Phase-contrast microscopy is among the oldest and the easiest methods to enlarge biological specimens. Dutch Scientist Frits Zernike first introduced it in the 1930s. He proposed that one can utilize the contrast-enhancing technique to develop a relatively higher contrast image of the set specimen. Typically, biologists use this unique microscopy to analyze living cells, thin tissues, microorganisms, organelles, glass fragments, fibers, and much more.
Let’s discuss the method to find out why biologists preferably use phase-contrast microscopy.
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Principle of Phase Contrast Microscopy
The entire working of phase contrast microscopy depends on phase shifts, a difference between two periodic signals. Once you’ve set a specimen to observe under a microscope type, the light will pass through it. In phase-contrast microscopy, small phase shifts take place that actively change the amplitudes or intensity of light. These are further observed as higher-contrast, enlarged images.
Such microscopes enhance image contrast in two ways. It might generate constructive interference between lights passing through the specimen or might work by lessening the background light reaching the plane. In either of the two, an observer can see a clear image.
What are the Types of Phase Contrast Microscopy?
As phase plates are connected to the microscope, they can be of several types. The most common of them are:
Positive Phase Contrast
It is among the most commonly used forms of phase-contrast microscopy. Positive phase contrast produces dark details and destructive interference in a light background. It works by augmenting direct light with ¼ waves.
Negative Phase Contrast
It is the exact opposite of positive. The negative phase contrast presents bright details on a dark background through constructive interference. All this process occurs by diminishing direct light by ¼ waves.
Phase Contrast Microscopy Working
- It has a tungsten-halogen lamp to produce partially transparent illumination that further passes through a collector lens from the annulus. The annulus is a specialized part of the microscope adjusted at the substage condenser of the front plane.
- Then, the type of wavefronts passing through the annulus decides if to pass through the specimen or directly diffract back. Either way, the sample is enlarged with moderately higher contrast. The sample’s diffraction could be due to the phase gradients or structures present in the set specimen.
- When the diffracted and undeviated light passes through the specimen, they are collected at the phase plate to create a focus in the image plane. All this process finally produces a high-contrast image observed by the eyepiece.
Why do Biologists Prefer a Phase Contrast Microscope?
Most of the time, microscopes run on the phenomenon of staining a specimen, which might kill the object to be observed. In a phase-contrast microscope, a biologist can easily view a clear/transparent image of a living cell without staining it, thus, saving time. So, it is one of the most precise and time-saving methods of observing unstained living cells by biologists.
Advantages of Phase Contrast Microscopy
There are numerous advantages of using a phase-contrast microscope, such as:
- It produces a more visible image with high transparency.
- No extra work is required to stain the specimen, preserving time.
- Biologists can observe living cell specimens in their actual state.
- The organelles, like vacuoles, chloroplasts, mitochondria, etc., could be observed in higher resolution.
Applications of Phase Contrast Microscopy
We have found numerous applications of phase contrast microscopy in the research and development area. Some of the uses include producing high-contrast results of objects, such as:
- Cultured living cells
- Different organelles
- Thin tissue slices
- Latex dispersions, etc.
Where do the Limitations of Phase Contrast Microscopy Appear?
Although it is among the most effective methods of observing transparent specimens, still, with the pros come cons. Sometimes the phase shift cannot produce an apparent light intensity due to the thick sample, making it challenging to pass light. Besides, the parts used in such microscopes are pretty expensive.
Whatever inventions have been made till now are for people’s ease and to improve technology. The Dutch physicist made it possible for biologists to clearly observe living cells without killing them, which is a victory! Despite having a few limitations, a phase-contrast microscope is still in use, is very beneficial in laboratories