Brightfield Microscope Definition
Brightfield Microscope (Compound Light Microscope). It is an optical microscope that uses light beams to generate a dark image against a sparkling background. Microbiological, cellular biology, as well as laboratory examinations of microorganisms, utilise the conventional microscope.
Utilizing fundamental stains that give contrast between the picture and the image background, this microscope is used to observe fixed and living specimens. It is specifically made with lenses, which are magnifying glasses, that change the specimen to create a picture seen via the eyepiece.
Principle of Brightfield Microscope
Under a brightfield microscope, a specimen must pass through a straight stream of light in order to be the focal point and generate an image. Differential absorption and differential refraction will provide a contrasting image through the microscope.
The specimens are first stained to add colour and facilitate easy contract characterization. The coloured specimens’ refractive indices will set them apart from their surroundings and provide a refractive contrast that combines absorption and refraction.
The operation of the microscope depends on its capacity to generate a high-quality, focused picture with high resolution from an acceptable light source.
A coverslip or oil immersion is used to observe the specimen that is mounted on a microscopic slide.
Parts of Brightfield Microscope
The brightfield microscope is made up of various parts, including
- Eyepiece (ocular) lenses:Two eyepiece (ocular) lenses are located at the top of the microscope to focus the picture produced by the objective lenses. Your eyes are the source from which you see the produced image.
- Objective lenses:The objective lenses, which include at least six glass lenses, produce a clean image of the specimen or object being focused.
- Two focusing knobs:On the microscope’s arm are two focusing knobs, the fine adjustment knob and the coarse adjustment knob, which may be used to move the stage or the nosepiece to concentrate on the picture. Their job is to make sure that a clear, crisp image is produced.
- The Stage:Just below the objectives is a stage where the specimen is placed. Using flexible knobs, the specimen may be moved about for better viewing, and this is also where the light is concentrated.
- The condenser:A light beam is focused on the specimen by the condenser, which is positioned below the stage. To change the light’s quality, it can be either fixed or mobile, although this solely relies on the microscope.
- The arm:The arm is the microscope’s durable iron backbone and is used to transport it from one location to another. Additionally, they support the base of the microscope, which serves as its stand. All the tiny components are contained in the base and the arm.
- It has a mirror or a light illuminator at the base or on the nosepiece of the microscope.
- About two to five distinct objective lenses with varying magnification powers are in the nosepiece. Depending on the objective lens being used to focus on the image, it can rotate to any position.
- An aperture diaphragmContrast: An aperture diaphragm regulates the size of the light beam that goes through the condenser. While the condenser is near to closing entirely, light enters the condenser’s core, producing strong contrast, and when it is almost completely open, the picture is extremely bright with very little contrast.
Magnification by Brightfield Microscope
- The primary lenses used to focus the picture on the condenser are the objective lenses. As a result, a clear picture is amplified and made larger. The eyepiece then magnifies the image once more to create the main image that is viewed by the eyes.
- The objective lenses stay parfocal throughout imaging, which means that even after switching lenses, the picture maintains focus. The virtual image, or expanded clear representation of the specimen, is what is visible via the eyepiece.
- The picture magnification is determined by the ratio of the objective’s magnification to the eyepiece lens’ magnification. The objectives of various types of brightfield microscopes contain magnification powers ranging from 40x to 1000x, while the eyepiece lens maintains a standard magnification power of 10x.
- Therefore, to calculate
- Total magnification power is equal to the sum of the magnifications of the objective and eyepiece lenses.
- For instance, if the eyepiece’s magnification is 10x and the objective’s is 45x, the specimen will be 450x magnified overall.
- Because the magnification is standard—that is, neither too high nor too low—it will vary between 40X and 100X depending on the lenses’ magnification capability.
- Resolution refers to how much the objective lens enlarges the viewable image. Prescott defined resolution as a lens’s capacity to differentiate or discriminate between tiny objects that are closely related to one another.
- Although the eyepiece magnifies the final picture, its magnification range is smaller than that of the objective lens, which has a range of 40X–100X and an 8X–12X (10X standard) magnification range. The objective lens is also largely responsible for the microscope’s resolution and magnification.
Applications of Brightfield microscope
Brightfield microscopes are employed in a variety of domains, including fundamental biology, cell biology, microbiology, bacteriology, and parasitology, where they are used for seeing parasitic organisms. The majority of the specimens that may be seen have been specially dyed to aid with vision. Negative staining and Gram staining are two of the staining methods employed.
Some of its applications include:
- Used to investigate and see animal cells.
- Used to examine and see plant cells.
- A tool for studying and observing the morphologies of bacterial cells.
- It is a term used to describe parasitic protozoans like Paramecium.
Advantages of the Brightfield Microscope
- It is easy to use and requires only a few tweaks to see the image.
- Both stained and unstained objects may be seen with it.
- The hue of the specimen is not changed by the microscope’s optics.
- For enhanced viewing, the microscope can be modified by adding a camera to form a digital microscope or by modifying the way the image is lighted, such as by applying fluorochromes to the specimen and observing in total darkness to produce a darkfield microscope.
Disadvantages of the Brightfield Microscope
- The iris diaphragm is preferable because the aperture diaphragm might produce excessive contrast, which could damage the final picture.
- The use of living specimens, such as bacterial cells, is not permitted. Brightfield microscopes can only be used to observe fixed specimens.
- The brightfield microscope’s maximum magnification is 100x, but with a few adjustments, the magnification can be changed to 1000x, which is the best magnification for seeing bacterial cells.
- Since there is little contrast, most specimens must be dyed in order to be seen.
- The picture may be distorted if oil immersion is used.
- The specimen might be harmed if a coverslip is used.
- The specimen may get contaminated or acquire extraneously undesirable features as a result of staining.
- The specimen must be tediously stained before being seen using a brightfield microscope.
- For magnification, the microscope requires a powerful light source, and occasionally, this light source may generate a lot of heat that might harm or destroy 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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