Calibration methods for quantitative image processing in electron microscopy

An image can be represented digitally as a matrix of numbers. When those numbers are linearly related to a property of the object, such as mass per unit area, a simple integration of an image area leads to a total of that property, such as the mass of a particle that is represented in a selected area. Following techniques pioneered by Bahr and Zeitler, we illustrate the use of photographic densitometry of films exposed in an electron microscope to measure electron scattering. The transmission of an electron micrograph will be linear with respect to mass thickness for a particular value of background brightfield density, hence allowing determination of the mass of microscopic particles. We show here a digital computer method for conveniently establishing the linear condition by quantitative image processing using micrographs of polystyrene spheres. The method also serves to produce calibration curves for cases where the transfer from transmission to mass thickness is not linear. We also illustrate how an inexpensive computer is used to display and integrate regions of micrographs to determine particle mass.