Phase Contrast Microscope: Short notes on Phase Contrast Microscope !
In recent years, remarkable advances have been made in the study of living cells (unstained) by the development of special optical techniques such as phase contrast and interference microscopy.
The biological specimens are highly transparent to visible light and they cause phase changes in transmitted radiations.
The phase contrast microscope has the same resolving power as the ordinary light microscope but it permits visualization of different parts of the cell due to differences in their refractive index (Refractive index is defined as the ratio of the velocity of light in a vacuum to its velocity in a transmitting medium).
Because light is transmitted through a structure at a velocity inversely proportional to the refractive index of the structure, light waves emerging from structures with different refractive index will be out of phase with one another.
The phase contrast microscope is able to convert these differences in phase to differences in light intensity, producing an image with good contrast. The phase-contrast microscope utilizes interference between two beams of light.
In the phase contrast microscope, the small phase differences are intensified. The most lateral light passing through the objective lens of the microscope is advanced or retarded by an additional l/4th wavelength (1/4λ.) with respect to the central light passing through the medium around the object, by an annular phase plate that introduces a 1/4 wavelength variation in the back focal plane of the objective.
In addition an annular diaphragm is placed in the substage condenser. The phase effect results from the interference between the direct geometric image given by the central part of the objective and the lateral diffracted image, which has been retarded or advanced to a total of 1/2 wavelength.
In bright or negative contrast, the two sets of rays are added and the object appears brighter than the surroundings. In dark or positive contrast, the two sets of rays are subtracted making the image of the object darker than the surroundings. Because of this interference, the minute phase changes within the object are amplified and intensified.
A transparent object thus appears in various shades of gray, depending upon the thickness of the object and the difference between the refractive indices of the object and the medium.
Phase microscopy is used to observe living cells and tissues. It is particularly valuable for observing the cells cultured in vitro during mitosis.
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Principle of the phase-contrast microscope:
This is to convert small phase differences into differences in contrast that can be detected visually. An annular phase plate is placed in the objective of the microscope and an annular diaphragm is placed in the condenser as shown in the figure 2. As light is transmitted through the lenses, some of the rays pass through in a direct path while others are diffracted laterally. Diffracted light rays are, thus out of phase with the direct light, and an image of strong contrasts is produced.
The annular diaphragm illuminates the object with a narrow cone of light, and the annular phase plate produces a variation of 1/4 A. between the diffracted lateral light and the direct light. The phase effect is the result of interference between the direct image in the centre of the objective and the diffracted lateral image.
If the diffracted image is retarded, negative contrast results, whereas if it is advanced, positive contrast results. When the refractive index of the medium is greater than that of the object, the object is dark, and when the refractive index of the medium is less than that of the object, the object is bright.