The phase-contrast microscope is a modified version of the bright-field microscope that helps visualize living cells without affecting the cells’ viability. It is termed phase-contrast because it consists of a unique phase contrast condenser (annular ring) and a phase contrast objective (phase plate). These parts amplify the phase difference of transparent specimens.
Principle of Phase Contrast Microscope
Let us consider light as a wave. Two identical beams of light releases in synchronization from a source. The first light beam strikes a material with a higher refractive index than air. The beam of light slows down, and the number of light waves increases in proportion to the refractive index and thickness of the material. But the second beam travels through the air only. Although the light rays were synchrony, a difference establishes in the beams’ phase after traveling through different mediums. It is termed phase difference.
Every specimen can diffract the beam of light. An image is formed due to the phase difference between diffracted and undiffracted beams (direct light). A stained or colored object has enough phase difference to create an image.
Since the phase difference between diffracted and direct light is insufficient in transparent specimens. A disk (annular ring) in the condenser separates direct and diffracted light. Likewise a special plate (phase plate) in the back of the focal plane of the objective lens increases the phase difference between diffracted light and direct light. The resulting increase in phase difference helps to form the image of transparent objects without staining.
A physicist, Frits Zernike, gave this principle in 1932 and used this principle in microscopy in 1935. He was awarded the Nobel Prize in physics for this work.
Diffraction: the scattering of waves as they touch the specimen’s edge.
Parts of Phase Contrast Microscope
The phase-contrast microscope has all the parts of the microscope and some additional parts. They are:
An annular ring or phase annulus is a clear ring in an opaque disk. The purpose of the apparatus is to produce a circle of light in the front focal plane of the condenser. Thus, the condenser focuses on the specimen plate. The annular ring is different for different objectives.
A phase plate is a transparent plate with a circular ring in it. The ring of the phase plate is filled with phase advancing and retarding material. The ring of the plate may appear lighter or darker than other parts of the plate. There is an addition of some material that changes light amplitude. The phase plate’s position is complementary to the annular ring. However, the position of the phase ring is over the image of the annulus ring. So the phase plate is a part of objective lens and has conjugate and complementary parts.
A phase telescope is a special eyepiece that focuses an objective’s back focal image onto a human retina. This procedure is necessary for aligning the annulus ring to the phase plate. Bertrand lens replaces the phase telescope on some microscopes.
Assembling the Apparatus of Phase Contrast Microscope
The proper functioning of the phase microscope requires proper assembling and alignment of the phase apparatus. Follow the steps below to assemble the phase apparatus:
- Kohler illumination is preferred for this type of microscopy because phase-contrast requires the condenser to produce nearly parallel light waves in the specimen.
- After setting set up the microscope on Kohler illumination, open the condenser’s iris fully.
- Then, focus specimen with low power objective with phase plate. And insert the adequately sized annulus ring.
- After that, replace an eyepiece with a phase telescope. And focus the telescope on the annulus ring.
- Align the phase plate with the annulus ring.
- Finally, observe the phase plate and annulus ring through the telescope.
Working mechanism of the Phase Contrast Microscope
- In phase-contrast microscopy, illumination of the specimen occurs by light passing through an annular ring. It produces a hollow cone of light.
- In this microscope, one set of light rays directly comes from the light source (direct light). In contrast, the other set comes from light reflected or diffracted from a particular structure in the specimen.
- Direct light rays strike a phase ring in the phase plate within the objective to give a bright background. At the same time, the diffracted beams miss the ring and pass through the rest of the plate to provide dark and well-defined structures.
- When the two sets of light rays, direct and diffracted beams, are brought together, they form an image of the specimen on the ocular lens with a bright background and different dark areas. This type of microscopy is called dark-phase-contrast microscopy.
Types of Phase Contrast
Depending upon the construction of the phase plates, there are several types of phase-contrast microscopy. The most common types are
Positive phase contrast
Positive phase contrast advances direct light by ¼ wave, producing destructive interference and dark details in a light background. It is the most commonly used form.
Negative phase contrast
Negative phase contrast retards direct light by ¼ wave, producing constructive interference and light details on a dark background.
Either positive or negative phase contrast
The phase plate in this form is of two types. Either the phase plate absorbs direct light, or it can absorb diffracted light.
Application of Phase Contrast Microscope
A phase-contrast microscope is applied in various biological researches to visualize transparent samples like living cells in culture, pieces of tissue, microorganisms, fibers, subcellular particles, glass fragments, and latex dispersions.
Limitation of Phase Contrast Microscope
There is some limitation to the phase-contrast microscope. These are:
- It works excellent for observing thin, colorless, and transparent specimens. However, a confusing phase image will be seen if the sample is thick.
- The assembly of the microscope parts makes the whole procedure more expensive.
- The phase plate limits the objective’s NA (numerical aperture).
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