Phase
Contrast Light Path
WHY PHASE CONTRAST MICROSCOPY?
Living cells and most cell organelles are often difficult if not impossible to see by brightfield or darkfield microscopy because they do not absorb, refract or diffract sufficient light to contrast with the surrounding medium. While staining of specimens with preferential stains (methylene blue for example stains nuclei blue) have allowed researchers to examine cells structures, staining generally kills the cells and in some cases distorts the cell structure.
The phase
contrast microscope was developed to improve contrast differences between
cells/organelles and the surrounding medium, making it possible to see
cells/organelles without staining. Thus we can view wet mounts of living
cells.
PHASE LIGHT PATH
Light rays
passing through a transparent specimen emerge as either direct rays or
diffracted rays. In phase microscopy, this effect is amplified by using
a microscope equipped with a special annulus (below the stage) and phase
plates (located in the objectives) which accentuate the phase changes
produced by the specimen. Direct rays, unimpeded by the phase plate (red
ray) are of higher intensity, making the background bright and the diffracted
rays (black ray)
impeded by the phase plate are of lower intensity, making parts of the
specimen darker. This results in improved contrast differences between
the specimen and the surrounding medium, making it possible to see cells
and cell organelles without staining.