Phase Contrast Microscopy (A Type of microscope )

Phase Contrast Microscopy

An advantage of this technique is its ability to show cell structure without using dyes or killing the cell or the organism. Thus it is possible to study life processes in situ. The phase contrast microscopy uses a modified light microscope that enhances the contrast between substances of different thickness or different density. 

In bright field microscopy, the unstained objects in the specimen are visible because of the change in the amplitude of the light ray passing through them producing a contrast with the surrounding medium. 

When a ray of light passes through a part of the specimen having a lower density, a very small amount of light is absorbed and the emerging light wave will have a comparatively higher amplitude or intensity. 

Another light ray, passing through a denser part of the specimen, will emerge with a lower amplitude or intensity due to higher absorption by the dense specimen. This part of the specimen, therefore, will appear darker. 

However, these differences in intensity are very subtle and the human eye is not sensitive enough to notice the minute differences. The purpose of phase contrast microscope is to convert optical densities or refractive indices into amplitude changes which are easily visible to the eye.

Principle A ray of light is made of waves travelling in a straight line. When two such waves travel together, they are said to be "in phase". Such a ray will appear bright to the observer. However, if one of the waves is held up or made to change the path, they will no longer travel together and they are said to "interfere with each other, differing in the intensity.

In a phase-contrast microscope, a special condenser and objective control the illumination in a way that accentuates the differences in densities. It causes light to travel different routes through the various parts of the cell. The result is an image with differing degrees of darkness and brightness, collectively called "contrast".

To achieve phase contrast, one needs: 

I. An annular diaphragm (annulus), appropriate for the objective.

2. A phase plate

3. A high intensity compound lamp 
4. An auxiliary telescope

The annulus is placed in the condenser and the phase plate is placed in the objective. The annulus allows only a small ring of light to pass into the microscope. The size of annulus required varies with the numerical aperture of the objective used. A low power objective requires the annulus of a smaller diameter than that for the high power objective. 

The phase plate has a circle engraved on it. This circle matches with the ring of light coming in from the annulus through the condenser.

The direct rays of light coming in through the annulus thus pass through the engraved circle and take the shortest route to the observer by 1/4th of a wavelength. However, some light rays are reflected by the object, the reflection being proportional to the density. These rays are diverted from the shortest route and interfere with the direct waves, producing variation in the intensity of illumination. 

This results in the increased contrast in the observed image. With this method, denser materials appear bright, while parts of the cell that have a density close to that of water (e.g., the cytoplasm) appear dark. Figure 3.26 shows the light path of a phase contrast microscope.

Setting up phase-contrast microscope 

1. With a high intensity lamp, illuminate the microscope. Turn the required objective into position and focus on the specimen.

2. Move the annulus matching with the objective into place.

3. Remove the eyepiece and insert the telescope; adjust until the two rings, one bright and one dark, are in focus.

4. Adjust the centering screws of the condenser until the bright ring of the annulus fits exactly into the darker ring of the phase plate.

5. Remove the telescope, replace the eyepiece, focus and examine the specimen. Some applications of phase contrast microscopy

1. For examining unstained bacteria e.g.vibrios in specimens.

2. For examining wet preparations of specimens like urine, vaginal swabs.

3. For examining faecal preparations for trophozoites of amoebae.

4. In searching for trypanosomes in blood and other body fluids.

5. In the detection of promastigotes of Leishmania in culture fluid.