunoenzymatic staining procedures, is described. The two wavelengths used were 500 nm and 620 nm, each with a 20 n bandpass to reduce glare and to provide independent sensing of typical immunohistochemical two-color stain spectra. The optical arrangement is such that the original multicolor stained image is separated into two components by two filters and projected simultaneously onto two separate CCD sensors that are in optical registration for the same spatial regions in each image. Independent measurements of individual stained subcomponents of each cell can be made, and the entire image is reconstructed digitally and displayed showing both of the sensed subcompartments in one image. Optical methods of calibration for obtaining accurate densitometry in two colors simultaneously are discussed, as well as various applications related to performing assays in surgical pathology for cancer prognosis.
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