Bright Field Microscope: Definition, Parts, Working Principle, Application.

Bright Field Microscope Definition

A brightfield microscope is a type of microscope that uses visible light and an objective lens to magnify and illuminate a sample. It is called a “brightfield” microscope because the sample is illuminated from below and appears bright against a dark background. Brightfield microscopes are commonly used in biology and medicine to observe cells, tissues, and other biological samples.

To use a brightfield microscope, the sample is placed on a transparent glass stage and illuminated from below by a light source, such as a bulb or LED lamp. The light passes through the sample and is focused onto the objective lenses by a condenser lens. The objective lenses magnify the image and the eyepieces, also known as ocular lenses, further magnify the image for the observer.

Brightfield microscopes are widely used in research, education, and other fields because they are relatively simple and inexpensive. However, they have some limitations, such as the inability to distinguish between different types of molecules or structures that are the same color. Other types of microscopes, such as fluorescence microscopes or electron microscopes, may be used to overcome these limitations.

Principle of Brightfield Microscope

The principle of a brightfield microscope is based on the use of visible light and an objective lens to magnify and illuminate a sample. When a sample is placed on the stage of a brightfield microscope and illuminated from below, the light passes through the sample and is focused onto the objective lenses by a condenser lens. The objective lenses magnify the image and the eyepieces, also known as ocular lenses, further magnify the image for the observer.

The sample appears bright against a dark background, hence the name “brightfield” microscope. This contrast is created by the difference in light intensity between the illuminated sample and the surrounding area. The contrast can be adjusted by using a diaphragm, which is an adjustable aperture located below the condenser that controls the amount of light that reaches the sample.

In order for a specimen to be the focal point and generate an image under a Brightfield Microscope, it must travel through a uniform beam of the illuminating light. The microscope will produce a contrasted image by differential absorption and differential refraction.

The specimens are initially stained to introduce colour for straightforward contracting characterisation. The coloured specimens will have a refractive index that distinguishes them from their surroundings through both absorption and refractive contrast.

The operation of the microscope is dependent on its ability to produce a high-resolution image from an appropriately supplied light source that is focussed on the image, thereby providing an image of high quality.

The item is seen under oil immersion or with a coverslip after being placed on a microscope slide.

Light Path of Brightfield Microscope

• A stained specimen or sample is placed over the specimen stage.
• A condenser lens containing an aperture diaphragm is located under the stage, will focus the light ray on the sample or specimen.
• The light rays will pass through the specimen sample and then it will be collected by an objective lens located over the stage.
• The objective lens will form a magnified image of the specimen and then transmit it to the eyepiece, where viewers will observe a dark image against the brightfield.
• During the transmission through the specimen, some of the light rays are absorbed by the stains, pigmentation, or dense areas of the specimen.

Types of Bright Field Microscope

Brightfield microscopy is a microscopy technique that uses a bright background to create contrast between the sample and the background, making it easier to see the sample. There are several types of brightfield microscopes that are commonly used, including the following:

1. Compound microscope: A compound microscope is a type of brightfield microscope that uses multiple lenses to magnify the image of the sample. It consists of two sets of lenses: an objective lens that is located close to the sample and a eyepiece lens that is located near the viewer. Compound microscopes are often used to study small samples such as cells, tissues, and microorganisms.
2. Stereo microscope: A stereo microscope is a type of brightfield microscope that uses two eyepieces to provide a three-dimensional image of the sample. It is often used to study large or three-dimensional specimens, such as plants or insects, or to perform tasks that require fine manipulation, such as dissection or microsurgery.
3. Inverted microscope: An inverted microscope is a type of brightfield microscope that is used to study samples that are mounted on a slide or other flat surface. The sample is placed on the stage of the microscope and viewed from below, rather than from above as in a regular microscope. Inverted microscopes are often used to study cells or tissues that are grown in culture or to study large samples that cannot be easily mounted on a slide.
4. Polarizing microscope: A polarizing microscope is a type of brightfield microscope that uses polarizing filters to study the optical properties of a sample. It is often used to study minerals, crystals, and other materials that have birefringence, or the ability to change the polarization of light as it passes through the sample.

Overall, brightfield microscopy is a widely used technique for studying small or transparent specimens, and there are several types of brightfield microscopes that are used for different applications. Each type of brightfield microscope is designed to meet the specific needs of the researcher and the sample being studied.

Bright field Microscope parts

A brightfield microscope is a type of microscope that uses visible light and an objective lens to magnify and illuminate a sample. The main parts of a brightfield microscope include:

1. Head: The top portion of brightfield microscope is known as the head.
2. Arm: The holding portion of the microscope is known as Arm. Which we used to carry the microscope from one place to another. This portion provides support to the both optical and mechanical components.
3. Body tube: A long tube connected the both ocular lens and objective lens, called as body tube.
4. Objective lenses: These lenses are located at the bottom of the microscope and are used to magnify the sample. Brightfield microscopes usually have multiple objective lenses with different magnification levels.
5. Stage: The stage is a platform located below the objective lenses where the sample is placed. It usually has a transparent glass bottom to allow light to pass through the sample.
6. Condenser: The condenser is a lens located below the stage that focuses light onto the sample. It helps to illuminate the sample and increase the contrast of the image.
7. Diaphragm: The diaphragm is a adjustable aperture located below the condenser that controls the amount of light that reaches the sample. It helps to fine-tune the contrast of the image.
8. Eyepieces: The eyepieces, also known as ocular lenses, are located at the top of the microscope and are used to magnify the image formed by the objective lenses.
9. Stage clips: The stage clips are located over the specimen stage. It holds the specimen slide.
10. Focus knobs: The focus knobs are used to adjust the distance between the objective lenses and the stage to bring the sample into focus. Bright-fields microscope also contains two focusing knobs, called fine adjustment knob and the coarse adjustment knob. These knobs help to adjust the focus of objective lens.
11. Illuminator: The illuminator is a light source located below the stage that provides the necessary light for the sample. It can be a bulb or a LED lamp.
12. Nosepiece: The nosepiece is a rotating mechanism that holds the objective lenses and allows the user to switch between different magnification levels.
13. Base: The base is the bottom part of the microscope that provides support and stability. It also houses the illuminator and the electrical components of the microscope.
14. Stage knob: This microscope also contains a stage knob, which controls the stage.

Bright field Microscope Magnification

The magnification of a brightfield microscope is determined by the combination of the objective lenses and the eyepieces. The objective lenses are located at the bottom of the microscope and are used to magnify the sample. Brightfield microscopes usually have multiple objective lenses with different magnification levels, such as 4x, 10x, 40x, and 100x. The eyepieces, also known as ocular lenses, are located at the top of the microscope and are used to further magnify the image formed by the objective lenses.

Total Magnification power = Magnification of the objective lens x Magnification of the eyepiece

To calculate the total magnification of a brightfield microscope, the magnification of the objective lens and the eyepiece must be multiplied together. For example, if a microscope has a 10x objective lens and a 10x eyepiece, the total magnification would be 10x * 10x = 100x.

It is important to note that the magnification of a microscope is not the only factor that determines the resolution of the image. The quality of the optics, the wavelength of the light used, and the sample itself also affect the resolution of the image. A microscope with a high magnification but poor optics or a low-quality sample may produce a blurry or low-contrast image.

Application of Bright-Field Microscope

Brightfield microscopes are widely used in a variety of applications, including:

1. Biology and medicine: Brightfield microscopes are commonly used in biology and medicine to observe cells, tissues, and other biological samples. They are used to study the structure and function of cells, tissues, and organisms, as well as to identify and classify different types of cells or tissues.
   • Used in blood counting.
   • Used to examine the bacterial cells.
   • Used to examine the fungal cells.
   • It also used in forensic laboratories.
   • Used in agricultural laboratories.
   • Used to study plant cells.
2. Pharmaceutical industry: Brightfield microscopes are used in the pharmaceutical industry to analyze the purity and quality of drugs. They can be used to identify contaminants or impurities in drug samples and to ensure that the drugs meet quality standards.
3. Food industry: Brightfield microscopes are used in the food industry to detect contaminants or defects in food products. They can be used to inspect food products for bacteria, fungi, or other contaminants that may pose a health risk.
4. Materials science: Brightfield microscopes are used in materials science to study the structure and properties of materials. They can be used to examine the microstructure of metals, ceramics, polymers, and other materials.
5. Geology: Brightfield microscopes are used in geology to study rocks and minerals. They can be used to identify different types of minerals and to examine the microstructure of rocks.
6. Forensic science: Brightfield microscopes are used in forensic science to analyze evidence from crime scenes. They can be used to identify fibers, hairs, and other small particles that may be used as evidence in criminal investigations.
7. Education: Brightfield microscopes are widely used in education to introduce students to the principles of microscopy and the structure and function of cells and tissues. They are relatively simple and inexpensive, making them a popular choice for educational purposes.

Advantages of bright field microscope

1. Simplicity: Bright-field microscopes are relatively simple to use and operate, making them a popular choice for many educational and research settings.
2. Wide availability: Bright-field microscopes are widely available and can be found in many research and educational labs, making them a convenient choice for many scientists and students.
3. Good for observing detail: Bright-field microscopes can produce high-resolution images and are good for observing fine details in specimens.
4. Versatility: Bright-field microscopes can be used to view a wide variety of specimens, including biological samples, minerals, and other materials. It is capable of displaying both stained and unstained images.
5. Low cost: Bright-field microscopes are generally less expensive than other types of microscopes, such as confocal microscopes or electron microscopes.
6. Easy to modify: The brightness and contrast of the image can be easily adjusted using the light source and condenser, making it easy to optimize the image for different samples. The microscope can be altered and modified for improved viewing, such as by inserting a camera to create a digital microscope or by using fluorochromes on the specimen and observing in a dark environment to create a darkfield microscope.
7. Color Change: The optics of the microscope have no effect on the specimen’s hue.

Disadvantages of Bright-Field Microscope

1. Limited contrast: Bright-field microscopy relies on the absorption of light by the sample, so it is not very effective for observing samples that do not absorb light well or that have similar refractive indices to the surrounding medium. This can make it difficult to distinguish between different structures within the sample.
2. Poor resolution: Bright-field microscopy has a relatively low resolution compared to other types of microscopy, such as phase contrast microscopy or electron microscopy. This means that it may not be possible to see very small structures or details within the sample.
3. Limited sample preparation: Bright-field microscopy requires samples to be thinly sectioned and mounted on a microscope slide, which can be time-consuming and may not be suitable for all types of samples.
4. Limited visualization of fluorescent or transparent samples: Bright-field microscopy is not well-suited for observing fluorescent or transparent samples, as it relies on the absorption of light by the sample. For these types of samples, other techniques, such as fluorescence microscopy or differential interference contrast microscopy, may be more appropriate.
5. Limited depth of field: Bright-field microscopy has a limited depth of field, which means that objects at different depths within the sample may be out of focus. This can make it difficult to get a clear image of samples with multiple layers or structures at different depths.
6. Aperture diaphragm: Therefore, the iris diaphragm is recommended over the aperture diaphragm, which may generate excessive contrast that distorts the image.
7. Living Sample: It cannot be used to observe living organisms such as bacterial cells. Under the brightfield microscope, only fixed specimens may be observed.
8. Magnification Power: The brightfield microscope’s maximum magnification is 100x, but it can be adjusted to 1000x, which is the optimal magnification for viewing bacterial cells.
9. Other Disadvantages:
   • Due to its poor contrast, the majority of specimens must be dyed to be visible.
   • The use of oil immersion may cause visual distortion.
   • Using a coverslip may harm the specimen.
   • Staining may introduce undesirable details or contaminate the specimen.
   • Staining the specimen prior to viewing it under a brightfield microscope is laborious.
   • For magnification, the microscope requires a powerful light source, which can produce excessive heat that can harm or kill the specimen.

Enhancements of Bright-Field Microscope

• The iris diaphragm reduces or increases the quantity of the light source.
• Employing an oil-immersion objective lens and covering the specimen with a glass cover containing a particular immersion oil. Immersion oil has the same refractive index as glass and improves the observed specimen’s resolution.
• Use of staining techniques for microbiological samples, such as simple stains (methylene blue, safranin, crystal violet) and differential stains (negative stains, flagellar stains, endospore stains).
• Use a coloured (often blue) or polarising filter on the light source to highlight elements that are not visible in white light. Filters are very beneficial for mineral samples.

Difference between dark field and bright field Microscope

Dark-field microscopy and bright-field microscopy are two different techniques used to visualize samples under a microscope.

In bright-field microscopy, light is shone directly onto the sample, and the image is formed by the light that is transmitted or scattered through the sample. This technique is best suited for observing samples that absorb light well, such as tissues or cells that have been stained with a dye.

In contrast, dark-field microscopy uses a special condenser and objective lens to scatter light around the sample, creating a bright image against a dark background. This technique is best suited for observing samples that are difficult to visualize using bright-field microscopy, such as transparent or low-contrast samples.

One key difference between these two techniques is the way that they create an image of the sample. Bright-field microscopy creates an image by transmitting or scattering light through the sample, while dark-field microscopy creates an image by scattering light around the sample. This means that dark-field microscopy is better suited for observing transparent or low-contrast samples, while bright-field microscopy is better suited for observing samples that absorb light well.

FAQ

Bright Field microscope image

References

• https://www.microscopemaster.com/brightfield-microscopy.html
• https://en.wikipedia.org/wiki/Bright-field_microscopy
• https://www.med.unc.edu/microscopy/files/2018/06/lm-ch-8-bright-field.pdf
• https://www2.hawaii.edu/~johnb/micro/m140/syllabus/week/handouts/m140.2.4.html
• https://www.rsc.org/publishing/journals/prospect/ontology.asp?id=CMO:0002529&MSID=C2LC21116E
• https://science.umd.edu/CBMG/faculty/wolniak/wolniakmicro.html