Errors in microdensitometry

Abstract

Microdensitometric errors can originate in the instrument, in the specimen or in the human operator. Instrumental sources of systematic error mostly reduce the apparent integrated absorbance, especially of relatively small and highly absorbing objects. They can be assessed, minimized or eliminated by available techniques, but with modern apparatus are in general important only if results of high accuracy are required. Instrument errors include: (a) distributional error, due to the use of too large a measuring spot or the specimen being out of focus; (b) glare (stray light), due mainly to multiple reflections in the microscope objective; (c) monochromator error (the use of insufficiently pure light); (d) calibration errors; and (e) errors resulting from lack of photometric linearity, or the specimen absorbance exceeding the measuring range of the instrument. Specimen errors, including the problems of specificity and stoichiometry, are now the most important obstacles to a wider use of microdensitometry. The following selected topics are briefly discussed: fading; rate of staining; Beer's law deviations and the microdensitometry of opaque particles. Human errors include faulty logic, and failing to attempt an investigation because of anticipated difficulties which are in fact exaggerated or imaginary. The significance of microdensitometric results should, in general, be assessed by biological criteria rather than merely statistically; the use is urged of appropriate internal biological controls and standards wherever possible.