Abstract
Non-absorbing colorless specimens (phase objects) can be visualized either by chemical staining (histology) or by the so-called optical contrasting (staining). The latter is achieved by converting optical phase shifts within the specimen, invisible to human eye, to intensity differences in the microscopic image.
Out of several available modes of microscopic phase visualization, conventional, apodized, and relief phase contrast are described in detail. A comparison with relief contrast alone, that is, the off-axis (schlieren) illumination mode is presented as well.
Images of various phase specimens of biological origin are shown, demonstrating the strong and weak points of each mode. Physiological aspects of image comprehension, facilitated by shading and other visual cues to depth structure are briefly discussed.
A phase-contrast microscope equipped with an objective hosting no phase annulus is presented; the latter is located in a pupil projection (optically relayed) plane, in an external attachment unit. This setup enables phase-contrast and, for example, conventional epi-fluorescence and total internal reflection fluorescence (TIRF) images to be acquired with a single objective lens.
Such modality is demonstrated in growth cones of neuroblastoma-glioma hybrid mouse-rat cells and touch receptor neurons of Caenorhabditis elegans. Examples of phase-contrast imaging in electron and X-ray (synchrotron radiation) microscopy are also presented.