During the early years of the development of microscopes for use in the medical and biology field, many researchers, doctors and scientists struggled to study live specimens. By 1930, a Dutch physicist named Frits Zernike developed the phase-contrast microscope. A phase-contrast microscope can be used to reveal the nature of living cells, and it does so quite well.
If you want to learn more about this type of microscope, read more on its working principles, applications and advantages below.
Understanding phase contrast microscopes
Phase-contrast microscopes are most often used for the study of the structure and components of living organisms. One advantage of using this method over other forms of microscopy is that bright-field microscopes can provide images of living matter in action. This is because living matter is not always in its absolute normal condition—it can distort and cause problems for the images if they’re viewed through regular lenses.
Phase contrast is a technique used to enhance the contrast of light microscopy images of specimens that are transparent and colourless. This enables researchers to visualize the cells and its components, which is difficult to achieve when an ordinary light microscope is used.
Using a phase-contrast microscope, the image contrast is increased in two ways. First, it generates constructive interference between scattered and background rays in the area of the field of view, which contains the sample. Next, it reduces the amount of background light that reaches the image plane.
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Part of a phase contrast microscope
To understand the working principle of the microscope, you should be first acquainted with its different parts. Some of the details of a phase-contrast microscope are the same as of the light microscope. These additional parts are specially designed so they can provide better imaging of the specimen:
Annular diaphragm
The annular diaphragm is located just beneath the condenser and is made of a circular disk that has annular grooves. The light rays are allowed to pass through the annular groove and land right on the specimen. Behind the focal plane, there is an annular phase plate, and the objective forms the image of the sample.
Phase plate
There are two types of phase plates: negative and positive. The former has a thick ring-shaped area while the latter has a thin disk-like groove. This area of the phase plate is referred to as the conjugate area. The combination of both phase plate and annular diaphragm helps create the phase contrast.
The contrast is achieved by splitting up the light rays from the diffracted beams. The former goes through the annular groove while the latter passes through the portion outside the groove.
Working principle of phase contrast microscopes
The phase-contrast type of microscope is different from other types of microscope like a stereo microscope. A stereo microscope is designed for observing specimens with low magnification. Typically, it utilizes the light that’s reflected on the surface of the sample rather than the light that goes through it. Meanwhile, the phase-contrast variant uses direct light and diffracted life to adjust the contrast of the specimen so you can study it even though it’s transparent.
When light passes through the cells contained in the specimen, small phase shifts occur. These phase shifts are invisible to the naked eye. The phase-contrast microscope converts these shifts into changes in amplitude, which can be observed as different contrasts in the images.
It has a partially coherent illumination which is produced by a tungsten-halogen lamp. The light is directed through a collector lens and focused on a specialized condenser annulus found in the front of the focal plant.
The wavefronts are passing through this annulus light into the specimen. Then the direct light is separated from the diffracted light. This creates the gradients present in the sample. The direct light and diffracted light are segregated at the rear focal plane by the phase plate. The light is then focused at the intermediate image plane to form the phase-contrast image of the specimen as observed in the eyepiece.
Applications Of phase contrast microscopes
The applications of the phase-contrast microscope are numerous in the field of biological research. It’s used to generate high-contrast photos of clear or transparent specimens like:
- Living culture cells
- Microorganisms
- Thin tissues
- Fibres
- Glass fragments
- Subcellular particles like nuclei and organelles
- Latex dispersions
- Lithographic patterns
Researchers can see these specimens without staining or killing the organism. Aside from that, they can observe the parts and behaviour of living cells with relatively high-definition images. One of the many uses of the phase-contrast microscope is that it’s used to gain more understanding on living cells and how they reproduce through the process of division.
Takeaway
Before robotics advanced the health care industry, apparatuses like the phase-contrast microscope were already developed. These microscopes helped people in the medical field understand how bacteria and virus cells reproduce, so they can work on medications and vaccines.
Phase-contrast microscope converts optical phase changes in light passing through a sample to visible light changes in the digital image. Phase-contrast microscopes employ fluorescent or contrast-enhanced microorganisms or other light sources to excite the phase contrast and to alter the colors of the organisms. The result is that the organisms are illuminated against a blue chromatic spectrum.