Variation on Zernike’s phase-contrast microscope

Abstract

We describe the design, construction, and testing of a variant of Zernike's phase-contrast microscope. The sample is illuminated with a white-light source through an annular aperture, which is projected onto the entrance pupil of the objective lens. In the return path the light diffracted by the sample and appearing in the interior of the objective's aperture (i.e., the test beam) is separated from the light returning in the annular region near the rim of the objective (i.e., the reference beam). The separated beams are relatively phase shifted and then combined to create an interferogram of the sample's surface on a CCD camera. It is fairly straightforward to use this system as a conventional bright-field or dark-field microscope, but its most interesting application is as a Zernike phase-contrast microscope with adjustable amplitude ratio and phase shift between test and reference beams. The ability to continuously adjust the phase of the reference beam also enables quantitative measurement of the phase imparted by the sample to the incident beam.