Using changes in refractive index to produce high-contrast images of transparent specimens
In phase contrast imaging, small changes in optical path length (as light moves through different materials of differing refractive index, e.g. water, cellular components) are optically translated into corresponding changes in light intensity, which can be observed as differences in image contrast.
Light from a tungsten-halogen lamp is directed through a collector lens and focused on a specialized annulus positioned in the substage condenser. Wavefronts passing through the annulus illuminate the specimen and either pass straight through or are diffracted and retarded by phase gradients in the specimen. Undeviated and diffracted light collected by the objective is separated by a phase plate and focused at the intermediate image plane to form the final image. One drawback of phase contrast imaging is the occurrence of "halos" around areas of high phase shift.
Phase contrast imaging is applicable to many transparent subjects, such as living cells in culture, micro-organisms, thin tissue slices, lithographic patterns, fibres, latex dispersions, glass fragments, and subcellular particles (including nuclei and other organelles) where the technique reveals structure that is not visible in brightfield imaging. An advantage of phase contrast microscopy is that living cells can be imaged in detail without the need for staining or use of fluorophores.
Phase contrast capability can be added to almost any brightfield microscope, provided the specialized phase objectives conform to the tube length parameters and the condenser will accept an annular phase ring of the correct size. Nikon, and all major microscope manufacturers, provide phase contrast accessories for their upright microscopes, inverted research microscopes and educational microscopes.
Introduction to Phase Contrast Microscopy
Phase Contrast Microscope Configuration