Definition of Phase Contrast Microscopy
A phase contrast microscopy converts slight differences in refractive index and cell density into easily detected variation in light intensity to observe living cells.
This microscope is used for visualization of cell culture and live cells. Living cells can be observed without any staining.
Unstained specimens have absorbed no light, as a result it creates extremely small differences in the intensity distribution in the image. Therefore in a bright field microscope, the specimen is not clearly visualized.
Because a small phase shifts occurred, when light passes through specimens, which we can’t see with our eyes.
In a phase contrast microscopy, these phase shifts are transformed into changes in amplitude, which can be observed as differences in image contrast.
In 1934, a Dutch physicist Frits Zernike was first described the Phase Contrast Microscopy. He was awarded Noble Prize in 1953, for Physics. In 1942, the commercial production of Phase contrast microscopy was started in Germany.
Principle of Phase Contrast Microscope
The condenser of a phase-contrast microscope has an annular stop an opaque disk with a thin transparent ring that produces a hollow cone of light.
As this cone passes through a cell some light rays are bent due to variation in density and refractive index within the specimen and are retarded by 1/4 wavelength. The deviated light is focused to form an image of the object.
The undeviated light rays strike a phase ring in the phase plate a special optical disks located in the objective, while the deviated rays miss the ring and passed through the rest of the plate. The undeviated light which strikes the phase ring gets advance by 1/4 wavelength when passing through this ring.
The deviated and undeviated waves become 1/2 wavelength to each other and will cancel each other to come together to form an image. Therefore deviated and undeviated lights from different image.
The background formed by undeviated light is bright while the unstained object appears dark and well-defined.
Light Path of Phase Contrast Microscope
- The light rays enter the annular diaphragm from its source.
- Then it passes through the condenser lens, which focused the rays on the specimen.
- The light transmitted through the specimen and then enter the objective lens where an image of the specimen is created.
- As the light transmitted through the specimen it creates a deviated and undeviated light rays.
- The deviated light rays miss the phase ring over the objective lens.
- Whereas the undeviated light rays strike a phase ring. As a result, deviated and undeviated rays formed different images.
- The undeviated light rays formed the background of specimen’s image.
Parts of Phase Contrast Microscope
A phase contrast microscope is basically a modified form of a simple microscope. It contains all the components of a normal microscope, except for two important parts that are missing in a simple light microscope such as, Annular diaphragm and Phase plate. This two component helps to form the phase contrast, by separating the direct rays from the diffracted rays.
- Annular diaphragm:
- Annular diaphragm is consists of a circular disc that had a circular annular groove.
- This annular groove passes the light rays to the object.
- It is located under the condenser lens.
- Phase plate
- It is a transparent disc.
- There are present two types of phase plates such as the positive phase plate and the negative phase plate.
- The positive phase plate contain a thick circular area where the negative phase plate contain a thin circular groove.
- These thin and thin areas in phase plate called conjugate area.
- The direct light rays and diffracted light are basically passed through the annular groove and through the region outside the groove.
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Other parts of a Phase contrast microscope;
- objective lense
- condenser lense
- specimen stage.
- stage clips
What is the negative and positive phase contrast?
- In the positive phase contrast, the object appears as dark gray on a brighter grey background.
- In the negative phase contrast, the object appears as brighter on a dark background.
Applications of Phase Contrast Microscope
- Phase contrast microscopy is specially useful for the detection of bacterial components such as endospore and inclusion bodies.
- Phase contrast microscopy also are widely used in studying Eukaryotic cells.
- It also used to visualize a thin tissue slice.
- Living cells are observed in the natural state. It will provide more information about the specimen than specimens that need to be killed, fixed or stain to view under a microscope.
- It produces a resolution and high contrast images of the specimen.
- It is useful for studying on thin specimen.
- The modern form of this microscope can capture photos or can record videos.
- Phase Contrast Microscopy can create a visible image of a highly transparent objects.
- No need for staining and fixation of the specimen.
- Intercellular components of living cells can be observed with high resolution.
- The Annuli or rings of phase contrast microscope limit the aperture to some extent, which decreases the resolution of image.
- Thick organisms or specimens can not be observed by this microscope, a distorted image can appear.
- With the uses of white or green lights Images appear as grey or green, which results a poor photomicrography.
- Shade-off and halo effects can occur in this microscope.