In bright field microscopes, transparent protoplasmic contents cannot be differentiated unless they are stained this is due to the fact that when light rays pass through both medium and the specimen, there is very little difference or contrast in their refractive indices.
As a result, the alteration in the rays that pass through the specimen or the medium will be almost same when viewed under light microscope.
In order to increase the contrast between the specimen and the background or to bring about differences in the refraction of the rays that pass through the specimen and those that pass through the medium, dyes (stains) are used that selectively stain different cell organelles.
Image Source: upload.wikimedia.org
ADVERTISEMENTS:
Staining however leads to cellular death and when we observe a stained specimen we will not be seeing a live specimen.
Phase contrast microscope is a special microscope that enables the viewer to see transparent protoplasmic components without staining and without killing.
Originally developed by Frederick Zernike (1933) hence called Zernike microscope, Phase contrast microscope is the ideal instrument for observation of living protozoan’s and other transparent microbes, without staining.
ADVERTISEMENTS:
Frederick Zerinke was awarded-Nobel Prize in physics in the year 1953 for the discovery of phase contrast principle. Objects in a microscopic field can be categorized into two – Amplitude objects and Phase objects.
Amplitude objects show up as dark objects under the microscopic view because of the reduction in the intensity (amplitude) of the rays that pass through. Phase objects which are transparent on the other Hand allow the light rays to pass through without any reduction in their intensity.
But when the light rays are passing through a transparent object, some rays will have retardation by about one quarter wave length.
This retardation is called phase shift. But the phase shift will not cause any change in amplitude; thus the objects appear transparent.
ADVERTISEMENTS:
The 1/4 wave length phase shift is utilized in the phase contrast microscope to create image contrast. In order to understand this, first we should understand what happens to the rays when they pass through a transparent object.
Behavior of Rays:
Light rays passing through a transparent object emerge out in two different ways – direct and diffracted. The rays that pass through the object in a straight line are called direct rays and are unaltered in amplitude and phase.
The rays that are bent and slowed down as they pass through due to differences in density of the medium, are called diffracted rays. It is this relationship between the rays that is made use of in the phase contrast microscope.
If the direct rays and diffracted rays of an object can be brought into the same phase (crest of both light waves coincide) with each other, the resultant increase in amplitude is due to the sum of both the converged rays and is called coincidence.
As a result of the increased amplitude, the object looks very bright in the field. On the other hand if the direct and diffracted rays are out of phase (i.e. the crest of one wave coincides with the trough and not the crest of another wave) it is said to be reverse phase and their amplitudes cancel each other and the object looks dark.
This is called interface. Both these phenomena (coincidence and interference) are used in a phase contrast microscope.
The Phase Contrast Setup:
The basic construction of a phase contrast microscope is like a bright field microscope only except for two special attachments. This are-
(i) A special type condenser and
(ii) A phase plate.
The condenser has a special diaphragm consisting of an annular stop. This annular stop can be compared to a solid rod kept loosely at the centre of a cylinder.
The annular stop allows only a hollow cone of light rays to pass through the condenser and light the object on the slide. The phase plate is a special optical disc located in the rear focal plane of the objective.
It has a special phase ring coated with a material that can either advance or retard the direct rays depending on its construction. The rays of light (direct rays) that emerge from the object as solid lines converge on the phase ring within the objective.
Here, depending on the coated material the desired phase shift (i.e. retardation or advancement) is produced. The diffracted rays (that have already undergone phase shift) that pass through the object on the slide miss the phase ring and are not affected by the phase plate.
The resultant image will depend on whether the diffracted and direct rays are allowed to converge and interfere.
If they converge (it depends on the type of phase ring) it will result in a bright image against a dark back ground {bright phase microscopy) or if they interfere the amplitude becomes zero and it results in a dark image against a bright background (dark phase microscopy).
In a phase contrast microscope the most important adjustment is the proper alignment of the two rings – the one in the condenser and the one in the objective. The ring of direct light that comes out of the object must correspond to the phase ring in the objective.
How an unstained preparation can be seen under a phase contrast microscope? In any specimen or cell there will be difference in thickness between the structures of components.
When the light rays pass through these there will be variable refraction of the rays and these phase changes are converted into visible differences of light intensity (due to the phase ring).
Even some structures which are difficult to stain and observe under light microscope become conspicuous in a phase contrast microscope because small phase changes result in interference of light waves resulting in high contrast images.