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UCL Home  /  Geography  /  Resources  /  Laboratory  /  Light Microscopy  /  Phase Contrast

Phase Contrast

Most of the detail of living cells is undetectable in bright field microscopy because there is too little contrast between structures with similar transparency and no color. Unless the specimen mount is extremely thin, dark field mode may distort details.

Highly refractive structures bend light to a much greater angle than do structures of low refractive index. The same properties that cause the light to bend also delay the passage of light by a quarter of a wavelength or so. In a light microscope in bright field mode, light from highly refractive structures bends farther away from the center of the lens than light from less refractive structures and arrives about a quarter of a wavelength out of phase.

Light from most objects passes through the center of the lens as well as to the periphery. If the light from an object to the edges of the objective lens is retarded a half wavelength and the light to the center is not retarded at all, then the light rays are out of phase by a half wavelength. They cancel each other when the objective lens brings the image into focus. A reduction in brightness of the object is observed. The degree of reduction in brightness depends on the refractive index of the object.

Phase contrast is preferable to bright field microscopy when high magnifications (400x, 1000x) are needed and the specimen is colorless or the details so fine that color does not show up well.

In phase contrast a phase plate is placed in the light path. Barely refracted light passes through the center of the plate and is not retarded. Highly refracted light passes through the plate farther from center and is held back another one quarter wavelength.

Using Phase Contrast

phase1Phase contrast condensers and objective lenses add considerable cost to a microscope. Most of the microscopes available in our microscope rooms have phase - the settings on these should not be changed unless you really know what you are doing!

To use phase contrast the light path must be aligned. An element in the condenser is aligned with an element in a specialized phase contrast lens. This usually involves sliding a component (a phase telescope) into the light path. The elements are either lined up in a fixed position or are adjusted by the observer until the phase effect is optimized. If in doubt please ask someone to do this for you.

Generally, more light is needed for phase contrast than for corresponding bright field viewing, since the technique is based on a diminishment of brightness of most objects.

There are some limitations of phase contrast microscopy:

  • The images are usually surrounded by "halos" around the outlines of details. These are optical artifacts, which may obscure the boundaries of details.
  • There may be a reduction in the image resolution.
  • Phase contrast does not work well with thick specimens because of shifts in phase occur from areas slightly below or slightly above the plane that is in focus. Such phase shifts confuse the image and distort image detail.