Phase-contrast microscopy is an optical microscopy illumination technique. In this technique, small phase shifts in the light when passing through a transparent object is converted into amplitude or contrast changes in the image.
This type of microscope does not need staining to view the object. This is a type of light microscopy that magnify the contrast of transparent and colorless objects by influencing the optical path of light.
The phase-contrast microscope adapters convert the minute difference in phase changes in transmitted light due to refractive indices of the cell into perceptible shades.
It enables visualization of cells and it is a composition that would be difficult to see using an ordinary microscope.
History
Dutch physicist- Frits Zernike in 1934 and won the Nobel prize in 1953.
Working Principle
When light passes through a cell, a small phase shift occurs which is invisible in the human eye.
In a phase-contrast microscope, these phase shifts converted into changes in amplitude, which can be observed as the difference in image contrast.
• Annular ring- between the condenser and light source.
• Phase ring- objective lens and image plane.
Both the rings allow partial light to pass through it and the rest is blocked, this mechanism creates contrast.
Working of phase contrast microscopy
• The illumination created by the tungsten halogen lamp which is directed through a collector lens and focuses on an annular ring.
• Waves passed through the annular ring fall on the specimen the light contacts the specimen get regarded and other unretired.
• The rays are collected by the objective and separated at the rear focal plane by the phase plate.
Parts of phase contrast microscopy
The Annular Diaphragm
• It is located below the condenser.
• It is made up of a circular disc having a circular annular groove in which the light rays are pass.
• The focal plane of the objective develops an image at the backside.
The Phase Plate
• If it is a negative phase plate then, it has a thick circular area or if it is a positive phase plate then, it has a thin circular groove. This phase plate is called the conjugate area.
• The phase plate is a transparent in nature.
• The light rays directly travel through the annular groove whereas the diffracted light rays pass through outside the groove.
• Depending upon the different refractive indices of different cell components, the object shows a different degree of contrast in this microscope.
Uses
• A living cell, microorganisms, thin tissue slices, fibers, glass fragments, etc.
• This type of microscope helps to study the cell cycle.
• The phase contrast microscope is also used for examination of biological tissues samples.
Advantages
• Living cells can be seen in their natural state without any changes.
• It makes a highly transparent object more visible.
• There is no requirement for sample preparation to study an object under a phase-contrast microscope.
• It saves a lot of time.
• It helps to examining intracellular components of living cells at high resolution.
• It made it possible for biologists to study living cells and cell division.
Limitations
• Phase-contrast condensers and objective lenses add cost to a microscope.
• Usually, Phase contrast is not used in teaching labs except in the health professions.
• To use a phase-contrast microscope the light path must be aligned.
• Generally, more light is needed for a phase-contrast microscope.
• The technique is based on the diminishment of the brightness of most objects.
