Loading

Phase Contrast Microscopy Susmita chakrabarty

Definition

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.

The principle of phase contrast microscopy is based on the wave nature of light rays, can be in phase or out of phase.

History of Phase Contrast Microscope

  • The Phase Contrast Microscope was developed by Zernike in early 1930s.
  • The invention of this microscope enables us to visualize live cells and cellular processes.
  • Due to the remarkable contribution of phase contrast microscopy in biological sciences, the inventor was awarded Nobel Prize in Physics in 1953.
Frederik "Frits" Zernike

Uses

  • To study unstained living cells.
  • Detailed examination of internal structures in living micro-organisms.
  • To study flagellar movements and motility of bacteria and protozoans.
  • To study intestinal and other live protozoa such as Amoeba and Trichomonas.
  • To examine fungi grown in culture.

Phase-Contrast Microscopy

  • The phase contrast microscopy made it possible to study living cells and how they proliferate through cell division.
  • Used to examine living organisms or specimens that would be damaged/altered by attaching them to slides or staining.
  • It uses a conventional light microscope fitted with a phase-contrast objective & phase-contrast condenser.
  • Light passing through one material & into another material of slightly different refractive index or thickness will undergo a change in phase. This change in are translated into variations in brightness of the structures.
Phase-contrast microscope

Principle of Phase Contrast Microscopy

  • Unstained bacteria have constituents of different refractive index.
  • Diffraction of light.
  • Phase contrast microscope employs an optical mechanism to translate minute variations in phase into corresponding changes in intensity of image.
  • When light passes through cells, small phase shifts occur, which are invisible to the human eye. In a phase-contrast microscope, these phase shifts are converted into changes in amplitude, which can be observed as differences in image contrast.
Principle of Phase Contrast Microscope
Principle of Phase Contrast Microscope

Parts of Phase contrast Microscopy

A phase contrast microscope

Phase-contrast microscopy is basically a specially designed light microscope with all the basic parts in addition to which an annular phase plate and annular diaphragm are fitted.

The condenser annulus (left) and the phase plate (right).

The annular diaphragm

  • It is situated below the condenser.
  • It is made up of a circular disc having a circular annular groove.
  • The light rays are allowed to pass through the annular groove.
  • Through the annular groove of the annular diaphragm, the light rays fall on the specimen or object to be studied.
  • At the back focal plane of the objective develops an image.
  • The annular phase plate is placed at this back focal plane.
Condenser Annulus

The phase plate

  • It is either a negative phase plate having a thick circular area or a positive phase plate having a thin circular groove.
  • This thick or thin area in the phase plate is called the conjugate area.
  • The phase plate is a transparent disc.
  • With the help of the annular diaphragm and the phase plate, the phase contrast is obtained in this microscope.
  • This is obtained by separating the direct rays from the diffracted rays.
  • The direct light rays pass through the annular groove whereas the diffracted light rays pass through the region outside the groove.
  • Depending upon the different refractive indices of different cell components, the object to be studied shows a different degree of contrast in this microscope.
Phase Plate

The working of Phase contrast microscopy

  • To make phase changes visible in phase contrast microscopy, separation of illuminating light (background) from the specimen scattered light are manipulated.
  • The ring shaped illuminates light passes the condenser annulus is focused on the specimen by the condenser. Some of the illuminating light is scattered by the specimen and forms the background light.
  • In a phase contrast microscope, image contrast is increased in two ways,
  1. By generative constructive interference between scattered and background light rays in regions of the field of view that contain the specimen.
  2. By reducing the amount of background light that reaches the image plane.
  • The background light is phase shifted by 90° but passing it through a phase shift ring. This phase shift causes background and scattered light rays originating from regions of field of view containing sample to constructively interfere resulting resulting in increase in brightness of sample.
  • The background is diminished to 70-90% by grey filter ring. This method maximizes the amount of scattered light generated by the illumination of light.
Working Principle

Applications of Phase contrast Microscopy

  • To produce high-contrast images of transparent specimens, such as
  1. living cells (usually in culture)
  2. microorganism
  3. thin tissue slices,
  4. lithographic patterns,
  5. fibres,
  6. latex dispersions,
  7. glass fragments, and
  8. subcellular particles (including nuclei and other organelles).
  • Examination of growth, dynamics, and behavior of a wide variety of living cells in cell culture.
  • Applications of phase-contrast microscopy in biological research are numerous.

Advantages

  • Living cells can be observed in their natural state without previous fixation or labeling.
  • It makes a highly transparent object more visible.
  • No special preparation of fixation or staining etc. is needed to study an object under a phase-contrast microscope which saves a lot of time.
  • Examining intracellular components of living cells at relatively high resolution. eg: The dynamic motility of mitochondria, mitotic chromosomes & vacuoles.
  • It made it possible for biologists to study living cells and how they proliferate through cell division.
  • Phase-contrast optical components can be added to virtually 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.

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 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.

Phase-contrast microscopy gallery

Ciliate (Oxytricha saprobia) in brightfield (left) and with phase contrast illumination (right)
Human Blood in phase contrast
Phase-contrast image of a cheek epithelial cell.
Cheek cells captured with phase contrast objectives.
Diatom Comparison - A couple of diatoms from Carolina Biological on a test slide, first at 40x Ph3, and on the right is a comparison of three different 100x Ph4 objectives, dark low, dark low low and dark medium (DL, DLL and DM), which have different optical densities in the phase rings.