Aspects of scanning microdensitometry. III. The monochromator system.

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, 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).