Brightfield Microscope (Compound Light Microscope)- Definition, Principle, Parts
A microscope is an instrument that magnifies small objects that are invisible to the naked eye, such as cells, bacteria, and viruses. There are different types of microscopes based on the source and nature of illumination, the magnification power, and the resolution of the image. One of the most common and simple types of microscopes is the brightfield microscope, also known as the compound light microscope.
A brightfield microscope 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.
The brightfield microscope works on the simple principle of absorption of light by the specimen. The specimen is placed on a stage and illuminated by a light source from below. The light passes through a condenser lens, which focuses the light beam onto the specimen. The specimen absorbs some of the light and reflects or transmits the rest. The objective lens collects the light that emerges from the specimen and magnifies the image. The ocular lens or the eyepiece further magnifies the image and allows it to be seen by the observer or captured by a camera.
The image produced by a brightfield microscope is a dark specimen against a bright background, hence the name. The contrast in the image depends on the difference in light absorption between the specimen and the surrounding medium. Most biological specimens are transparent or translucent and do not absorb much light. Therefore, staining is often required to increase contrast and make the specimen visible. Staining introduces color and chemical specificity to the specimen, allowing different structures or molecules to be distinguished.
The brightfield microscope is based on its ability to produce a high-resolution image from an adequate light source. Resolution is the ability of a lens to separate or distinguish between small objects that are close together. The resolution of a brightfield microscope is limited by the wavelength of visible light, which is about 400-700 nanometers (nm). The maximum resolution of a brightfield microscope is about 0.2 micrometers (μm), which means that two objects closer than 0.2 μm cannot be resolved as separate entities.
The magnification of a brightfield microscope is determined by the product of the magnification of the objective lens and the magnification of the ocular lens. For example, if the objective lens has a magnification of 40x and the ocular lens has a magnification of 10x, then the total magnification is 40x10 = 400x. The magnification of a brightfield microscope can range from 40x to 1000x, depending on the type and number of lenses used. However, magnification alone does not improve resolution; it only makes the image larger. To achieve higher resolution, shorter wavelengths of light or different types of microscopy are needed.
The brightfield microscope is made up of various parts that work together to produce a magnified image of the specimen. Figure 1 shows a diagram of a typical brightfield microscope and its main components.
Figure 1: Parts of Brightfield Microscope (Compound Light Microscope). Image created using biorender.com
The main parts of a brightfield microscope are:
- Eyepiece (Ocular lens): It is the lens that is closest to the eye of the observer. It usually has a magnification power of 10x and contains a pointer or a scale to measure the size of the specimen. Some microscopes have two eyepieces (binocular) while others have one (monocular).
- Objective lens: It is the lens that is closest to the specimen on the stage. It collects and focuses the light rays from the specimen and forms a real image that is magnified by the eyepiece. There are usually three or four objective lenses on a rotating nosepiece, each with different magnification powers ranging from 4x to 100x. The higher the magnification, the smaller the field of view and the lower the resolution.
- Focusing knobs: They are used to adjust the distance between the objective lens and the specimen to obtain a clear and sharp image. There are two types of focusing knobs: coarse adjustment knob and fine adjustment knob. The coarse adjustment knob moves the stage or the nosepiece up or down in large increments, while the fine adjustment knob moves them in small increments. The coarse adjustment knob should only be used with low-power objective lenses to avoid damaging the specimen or the lens.
- Stage: It is a flat platform that holds the specimen on a glass slide. It has clips or clamps to secure the slide in place and prevent it from moving. It also has knobs or screws that allow horizontal movement of the slide for scanning different areas of the specimen.
- Condenser: It is a lens system that is located below the stage. It collects and concentrates the light rays from the light source and directs them onto the specimen. It can be fixed or movable, depending on the type of microscope. It also has an iris diaphragm that controls the amount and angle of light entering the objective lens, affecting the contrast and resolution of the image.
- Light source: It is an electric lamp or a mirror that provides illumination for viewing the specimen. The brightness and intensity of the light can be adjusted by a rheostat or a switch. The light source should be aligned with the condenser and the objective lens for optimal image quality.
- Arm: It is a sturdy metal structure that supports and connects the upper parts of the microscope (eyepiece, objective lens, nosepiece) with the lower parts (stage, condenser, light source). It also serves as a handle for carrying and moving the microscope safely.
- Base: It is a flat and heavy part that supports and stabilizes the microscope on a table or a bench. It also contains some electrical components such as wires, switches, and batteries for powering the light source.
These are some of the basic parts of a brightfield microscope that are essential for its functioning and operation. However, some microscopes may have additional or modified parts depending on their design and purpose.
The brightfield microscope uses two sets of lenses to magnify the image of the specimen: the objective lens and the eyepiece lens. The objective lens is mounted on a rotating nosepiece and collects light from the specimen to form a magnified real image. The eyepiece lens is located at the top of the microscope and further magnifies the real image to form a virtual image that can be seen by the eye or captured by a camera.
The total magnification of the brightfield microscope is calculated by multiplying the magnification of the objective lens by the magnification of the eyepiece lens. For example, if the objective lens has a magnification of 40x and the eyepiece lens has a magnification of 10x, then the total magnification is 40x * 10x = 400x. This means that the specimen appears 400 times larger than its actual size.
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, the maximum total magnification that can be achieved by a brightfield microscope is 1000x * 10x = 10,000x. However, this does not mean that the image quality is improved by increasing the magnification. The resolution of the microscope, which is the ability to distinguish between two closely spaced points, depends on other factors such as the wavelength of light, the numerical aperture of the lens, and the quality of the optics.
The objective lens also determines the amount of light that reaches the eyepiece lens. The numerical aperture (NA) of the objective lens is a measure of its light-gathering ability and is related to its diameter and focal length. The higher the NA, the more light is collected and the better the resolution. However, higher NA also means smaller working distance (the distance between the front surface of the lens and the specimen) and smaller depth of field (the thickness of the specimen that is in focus). Therefore, there is a trade-off between resolution and ease of use.
The brightfield microscope produces low contrast images with most biological specimens, as few absorb light to a great extent. Staining is often required to increase contrast, which prevents use on live cells in many situations. Brightfield illumination is useful for samples that have an intrinsic color, such as chloroplasts in plant cells or blood cells. However, for transparent or colorless specimens, other techniques such as phase contrast or differential interference contrast may be more suitable.
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 be viewed are stained using special staining techniques to enable visualization. Some of the staining techniques used include negative staining and Gram staining. Some of its applications include:
- Used to observe, analyze, and study plant cells
- Used to view, magnify, and study animal cells
- Used to clearly study the morphologies of bacterial and viral organisms
- Also used in the study of parasites like paramecium
- It finds use in agricultural laboratories to study soil samples and microorganisms in the soil
- Used to examine blood smears and urine samples for medical diagnosis
- Used to observe the effects of drugs or chemicals on cells or tissues
Brightfield microscope is a simple and widely used technique that has many advantages for various fields of microscopy. Some of the advantages are:
- It is inexpensive and easy to use with few adjustments involved while viewing the image.
- It is found in nearly every lab, perfect for research or clinical work.
- It is great for histology, tissue culture, cell biology, microbiology and other disciplines that require observation of stained or unstained specimens.
- It can produce high-resolution images with a maximum magnification of 1000x using oil immersion objective.
- The optics of the microscope do not alter the color of the specimen, allowing for an accurate analysis.
- It can be modified and enhanced by using different staining techniques, polarizing filters, darkfield or phase contrast illumination, or digital cameras.
Despite its simplicity and versatility, brightfield microscopy also has some limitations and drawbacks. Some of the disadvantages of brightfield microscopy are:
- It has low contrast, especially for transparent or colorless specimens. Most biological samples require staining to enhance the contrast and visibility of their structures. However, staining can introduce artifacts, alter the natural state of the specimens, or even kill them if they are alive.
- It cannot be used to observe live specimens such as bacterial cells, which are too small and transparent to be seen under brightfield illumination. Only fixed specimens can be viewed under the brightfield microscope.
- It has a limited resolution, which depends on the wavelength of light and the numerical aperture of the objective lens. The maximum resolution of a brightfield microscope is about 0.2 micrometers, which means that structures smaller than that cannot be distinguished.
- It requires a strong light source for high magnification, which can produce heat and damage the specimen or cause photobleaching. The light source also needs to be adjusted carefully to avoid overexposure or underexposure of the image.
- It uses an aperture diaphragm to control the contrast, but this can also introduce distortion and aberration to the image. An iris diaphragm is preferred for better control of contrast and quality of the image.
- It needs oil immersion for high magnification, which can be messy and inconvenient. Oil immersion can also distort the image or damage the objective lens if not used properly.
- It needs a coverslip for thin specimens, which can affect the focus and alignment of the image. A coverslip can also damage or compress the specimen if not applied carefully.
These disadvantages limit the applicability and usefulness of brightfield microscopy for some types of specimens and research purposes. Therefore, other types of microscopy techniques, such as phase contrast, darkfield, fluorescence, or electron microscopy, may be more suitable for certain situations.
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