Phase Contrast Microscope: Introduction, Principle, Parts, Uses

Introduction of Phase Contrast Microscope

Phase-contrast Microscope with its various parts as shown above picture. It is a modification of a light microscope with the addition of an annular diaphragm and phase plate. This optical methodology was introduced by Dutch physicist, Frits Zernicke in 1932. Living cells can be observed in their natural state without previous fixation or labeling or staining. It makes a highly transparent object more visible.

Principle of Phase Contrast Microscope

Unstained test specimens ( bacteria, cell organelles, etc) have constituents of different refractive indexes. Diffraction of light creates in phase Contrast microscope which employs an optical mechanism to translate minute variations in phase into corresponding changes in the intensity of the image.

Parts of Phase Contrast Microscope

The phase-contrast microscope is similar to a light microscope with all the basic parts in addition to which an annular phase plate and annular diaphragm are fitted.

The annular diaphragm: It is present below the condenser and made up of a circular disc having a circular annular groove. The light rays are allowed to pass through the annular groove where the light rays fall on the specimen or object to be studied and at the back focal plane of the objective develops an image.

The phase plate: It is a transparent disc and It is either a negative phase plate having a thick circular area ( also called conjugate area) or a positive phase plate having a thin circular groove. With the use of the annular diaphragm and the phase plate, the phase contrast is obtained in this microscope and this is possible by separating the direct rays from the diffracted rays. Depending upon the variation of refractive indices of different cell components, the object to be studied shows a different degree of contrast in this microscope

Uses of Phase contrast Microscope

The magnification and resolution are similar to that of a light microscope.
Even though, it has widely applicable in biological sciences. e.g.

To produce high-contrast images of transparent specimens, e,g. microorganisms (Capsular Serotyping of Streptococcus pneumoniae Using the Quellung Reaction), living cells (usually in culture), thin tissue slices, lithographic patterns, fibers, latex dispersion, glass fragments, and sub-cellular particles (including nuclei and other organelles).
It helps to study cellular events such as cell division, phagocytosis, cyclosis, and so on.
It enables the visualization of living cells.
It enables the visualization of unstained cells.
It is also used to visualize all types of cellular movement. e.g. chromosomal and flagellar movement
It is also useful to study of membrane permeability of cells and different organelles.
Its more common use on living cells in tissue culture to monitor their growth.
Its use in biological research is very common.

Advantages of Phase Contrast Microscope

It provides a clear image of unstained cells.
It also avoids damages to the cells due to chemical preparation and staining.
It provides high-contrast images highlighting the fine details of cells.
The optical construction is relatively simple.
A compound microscope can be changed to phase contrast with minor additions.
It enables prolonged observation of living cells without losing their viability of cells.
Using this microscope, live-cell imaging and live process monitoring is possible.

Disadvantages of Phase Contrast Microscope

It is only useful for observing individual cells or thin layers of cells.
It produces a bright halo around the images. This halo formation is due to partial or incomplete separation of direct and deviated rays.

Keynotes on Phase Contrast 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. Phase shifts themselves are invisible but become visible when shown as brightness variations.
One major advantage is that phase-contrast microscopy can be used with high-resolution objectives, but it requires a specialized condenser and more expensive objectives.
Phase-contrast microscopy was the first accurate method used to count platelets in lysed blood diluted within counting chambers and was the gold standard earlier to the application of colter or impedance principle.

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