Phase Contrast Microscopy

Phase Contrast Microscopy

Introduction to Phase Contrast Microscopy

  1. Phase contrast microscopy, first described in 1934 by Dutch physicist Frits Zernike.
  2. Contrast-enhancing optical technique that can be utilized to produce high-contrast images of transparent specimens.
  3. Such as living cells (usually in culture), microorganisms, thin tissue slices, fibers, glass fragments, and sub cellular particles (including nuclei and other organelles).
  4. The phase contrast microscopy is a special adaptation of the light microscopy & helps to obtain a clear picture of living or unstained cells.
  5. The adaptors convert minute difference in phase changes in transmitted light due to refractive indices of all cell organelles in to perceptible shades of grey.
  6. This allow organelles of the living cell to become visible with fair contrast in them. 
Phase Contrast Microscopy


Annular Ring and Phase Ring

ANNULAR RING : is between condenser and light source.
PHASE RING : is between objective lense and image plane.
SPL PROPERTY : both the rings allow partial light to pass through it and the rest is blocked.

Annular ring and Phase ring


Working principle

  1. Regions of different composition likely to have different Refractive indices. Normally such differences cannot be detected by our eyes. However, PCM converts differences in μ into  differences in intensity (relative brightness and darkness) which are visible to eye.
  2. Interference of light: interaction of two light waves which leads to the formation of resultant wave  Constructive interference  Destructive interference 
  3. Light passes through the condenser via annular ring.
  4. After reaching the specimen plate two types of beams are formed.
  5. If there is no specimens in lens : Surrounding waves (S), Particle wave (P)  P=S  NO INTERFERENCE.
  6. If lens contain sample: Light beams gets diffracted because of different density at different regions of sample.  1) Surrounding wave (S)  2) Diffractive wave (D),  P=S+D  Either constructive interference or destructive interference may occur.
  7. The ring shaped illuminating light that passes the condenser annulus is focused on the specimen by the condenser.
  8. Some of the illuminating light is scattered by the specimen, The remaining light is unaffected by the specimen and forms the background light in a phase contrast microscope.
  9. The image contrast is improved by two different methods: 1) Negative Phase Contrast 2) Positive Phase Contrast. 


Negative Phase Contrast produces destructive interference: Thus, the image obtained is:
* Inner region of the sample - Bright
* Outer region of the sample - Darker
* Surrounding lens - Opaque

Positive Phase contrast produces constructive interference, Thus the image of the specimen obtained is:
* Inner region of the sample -Darker
* Outer region of the sample - Brighter
* Surrounding lens - Opaque

Interference


Advantages

  1. Small unstained specimens such as a living cell can be seen.
  2. It makes highly transparent objects more visible.
  3. Examining intracellular components of living cells at relatively high resolution.
  4. Example:- The dynamic motility of Mitochondria, Mitotic chromosomes & vacuoles.
  5. It made it possible for biologists to study living cells and how they proliferate through cell division.

Phase Contrast Microscopy

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