Aspects of scanning microdensitometry: II. Spot size, focus and resolution

SUMMARY In scanning microdensitometry increasing the size of the measuring spot, or throwing the specimen out of focus, decreases the apparent integrated absorbance A p E p of a discrete specimen. Both experimental observations and elementary geometrical theory (i.e. ignoring diffraction effects) show that with moderate spot sizes the relative error in A p E p is greater with small objects or objects of high absorbance, and that with a given object the absolute error is approximately proportional to the spot diameter (round spot) or spot width (square spot). From the observed apparent integrated absorbances AE 1 and AE 2 obtained using measuring spots of width s 1 and s 2 respectively, the true integrated absorbance AE 0 corresponding to zero spot size can therefore be calculated from the approximate expression which reduces to With very large spot sizes, or with the specimen grossly out of focus, the apparent integrated absorbance of a specimen of radius R and transmittance I t tends theoretically to a limit: A p E p = 0.4343π R 2 ( I‐I t ). Provided the true absorbance of the specimen does not exceed about 0.5 at any point, the true integrated absorbance could in principle be estimated with less than 3% error from the expression but in practice A p E p can probably not be measured with sufficient accuracy. In scanning measurements of apparent specimen area, the results depend both on the threshold absorbance used and the size of the flying spot. With low thresholds the apparent area of the specimen at first increases and later decreases, as the spot size is increased or as the focus is changed.