What Is Contrast In Microscope

In the scientific world, microscopy techniques are of great importance. Without using any microscopy technique, detailed observations can be made.   The microscopes are used in the technical world, to view and study objects and biological specimens using the source of light. There are different types of microscopes containing different modes of illumination or contrast.  In all such microscopes, the researcher or scientist can adjust the focus and concentrate the whole of the light on the surface of the biological specimen.  The contrast in microscopes refers to the darkness of the background relative to the specimen placed on the stage.  In recent times, microscopy techniques are becoming advanced and have been stacked up with all the latest advancements to very minutely observe transparent biological specimens. 

Optical microscopy with phase contrast features is a well-known name of today’s microscopy techniques. Many optical microscopy techniques are based on their respective contrast feature that helps in viewing the samples with full details and structure. It is seen that the lighter biological samples are easier to view on the darker backgrounds than the heavy specimens.  To see colorless or transparent samples, the researcher or scientist needs a specialized microscope, which can easily help in viewing objects. In this context, phase-contrast microscopy techniques are used.  Electron microscopy techniques are also useful in viewing the transparent phase objects through the execution of optical pathways, like reflection, diffraction, refraction of light rays from the light source. Electromagnetic radiation is also used for producing contrast-rich images of the respective samples.  Here, the beams of light scatter over the surface of the biological specimen and then again collect to create a contrasted image . 

This process of image creation can also be used by other microscopy techniques.  Some  other optical microscopy techniques that manipulate the optical light rays or beams  are as follows:

• Brightfield Microscopy
• Darkfield Microscopy
• Transmission Electron Microscopy
• Scanning Electron Microscopy
• Confocal Laser Scanning Microscopy

In all these above microscopy techniques,  the optical light beams scatter and collect at one point near the objective lens to form the images. The different refractive indexes of the specimens and the incident light help in the process of image formation. In the scanning probe microscopy technique,  the interaction of the probe and surface of the microscope’s stage leads to the generation of contrasted images. This advent of scanning probes has revolutionized microscopy techniques in the biological world as well as in physical sciences.

Contrast In Microscopes

For viewing  all types of biological specimens,  the microscope should be able to produce a distinct contrast. This contrast enables the observation of transparent specimens and structures very well. Such microscopes have the feature of adjusting the contrast to enable good observations. Contrast refers to the relatively darker background concerning the biological surface of the specimen.   When a biological specimen is mounted on the stage,  the light rays scatter over the surface of the biological specimen to view the samples.   All internal structures are well defined in the observations in the contrast microscopes. Such microscopes are also called cell culture microscopes, culture microscopes,  and tissue culture microscopes. 

Types Of Microscopes For Contrasting Image Formation

Generally, there are two types of microscopes  that produce the contrast microscope :

• Bright-field Microscopes
• Phase contrast microscopes

Brightfield microscopes aim the light rays up through a condenser from beneath the pointing stage.   All light rays from the light source bend through the underside of the biological specimen. The lens and eyepiece reflect the light rays before reaching the viewer’s eye.  At the eyepiece, the light rays get magnified and as a result,  the magnified image is produced.

In phase-contrast microscopy, it works by shifting light waves or interfered waves to change the contrast.  Light rays pass through both the centers of the lens and the sides of the phase-contrast microscope.   Here, the light rays on the sides hit a phase plate in the inverted phase-contrast microscope.  It delays the part of the light wave that generates the high-contrasted images. Further, the light waves are manipulated to reduce the excessive brightness of the phase objects. while viewing the images,  the researchers can easily adjust the contrast of the generated images in the phase-contrast microscopy. 

Uses Of Phase Contrast Microscopy Techniques

Since its discovery,  the phase contrast microscope has been used to illuminate details of all living cells and other transparent micro-organisms.  In this microscopy technique,  little contrast is seen between the organelles within the living cells. This makes the view of the cell’s structure a little difficult.  And for this, the brightfield microscope is used.  But still, a phase contrast microscope helps in illuminating the whole structure of cells. A phase contrast microscope shows more dramatic differences in the contrast  making all these organelles easily viewable using the inverted phase microscope.

Steps To Adjust The Contrast With A Phase Contrast Microscope

 Following steps are required to follow with a  phase contrast microscope to generate contrasted images :

• The condenser turrets should be rotated in a very subtle manner to get access to all light rays.
• Rotate the eyepiece while looking through it.  Stop rotating the condenser, when the images generated are sharp and bright.
• Adjust the focus of the microscope to a good level for imaging the specimen.
• Use a light source of good electromagnetic radiation for absorption of light rays.
• Use the un-labeled biological specimens for the observations process to get a better view of the samples.

Steps To Adjusting Contrast In A Brightfield Microscope

Following are the steps required to adjust the contrast in a brightfield microscope :

• Move the condenser of the microscope in such a way that it becomes close to the stage itself.
• Close the aperture in the microscope in the whole process of magnification in the brightfield microscopy.
• Look at the observations through the eyepiece and check the contrast as needed.  You can also adjust the contrast as per your choice in the brightfield microscope. Meanwhile, continue viewing the specimen from the eyepiece for image generation.
• Stop viewing the images when it appears bright and clear. If the image of the concerned biological specimen appears distorted then open up the aperture and readjust the focus for a clear image.

Some Limitations Of Brightfield Microscope

Despite its ability to form contrast images, the brightfield microscopes also have some sort of limitations.  This type of inverted microscope could not adjust the contrast beyond the ideal viewing point. The sample object appears distorted after a threshold region. This does not happen.  However, the brightfield microscopy technique is included in the best trinocular microscope for examining the biological specimens with good angle and contrast.

Some fluorescent microscopes also work in a good way by using Ultraviolet rays to form high contrasted images. These are also called confocal microscopes which excite an electron from the UV light source.  Here, the electrons hit the biological specimen’s surface that illuminates the images.  The formed images can be seen through confocal dissecting scopes. This all increases the formation of contrasted images in the microcopy techniques.

Conclusion

All microscopy techniques are well structured to generate high contrasted images of the biological specimens.  They have different sources of light, but the image formation is of high contrast.  Phase contrast microscopes are specially developed to form high contrasted images of the transparent biological sample, while the brightfield microscopes are useful in generating images in good contrast upto a threshold level. You can check the price of a trinocular microscope in offline as well as online medium.