The Most Favorable Absorption of the Phase Plate in Phase Contrast Microscopy

Abstract

Accepting the conclusion of Zernike that phase structures in an object may be converted into apparent amplitude structures in its image by alteration of phase of portions of the light at the Fraunhofer diffraction spectrum, a simple relationship between intrinsic object contrast and optimum absorption of the phase plate is developed. For this purpose, the definition of contrast as the ratio of the brightness difference between adjacent small areas in the image to the greater brightness of the two is employed, and considered more suitable than the simple ratio of the brightest area to the darkest. The departure of biologic structures from the periodicity and grating constants characteristic of perfect object gratings is handled by means of a parameter defined as the ratio of the energy transmission by the null order of the diffraction spectrum to the total energy transmission by all orders admitted by the limited apertures of the microscope. Calculations based on the formula developed are given in the form of tables and graphs.