| Abstract: | In ‘ideal’ phase-contrast microscopy all the direct light and none of the diffracted light is influenced by the phase plate in the back focal plane of the objective. Contrary to almost all previous work, it appears that the intensity of an ideal phase-contrast image is affected not only by the transmittance and retardation of the object and of the phase plate, but also by the width of the specimen (or total width of multiple specimens) relative to the microscopic field. Equations and computer code are presented with which the intensity of such images can be calculated. Previously published equations are special cases, and implicitly or explictly assume either that the object is of negligible width, or occupies precisely half the microscopic field. The absolute brightness of an image in ideal central dark-field microscopy is a function of the object retardation, but the intensity of the image relative to the background is a function only of the width of the object(s) relative to the field. The equations give results for ideal phase-contrast microscopy identical with those of a computer program simulating microscopic imaging. The program can in addition take into account non-ideal factors including a finite width of phase plate, finite objective aperture, deviations from best focus, glare, primary spherical aberration and obliquity of the coherent illumination. |
