Phase contrast microscopy

Phase-contrast microscope

A phase-contrast microscope
Uses	Microscopic observation of unstained biological material
Inventor	Frits Zernike
Manufacturer	Zeiss, Nikon, Olympus and others
Related items	Differential interference contrast microscopy, Hoffman modulation-contrast microscopy, Quantitative phase-contrast microscopy

Phase-contrast microscopy is an optical-microscopy technique that converts phase shifts in light passing through a transparent specimen to brightness changes in the image. Phase shifts themselves are invisible, but become visible when shown as brightness variations.

When light waves travel through a medium other than vacuum, interaction with the medium causes the wave amplitude and phase to change in a manner dependent on properties of the medium. Changes in amplitude (brightness) arise from the scattering and absorption of light, which is often wavelength-dependent and may give rise to colors. Photographic equipment and the human eye are only sensitive to amplitude variations. Without special arrangements, phase changes are therefore invisible. Yet, phase changes often carry important information.

Phase-contrast microscopy is particularly important in biology. It reveals many cellular structures that are not visible with a simpler bright-field microscope, as exemplified in the figure. These structures were made visible to earlier microscopists by staining, but this required additional preparation and killed the cells. The phase-contrast microscope made it possible for biologists to study living cells and how they proliferate through cell division. After its invention in the early 1930s, phase-contrast microscopy proved to be such an advancement in microscopy, that its inventor Frits Zernike was awarded the Nobel prize (physics) in 1953.

The basic principle to making phase changes visible in phase-contrast microscopy is to separate the illuminating (background) light from the specimen-scattered light (which makes up the foreground details) and to manipulate these differently.

The ring-shaped illuminating light (green) that passes the condenser annulus is focused on the specimen by the condenser. Some of the illuminating light is scattered by the specimen (yellow). The remaining light is unaffected by the specimen and forms the background light (red). When observing an unstained biological specimen, the scattered light is weak and typically phase-shifted by −90° (due to both the typical thickness of specimens and the refractive index difference between biological tissue and the surrounding medium) relative to the background light. This leads to the foreground (blue vector) and background (red vector) having nearly the same intensity, resulting in low image contrast.