Aspects of scanning microdensitometry I. Stray light (glare)

Glare or stray light, an important source of error in microdensitometry, is mainly due to reflections at glass-air surfaces in the microscope objective, and much less to imperfections in other parts of the optical system. It is only slightly affected by the numerical aperture of the microscope condenser, but is closely related to the area of specimen-free field illuminated. If F-f is the amount of glare (expressed as a fraction of the intensity of the light incident on the specimen) with a given object and area of field illuminated, and F-infinity is the glare with an infinite field size or an infinitesimally small object, F-infinity approximately equals F(f)A/(A - a), where A is the area of the whole field illuminated and a is the area of the specimen itself. Glare with a given specimen and under given conditions of illumination may be taken to equal the apparent transmittance of an opaque object of the same size as the specimen to be measured, provided the opaque object is light absorbing (of low reflectance) and not too small (at least 5-10 mu m diameter). The true absorbance E-t of the specimen equals log [(I - F)/(I-p - F)] where the intensity of the incident light is unity, and I-p is the apparent transmittance of the specimen in the presence of F glare. The true absorbance of a specimen is never less than F% higher than its apparent absorbance, in the presence of F% glare, and the error rises sharply with increasing absorbance. Apparent differences in the amount of dye taken up by nuclei of different sizes, stained by the Feulgen method, which have been reported by various workers and attributed to differences in DNA content, are of an order of magnitude which could be due to the presence of glare in the system. This factor should be allowed for in critical work. Two methods of correcting the error due to glare in measurements of integrated absorbance are described. The first method utilizes data on the area of specimen containing material with an absorbance greater than a set threshold value, as provided for example by the area-measurement facility on the Vickers M85 integrating microdensitometer. In the second, and generally preferable method, glare is compensated for electronically in a way analogous to the offsetting of the dark-current of the photomultiplier tube of the instrument.