Study of Phase Contrast Microscopy.

 

Introduction:

•  It was first described in 1934 by Dutch physicist Frits Zernike for which he received a Nobel Prize.

• Unstained living cells absorb practically no light. 

• Poor light absorption results in extremely small differences in the intensity distribution in the image. 

• This makes the cells barely, or not at all, visible in a brightfield microscope. 

• Phase-contrast microscopy is an optical microscopy technique that converts phase shifts in the light passing through a transparent specimen to brightness changes in the image.

Principle:

• When light passes through cells, small phase shifts occur, which are invisible to the human eye. 

• In a phase-contrast microscope, these phase shifts are converted into changes in amplitude, which can be observed as differences in image contrast.

Parts of Phase contrast Microscope:

• Phase contrast microscope is a specially designed light microscope which contains all basic parts of compound microscope with addition of,

• Annular phase plate.

• Annular diaphragm.

• Annular diaphragm:

• It is placed below the condenser.

• It is made up of a circular disc having a circular ring shaped groove.

• The light rays are allowed to pass through the annular groove.

• Through the annular groove of the annular diaphragm, the light rays fall on the specimen or object to be studied.

• At the back focal plane of the objective develops an image.

• Annular phase plate:

• The annular phase plate is placed at the back focal plane of the objective.

• The phase plate is a transparent disc.

• It may be a,

• Positive Phase plate. (Thin circular groove).

• Negative Phase Plate. (Thick circular area).

• This thick or thin area in the phase plate is called the conjugate area.

• The direct light rays pass through the annular groove whereas the diffracted light rays pass through the region outside the groove.

• On the left, the objective phase ring and condenser annulus are mis-aligned. On the right, the components are correctly aligned, as the segmented circle of light created by the condenser annulus sits on the black ring.

Working of Phase contrast Microscopy:

• Illumination produced by the tungsten-halogen lamp is directed through a collector lens and focused on a condenser annulus positioned below the objective.

• Wavefronts passing through the annulus illuminate the specimen and either pass through undeviated or are diffracted and retarded in phase by structures present in the specimen.

• Undeviated and diffracted light collected by the objective is separated at the rear focal plane by a phase plate and focused at the intermediate image plane to form the final phase-contrast image observed in the eyepieces.

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Applications of Phase contrast Microscopy:

• Phase contrast microscopy is used for taking high contrast images of the transparent specimens in,

1. living cells (usually in culture),

2. microorganisms,

3. thin tissue slices,

4. lithographic patterns,

5. fibers,

6. latex dispersions,

7. glass fragments, and

8. subcellular particles (including nuclei and other organelles).

Advantages:

1. Living cells can be observed in their natural state.

2. It makes a highly transparent object more visible.

3. Saves Time: No special preparation of fixation or staining.

4. Examining intracellular components of living cells at relatively high resolution.

5. Phase-contrast optical components can be added to virtually any brightfield microscope, with some prerequisites.

Limitations:

1. Very costly. 

2. To use phase-contrast the light path must be aligned.

3. Generally, more light is needed for phase contrast than for corresponding bright-field microscopes.