Principle And Applications Of Phase Contrast Microscopy

Phase-contrast microscopy falls in the category of light microscopy that offers powerful label-free imaging of biological molecules. These biological molecules can be physical as well as soft materials.  With a good trinocular microscope, you can get the right images of the desired biological molecules. The standard light microscope has some limitations of low contrast that does not give out a perfect image of the biological samples. To eradicate this limitation, phase-contrast microscopy is used. Here, the biological sample gets illuminated with good contrast and light. The samples used here, tend to execute the diffraction process which gives out the changes in the various refractive indexes which reveal many structures of the biological samples. However, in some cases, the refractive index differences are so slim that the structures cannot be revealed. But with a perfect inverted phase-contrast microscope, the images can be formed in the microscope’s objective area. 

The discovery of the phase-contrast microscope led the discoverer Frits Zernike to the  Nobel prize.  Phase-contrast microscopy is defined as the process that converts phase shifts of light thrown to the samples to the light entering the transparent sample. Hence, the whole sample gets illuminated with the light entering it.  The high contrast label-free images are obtained at the end of the phase-contrast microscopy.  The best trinocular microscope of phase-contrast nature is present in the market that can be used to give out the perfect phase-contrast images.  Here, the phase shifts are not visible, unless their trajectories are recorded in the form of images. The trinocular microscope is used in this reference. The use of inverted microscope is to give out perfect images. 

A phase-contrast microscope does not record the phase variations at a time.  But can certainly give out the results in the form of high contrast images.  The images which are given out with this type of microscope, are the mere results of amplitude changes between the light shifts.  This is called amplitude contrast. Generally, the phase variations are not visible.  It needs modifications in a  tissue culture microscope of phase-contrast nature to give out the perfect illuminations. The adjustments are done in several parameters before getting the phase-contrast images of the biological samples.

Principle Of Phase Contrast Microscope

A standard phase contrast culture microscope runs on a certain principle which gives out the perfect illuminated images. All transparent unstained biological samples do not absorb light. So, they are called phase objects. When light passes through samples in a phase-contrast microscope with no phase object, there is no significant change in refractive indexes. The optical path length remains the same with no change.  Here, the non-diffracted light is referred to as zero-order light as it continues to be unchanged throughout the process unless there are no phase objects.

When the light passes through a transparent area of the biological sample, then small changes in refractive indexes are measured.  The phase objects here diffract the light and scatter the light at the same time. These changes in the refractive indexes of the light phases cause changes in the optical path length of the light and result in the illuminated bio-images. A culture microscope uses this principle to get well-structured images of the culture samples.

Here, the diffractions happen as per the thickness of each structure present in the sample.  It is seen that the thicker the structure of the biological sample, the greater is the diffraction of the light. In a phase-contrast microscope, the diffracted light represents only a very small part of total light passing through the sample. A large proportion of light passing through the biological sample is lost to the environment or helpful to the illumination process.

In phase-contrast microscopy,  the trinocular head is present which enables the light passing.  As the diffracted light arrives at the detector of the microscope,  it becomes out of phase with the direct light. The main core of phase contrast microscopy is the interference caused by the phase shifts of light. All small -small phase shifts of light created by the light piercing the transparent structures are not capable of creating the interferences between the phase lights. In addition to these, the feature of low absorption also causes negligible amplitude differences between the phase of lights.

To put it in simple words, phase contrast microscopy is a method or process that manipulates the nature of phase objects to introduce additional interferences. These interferences are introduced between the direct and diffracted light phases in the inverted phase microscopes. These additional interferences transform the diffraction differences into brightness or contrast.  It all increases the contrast, which can be seen in the non-absorbing samples. By this principle of a phase object, the phase-contrast microscope can give out illuminated images of the biological samples.

An inverted phase microscope can also be used in the process to get enlarged biological images. The phase-contrast microscope components can be fitted in this, to get the illuminated images to a good degree. The trinocular microscope price can be gathered from the offline as well as an online medium. 

Applications Of Phase Contrast Microscopy

Phase-contrast microscopy is useful in various methods of biological light microscopy processes.  In the biological and scientific world, it has a wide variety of applications which are used for cell imaging.  Because of the use of a trinocular compound microscope with phase contrast nature in the cell cultures, it is called the culture microscope.

Following are the key areas where the  phase-contrast microscopy is used and applied:

1. To View the living cells used up in cultures
2. To  know about the structures of microorganisms that prevailed in the biological samples
3. To cut out extremely thin slices of tissues and to visualize  the curt portions
4. To obtain the lithographic patterns  in the fossils and other related specimens of biology
5. To seek out the designs of fibers used up in the process of fiber generation.
6. To visualize the latex dispersions in the cells
7. To  know about the glass fragments 
8. To view the sub-cellular  particles like nuclei, other organelles of cells
9. To know about structures of cells and other types of cells

Like, these many types of applications are of phase-contrast microscopy. In a biological lab, it finds many applications in the research field.

Advantages Of Phase Contrast Microscopy

• All Living cells can be observed in their natural state without previous fixation or labeling process. The images are all label-free.
• A phase-contrast microscope makes a highly transparent object more visible due to the additional interference process.  The whole object gets illuminated.
• No special preparation of fixation or staining is needed to observe a biological sample under a phase-contrast microscope. It saves a lot of time for researchers and scientists.
• The microscopy technique examines intracellular components of living cells at relatively high resolution. eg: mitochondria of cells, mitotic chromosomes,  vacuoles, etc.
• The technique of phase microscopy has made it possible for biologists to study living cells and how they multiply through cell division at their respective stages of life.
• Phase-contrast microscope components can be added to virtually any standard light microscope, provided the specialized phase objectives conform to the tube length parameters, and the condenser will accept an annular phase ring of the correct size. 

Conclusion

A phase-contrast microscope is all good for getting an illuminated image of the biological samples.  It is all good for observing the small microscopic organelles in full brightness. The thicker the sample, the more light gets diffracted. This ensures that the biological sample works best in the lab for performing the biological research.