Phase Contrast Microscopy

Microscopy Methods in Microbiology
Microroscopy Methods in Microbiology Part 2
Microroscopy Methods in Microbiology - Introduction
Observation of Microbes in the Living State
Observation of Microbes in Smears
Dyes and Staining
Useful pH Range and Colour Change of Some Indicator Dyes
Factors Affecting Staining
Microscopy and Micrometry
Microscopy and Micrometry - Introduction
Behaviour of Light
Light Theories
Resolving Power
Refraction and Reflection
Bright Field Microscopy
Use of Bright Field Microscope

Phase Contrast Microscopy

	Phase Contrast Microscopy- Phase-contrast microscopy is quite useful in general biology, but has less applications in bacteriology. However, it is useful in examination of growth and cell division in bacteria, flagellar movements, spore and capsule formation and cytopathic effect of viruses in tissue culture. In broad sense, phase microscope is an ordinary bright field microscope with two additional plates, namely, annular diaphragm and phase shifting plate,which enable some of the image forming rays to be Phase-shiftedwith respect to others.
Annular diaphragm is attached in place of iris-diaphragm to the ordinary condenser. This attachment allows only a ring of light to pass through the condenser and then to the object. Each objective requires different size or annulus according to its numerical aperture. The phase-shifting plate is a attached at the back of objective, which is composed of a disk of glass having circular trough etched in it and such a depth that light after passing through it creates a phase difference of a quarter of a wavelength as compared with the light from rest of the plate.
The phase contrast principle was discovered by Fritz Zernike and for which he was awarded Nobel prize in Physics, of 1953. According to this, light waves have variable character for frequency and amplitude. Human eye cannot perceive a phenomenon when two light rays have similar amplitude and frequency, but different phase. Similar situation Takes place when diffraction grating is examined under ordinary microscope. If one considers the unstained bacteria as an alternative strips of material with slightly different refractive indices, though there is slight phase difference, it is totally obscured by the intensity of light and no detail is observed
In phase contrast microscope, when a single ray of incident light falls on a specimen, two rays develop, one of these is direct or transmitted light ray from annular diaphragm and passing through the object and is focused on the phase-shifting plate. Please note that this plate will either delay or advance this ray. The second ray is a scattered or diffracted ray, as a result of passing through the margin of specimen and is not striking the phase shifting element, therefore, its phase is not affected. When these two beams (direct and diffracted ) unite, they are not in phase and the phase differences become apparent. Two sets of rays emanating from the same point of specimen return to the same point in the image having phase difference with respect to one another interfere to produce variation in intensity of illumination, and therefore, contrast in the image formed Depending upon the phase shifting element employed, the specimen may appear lighter against dark background (dark contrast) or darker against light background (bright contract)

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