Phase contrast

Phase contrast microscopy allows the viewing of unstained specimens by using the light phase amplitude differences within microscopic objects. When an unstained biological specimen is observed in the normal brightfield microscope, it is quite difficult to see because most biological material is uncoloured and transparent, providing little contrast to the illuminated background. By converting the phase differences, between light passing through a specimen and that passing through the surrounding medium, into amplitude (brightness) differences, phase contrast microscopy provides a difference in brightness between the object and the background, which the eye can then see.

Phase contrast microscopy uses an annular stop in the condenser and a phase plate within the objective lens, which is aligned with the annular stop. In this configuration, the light path can be split and each of the separated beams will pass through the same transparent medium at the specimen stage. The light passes through the annular stop and forms a cone of light, which comes to its vertex at the focal point of the specimen. Any background light, which is un-deviated by the specimen, will go through the phase ring in the phase plate and is advanced by a quarter of a wavelength. Deviated light passing through the specimen is retarded by a quarter of a wavelength and passes through the phase plate without going through the ring. When the beams are recombined further along the light path, the differences in the phase of the deviated and un-deviated light beams become additive and subtractive. The resultant wave is the sum of the two waves which have their crests and troughs opposite each other. The difference in amplitude can be seen as a change in brightness, since brightness is proportional to the square of the amplitude. The net result is that features of the object are either lighter or darker than the surrounding field.