Aspects of scanning microdensitometry: III. The monochromator system
Authors: Goldstein D. J. 
Published: August 2011

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. Certain types of potential error are characteristic of particular instruments. Thus in the Vickers M85 microdensitometer, where the flying spot is the reduced image of a hole situated at the monochromator exit aperture, the interpretation of results obtained with different spot sizes is complicated by the fact that the hole size affects both the spatial resolution and the spectral bandwidth of the system. Similarly, in instruments in which the monochromator exit slit lies in an aperture plane the numerical aperture of the whole system may be affected by the spectral bandwidth and vice versa.

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 tungsten 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, λmax, 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 λmax.

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. Deviations from proportionality between pathlength and apparent absorbance have been found using the graded‐spectrum interference filter of the Barr & Stroud GN2 microdensitometer, but have not so far been demonstrated with the refracting monochromator of the Vickers M85 microdensitometer. Such deviations could result in systematic errors in the comparison of densely‐ and lightly‐stained specimens, and two methods to correct these errors are described. Where the error is a known function of the monochromator slit‐width it may sometimes be possible to use data obtained with two 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.