Aspects of scanning microdensitometry: III. The monochromator system

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Abstract
Microdensitometric errors can result from various factors associated with the monochromator system, including imperfect monochromaticity of the light, incorrect setting of the wavelength, and non-uniform illumination of either the microscopic field or the objective aperture. Cergain types of potential error are characteristic of particular instruments. Overall instrumental sensitivity is mainly limited at the blue and red ends of the spectrum, respectively, by the lamp output and the photomultiplier tube sensitivity. Quartz-iodine lamps are slightly brighter than conventional W sources, especially at short wavelengths, but tend to be less stable photometrically and are more expensive. Simple refracting monochromators and graded-spectrum interference filters in general pass more light, in the visible spectrum, than do grating monochromators of similar bandwidth. Most errors of wavelength setting can be avoided by routinely measuring at that wavelength, .lambda.max, found empirically to give the maximum absorbance or integrated absorbance. Off-peak wavelengths can be set reproducibly with the aid of an eyepiece spectroscope, or by adjusting the wavelength so that the absorbance of a given specimen is some precise fraction of that at .lambda.max. The monochromator bandwidth affects the apparent absorbance spectrum of a given specimen, but spectra are not very helpful in assessing possible microdensitometric errors at a fixed wavelength. In probably the best single test of monochromator performance, advisable before any microdensitometry using unfamiliar instrumental settings or staining methods, the apparent absorbance at a given wavelength is plotted against pathlength through a solution having an absorbance spectrum identical with or very similar to, that of the microscopic specimens of interest. Where an error is a known function of the monochromator slit-width it may sometimes be possible to use data obtained with 2 slit-widths to estimate the ideal result corresponding to monochromatic light. More generally, non-linearity can be corrected by electronic off-setting of the photomultiplier tube output. This procedure is analogous with, and if necessary should be carried out together with, a method previously described for the correction of error due to glare. Provided the pathlength and apparent absorbance are proportional over the range of absorbances of interest, results obtained with different monochromator bandwidths can be standardized by multiplication by an empirical calibration factor.

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