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Phase Contrast Microscopy Principle and Working

  • In this phase contrast microscopy principle and working post we have briefly explained about definition, principle, parts, working, applications, and limitations.

Phase Contrast Microscopy Principle and Working

  • The Phase Contrast Microscope was designed by Frits Zernike, a Dutch physicist who was awarded the Nobel Prize in 1953. It is the microscope that permits living cells to be observed. To achieve contrast, this microscopy employs unique optical components that take advantage of minute changes in the refractive indices of water and cytoplasmic components of living cells.

Principle

Microscope

Phase Contrast Microscopy Principle and Working

  • The phase contrast microscopy is based on the principle that small phase changes in the light rays, induced by differences in the thickness and refractive index of the different parts of an object, can be transformed into differences in brightness or light intensity. The phase changes are not detectable to human eye whereas the brightness or light intensity can be easily detected.

Parts

  • The phase contrast microscope is similar to an ordinary compound microscope in its optical components. It possesses a light source, condenser system, objective lens system and ocular lens system. A phase contrast microscope differs from bright field microscope in having,

Phase condenser

  • An annular diaphragm in the sub-stage (phase condenser) In the focal plane of the sub-stage, an annular aperture in the diaphragm regulates the illumination of the object. This is found behind the microscope’s condenser. This annular diaphragm serves to highlight the object by forming a narrow, hollow cone of light.

Phase Plate

  • Plate phase (diffraction plate or phase retardation plate). The back focal plane of the objective lenses houses this plate. The phase plate is divided into two sections, one of which is covered with a light-retarding material (Magnesium fluoride) and the other of which is light-absorbing but not light-retarding. This plate aids in the reduction of incident light phase.
Microscope

Working

  • The unstained cells cannot create contrast under the normal microscope. However, when the light passes through an unstained cell, it encounters regions in the cell with different refractive indexes and thickness.
  • When light rays pass through an area of high refractive index, it deviates from its normal path and such light rays experience phase change or phase retardation (deviation). Light rays pass through the area of less refractive index remain non-deviated (no phase change).
Microscope
  • The difference in the phases between the retarded (deviated) and un-retarded (non-deviated) light rays is about ¼ of original wave length (i.e., λ/4). Human eyes cannot detect these minute changes in the phase of light.
  • The phase contrast microscope has special devices such as annular diaphragm and phase plate, which convert these minute phase changes into brightness (amplitude) changes, so that a contrast difference can be created in the final image. This contrast difference can be easily detected by human eyes.
  • In phase contrast microscope, to get contrast, the diffracted waves have to be separated from the direct waves. This separation is achieved by the sub-stage annular diaphragm.
  • The annular diaphragm illuminates the specimen with a hollow cone of light. Some rays (direct rays) pass through the thinner region of the specimen and do not undergo any deviation and they directly enter into the objective lens.
  • The light rays passing through the denser region of the specimen get regarded and they run with a delayed phase than the non-deviated rays.
  • Both the deviated and non- deviated light has to pass through the phase plate kept on the back focal plane of the objective to form the final image. The difference in phase (Wavelength) gives the contrast for clear visibility of the object.

Applications

  • Phase contrast microscope enables the visualization of unstained living cells.
  • It makes highly transparent objects more visible.
  • It is used to examine various intracellular components of living cells at relatively high resolution.
  • It helps in studying cellular events such as cell division.
  • It is used to visualize all types of cellular movements such as chromosomal and flagellar movements.

Limitations

  • Phase-contrast condensers and objective lenses add considerable cost to a microscope, and so phase contrast is often not used in teaching labs except perhaps in classes in the health professions.
  • To use phase-contrast the light path must be aligned.
  • Generally, more light is needed for phase contrast than for corresponding bright-field viewing, since the technique is based on the diminishment of the brightness of most objects.

Further Readings

Reference