Brightfield Microscope Definition
Brightfield Microscope is also known as the Compound Light Microscope. It is an optical microscope that uses light rays to produce a dark image against a bright background. It is the standard microscope that is used in Biology, Cellular Biology, and Microbiological Laboratory studies.
This microscope is used to view fixed and live specimens, that have been stained with basic stains which gives a contrast between the image and the image background. It is specially designed with magnifying glasses known as lenses that modify the specimen to produce an image seen through the eyepiece.
Principle of Brightfield Microscope
For a specimen to be the focus and produce an image under the Brightfield Microscope, the specimen must pass through a uniform beam of the illuminating light. Through differential absorption and differential refraction, the microscope will produce a contrasting image.
The specimens used are prepared initially by staining to introduce color for easy contracting characterization. The colored specimens will have a refractive index that will differentiate it from the surrounding, presenting a combination of absorption and refractive contrast.
The functioning of the microscope is based on its ability to produce a high-resolution image from an adequately provided light source, focused on the image, producing a high-quality image.
The specimen which is placed on a microscopic slide is viewed under oil immersion or/and covered with a coverslip.
Parts of Brightfield Microscope
Figure: Parts of Brightfield Microscope (Compound Light Microscope). Image created using biorender.com
The brightfield microscope is made up of various parts, including
- Eyepiece (Ocular lens) – it has two eyepiece lenses at the top of the microscope which focuses the image from the objective lenses. this is where you see the formed image from, with your eyes.
- The objective lenses which are made up of six or more glass lenses, which make a clear image clear from the specimen or the object that is being focused.
- Two focusing knobs i.e the fine adjustment knob and the coarse adjustment knob, found on the microscopes’ arm, which can move the stage or the nosepiece to focus on the image. Their function is to ensure the production of a sharp image with clarity.
- The stage is found just below the objectives and this is where the specimen is placed, allowing movement of the specimen around for better viewing with the flexible knobs and it is where the light is focused on.
- The condenser: It is mounted below the stage which focuses a beam of light onto the specimen. It can be fixed or movable, to adjust the quality of light, but this entirely depends on the microscope.
- The arm: This is a sturdy metallic backbone of the microscope, used to carry and move the microscope from one place to another. They also hold the microscope base which is the stand of the microscope. The arm and the base hold all the microscopic parts.
- It has a light illuminator or a mirror found at the base or on the microscope’s nosepiece.
- The nosepiece has about two to five objective lenses with different magnifying power. It can move round to any position depending on the objective lens to focus on the image.
- An aperture diaphragm (contrast): It controls the diameter of the beam of light that passes through the condenser. When the condenser is almost closed, the light comes through to the center of the condenser creating high contrast and when the condenser is widely open, the image is very bright with very low contrast.
Magnification by Brightfield Microscope
- The objective lenses are the main lenses used for focusing the image, on the condenser. This produces an enlarged clear image that is then magnified again by the eyepiece to form the primary image that is seen by the eyes.
- During imaging, the objective lenses remain parfocal in that, even when the objective lens has changed the image still remains focused. The image seen at the eyepiece is the enlarged clear image of the specimen, known as the virtual image.
- The magnification of the image is determined by the magnification of the objective against the magnification of the eyepiece lens. The objectives have a magnification power of 40x-1000x depending on the type of brightfield microscope while the eyepiece lens has a standard magnification power of 10x.
- Therefore to calculate:
Total Magnification power = Magnification of the objective lens x Magnification of the eyepiece
- For example: if the magnification of the objective is 45x and that of the eyepiece is 10x, the total magnification of the specimen will be 450x.
- The magnification is standard, i.e not too high nor too low, and therefore depending on the magnification power of the lenses, it will range between 40X and 100oX.
- The objective lens enlarges the image which can be viewed, a characteristic known as resolution. Resolution according to Prescott, is the ability of a lens to separate or distinguish between small objects closely linked together.
- Whereas the eyepiece magnifies the image at the end of the viewing, its magnification range is lower than that of the objective lens at 8X-12X (10X standard) and that of the objective lens at 40X-100X, magnification, and resolution of the microscope is highly dependant on the objective lens.
Applications of Brightfield microscope
Brightfield Microscope is used in several fields, from basic biology to understanding cell structures in cell Biology, Microbiology, Bacteriology to visualizing parasitic organisms in Parasitology.
Most of the specimens to viewed are stained using special staining to enable visualization. Some of the staining techniques used include Negative staining and Gram staining.
Some of its applications include:
- Used to visualize and study the animal cells
- Used to visualize and study plant cells.
- Used to visualize and study the morphologies of bacterial cells
- Used to identify parasitic protozoans such as Paramecium.
Advantages of Brightfield Microscope
- It is simple to use with few adjustments involved while viewing the image.
- It can be used to view both stained and unstained.
- The optics of the microscope do not alter the color of the specimen.
- The microscope can be adjusted and modified for better viewing such as installing a camera, to form a digital microscope or in the way image illumination is done such as by use of fluorochromes on the specimen and viewing under a dark environment, forming a darkfield microscope.
- The aperture diaphragm may cause great contrast which may distort the outcome of the image, therefore iris diaphragm is preferred.
- It can not be used to view live specimens such as bacterial cells. Only fixed specimens can be viewed under the brightfield microscope.
- Maximum magnification of the brightfield microscope is 100x but modification can readjust the magnification to 1000x which is the optimum magnification of bacterial cells.
- It has low contrast hence most specimens must be stained for them to be visualized.
- Use of oil immersion may distort the image
- The use of coverslip may damage the specimen
- Staining may introduce extraneously unwanted details into the specimen or contaminate the specimen.
- It is tedious to stain the specimen before visualizing it under the brightfield microscope.
- The microscope needs a strong light source for magnification and sometimes the light source may produce a lot of heat which may damage or kill the specimen.
Reference and source
- Willey, J. M., Sherwood, L., & Woolverton, C. Prescott’s Microbiology. New York: McGraw-Hill (Page# 19-22).
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