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Phase Contrast Microscope

Phase contrast microscopy produces high-contrast images of transparent specimens like living cells without staining. It works by converting slight differences in a specimen's refractive index and density into variations in light intensity. Light passing through a specimen is split into deviated rays, which pass through a phase ring and are advanced in phase, and undeviated rays, which miss the ring. The out-of-phase recombination of these rays causes specimens to appear dark against a bright background, allowing living cells and other transparent structures to be readily observed. This technique is especially useful for studying microbial motility and structures with differing refractive indexes from water.

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20BBC539 RAJILA JANANI .L
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138 views

Phase Contrast Microscope

Phase contrast microscopy produces high-contrast images of transparent specimens like living cells without staining. It works by converting slight differences in a specimen's refractive index and density into variations in light intensity. Light passing through a specimen is split into deviated rays, which pass through a phase ring and are advanced in phase, and undeviated rays, which miss the ring. The out-of-phase recombination of these rays causes specimens to appear dark against a bright background, allowing living cells and other transparent structures to be readily observed. This technique is especially useful for studying microbial motility and structures with differing refractive indexes from water.

Uploaded by

20BBC539 RAJILA JANANI .L
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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Introduction

• Phase contrast microscopy, first described in 1934 by Dutch physicist Frits


Zernike, is a contrast-enhancing optical technique that can be utilized to
produce high-contrast images of transparent specimens, such as living cells
(usually in culture), microorganisms, thin tissue slices, lithographic patterns,
fibers, latex dispersions, glass fragments, and subcellular particles (including
nuclei and other organelles).
• Unpigmented living cells are not clearly visible in the brightfield microscope
because there is little difference in contrast between the cells and water.
• Thus microorganisms often must be fixed and stained before observation to
increase contrast and create variations in color between cell structures.
• A phase-contrast microscope converts slight differences in refractive index and
cell density into easily detected variations in light intensity and is an excellent
way to observe living cells
• The condenser of a phase-contrast microscope has an annular stop, an opaque
disk with a thin transparent ring, which produces a hollow cone of light
Working
• As this cone passes through a cell, some light rays are bent due to variations in density and refractive
index within the specimen and are retarded by about 1 /4 wavelength.
• The deviated light is focused to form an image of the object.
• Undeviated light rays strike a phase ring in the phase plate, a special optical disk located in the
objective, while the deviated rays miss the ring and pass through the rest of the plate.
• If the phase ring is constructed in such a way that the undeviated light passing through it is advanced
by / 4 wavelength, the deviated and undeviated waves will be about 1/ 2 wavelength out of phase and
will cancel each other when they come together to form an image.
• The background, formed by undeviated light, is bright, while the unstained object appears dark and
well-defined.
• This type of microscopy is called dark-phase-contrast microscopy.
• Color filters often are used to improve the image
Application
• Phase-contrast microscopy is especially useful for studying microbial motility,
determining the shape of living cells, and detecting bacterial components
such as endospores and inclusion bodies that contain poly- -hydroxybutyrate,
polymetaphosphate, sulfur, or other substances
• These are clearly visible because they have refractive indexes markedly
different from that of water.
• Phase contrast microscopes also are widely used in studying eucaryotic cells.

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