phase contrast microscope

Refractive index (µ): It is the ratio of Velocity

of light in air to that of in given medium

Diffraction: The process by which a beam of

light is spread out as a result of passing

through a narrow aperture or across an edge

typically accompanied by interference b/w the

wave forms produced.

Fritz Zernike (1888-1966)

Advantages1.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.

eg: The dynamic motility of

Mitochondria,

mitotic chromosomes & vacuoles.

4. It made it possible for Biologists to study

living cells and how they proliferate

through cell division.

•The phase contrast microscopy is a special

adaptation of the light microscopy & helps to

obtain a clear picture of living or unstained

cells.

•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

•This allow organelles of the living cell to

become visible with fair contrast in them.

Working Principle

• 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.

• PCM convert invisible small phase

changes caused by the cell component in

to visible intensity changes

Phase contrast is obtained with the help of

the

annular diaphragm by separating the central

& direct ray from the diffracted rays

• The ring shaped illuminating light that passes the

condenser annulus is focused on the specimen by

the condenser

• 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, the image contrast

is improved by two different methods

• Negative Phase contrast

• Positive Phase contrast

Negative Phase contrast:• The background light is phase shifted -90° by

passing it through a Phase shift ring. This

eliminates the phase difference between the

background and the scattered light.

• To further increase contrast, the background is

dimmed by a gray filter ring

Positive phase contrast:• the background light is instead phase shifted by

+90°. The background light will thus be 180° out of

phase relative to the scattered light.

• Leads to formation of an image where the

foreground is darker than the background.

1. GROWING CELLS

2. FIXING CELLS

3. SECTIONING SPECIMEN

Preparation of slide

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