History of Microscopy: Light Microscope Overview
- The development of the discipline of microbiology brought into focus the significance of the identification, seeing, observation, and comprehension of microbes, including their structural morphologies and processes. The study of tiny creatures and agents that can only be seen and investigated under a microscope falls within the purview of microbiology.
- Despite the fact that the earliest basic microscope was created by two Dutch scientists, Zaccharias Janssen and his father, Hans, who produced eyeglasses, was the first one to research with their lenses by merging them in a tube. They observed that adjacent things seemed closer and bigger. This action set the groundwork for scientific evolution while not being considered a scientific discovery.
- According to the History of Microbiology, an amateur microbiologist named Antony Van Lewnehoueek created the initial straightforward microscope, which allowed him to see the existence of microscopic, dot-shaped organisms inhabiting pond water. His basic microscope consisted of two silver plates holding a double convex glass lens in place.
- By enlarging the pictures of the cells and microorganisms, the use of microscopy in microbiology improved the viewing of these objects.
An optical microscope is another name for a light microscope.
What is a light microscope?
- A light microscope is a device or instrument used in biology laboratories that uses visible light to locate, magnify, and expand micro objects.
- Using lenses, they focus light on the specimen and magnify it to generate a photograph. Typically, the specimen is positioned close to the microscopic lens.
- The kinds and quantity of lenses that make up the microscope have a significant impact on the microscopic magnification.There were two types of microscopes: Simple light microscopes (with restricted magnification due to its single lens) and Compound light microscopes (that have several lenses) (A compound microscope has a greater magnification than a basic microscope since it utilises at minimum two pairs of lenses, an objective lens and an eyepiece). The lenses are placed in such a way that they may deflect light to magnify images effectively.
- The ability of the light microscope to create an image by concentrating a ray of light through a small, clear object is fundamental to its operation. The picture is then magnified for viewing via one or more lenses. The specimen’s transparency makes it simple and fast for light to enter. Bacteria, cells, and other microbial particles may all be included in specimens.
Principles of a light microscope (optical microscope)
As previously stated, light microscopes utilise a glass lens to view an image, and magnification is determined by the lens’s ability to curve light and focus it over the specimen to generate an image. A light beam bends at the interface as it travels from one medium into another, generating refraction. How light bends is determined by the index of refraction, a measurement of how much a substance slows down the speed of light. The direction and magnitude of light curving are determined by the indices of refraction of the two media composing the contact.
While light is transmitted via a material having a higher index of refraction, like air to glass, it normally slows down and curves perpendicular to the surface. A material having a lower refractive index, like glass compared to air, often accelerates the penetration of light and causes it to bend far from the usual.
In a prism, in this scenario, light will bend at an angle if put between these two substances, or between water and air. In order to work, tiny lenses must bend light at an angle. When light rays enter the convex lens, they are focused at the focal point, which is a particular point (F-point). Focal length is the distance between the centre of the lens and its focal spot.
Since the power of a lens is exactly proportional to its focal length, shorter focal lengths magnify more than longer focal lengths.—a microscope utilises lenses with fixed strengths.
The only determining element in microscopy is resolution. This refers to a lens’s ability to discern between closely packed, microscopic objects. Resolution is determined by the numerical aperture of a light microscope’s lens system and also the frequency of the light it employs; numerical openness is a specification of the light wavelengths created while the specimen is illuminated.
The Abbe equation, which uses the wavelength of the light that illuminated the specimen (Lambda, λ) and the numerical aperture (NA, n sin Ɵ) can be used to calculate the minimum distance (d) between two objects that distinguishes them as two separate entities and is determined by the wavelengths of the light, i.e. d=0.5 λ/n sin Ɵ
Types of light microscopes (optical microscope)
The microscopes have progressed along with the science of microbiology.
Simple and compound light microscopes both employ lenses to observe specimens. The contrast is that, whereas compound lenses use two or more lenses for magnification, single-lens lenses do not. Basic light microscopes only use one lens for that purpose. This suggests that a sequence of lenses is positioned so that the last lens enhances the image as much as the first lens.
The modern types of light microscopes include:
- Bright Field Light Microscope
- Phase-Contrast Light Microscope
- Dark-Field Light Microscope
- Fluorescence Light Microscope
Brightfield Light Microscope (Compound light microscope)
The simplest optical microscope, which creates a dark picture on a light backdrop, is employed in microbiology labs. Following simple staining, it is often employed to observe plant and animal cell organelles, along with some parasites such as Paramecium. It is composed of two lenses. Its ability to provide a high-resolution picture is the foundation of its functioning, which heavily relies on the right handling of the microscope. This indicates that enough light will allow for effective picture focusing, resulting in high-quality images. An alternative name for it is a compound light microscope.
Parts of a bright-field microscope (compound light microscope)
It is composed of:
- There are two lenses; the eyepiece or ocular lens and the objective lens.
- The objective lens, which has six or more glasses, creates a clean picture of the object.
- A light beam is directed towards the specimen by the condenser, which is positioned below the stage. To change the light’s quality, it may be either fixed or mobile, although this solely relies on the microscope.
- The base of the microscope has a robust metallic curving back that serves as both a support and an arm to hold them all together. All of the components of the microscope are held by the arm and base.
- The stage on which the specimen is set up is where the light is concentrated. Thanks to the adjustable knobs, the specimen may be moved for optimum viewing.
- On the arm of the microscope are two focusing knobs; the finest adjustment knob and also the coarse adjustment knob, that can be used to focus the image by moving the stage or nosepiece. The visual acuity is improved.
- It has a light source or mirror at the bottom or on the microbes of the nosepiece.
- Between three and five objective lenses with varying magnification powers are located in the nosepiece. It may spin in any position, based on the objective lens used to focus the image.
- The diameter of the beam of light that passes via the condenser is regulated by an opening diaphragm, also known as the contrast, as light enters the condenser’s core when the condenser is almost shut, producing great contrast. However, the picture is very brilliant and has very little contrast when the condenser is widely open.
Magnification by Bright Field Microscope (Compound light microscope)
When the objective lens is parfocal during visualisation, when the objective lens is changed, the image remains in focus. An essential purpose of the objective lens is to focus the image onto the condenser, thereby producing a magnified, clear picture inside the microscope, which will then be amplified by the eyepiece to form the primary picture.
The digital picture is the magnified, crystal-clear picture of the thing that is seen via a microscope. The magnification is calculated by multiplying the objective and eyepiece objective magnifications combined. Due to the usual magnification, which is neither extremely high nor very low, the magnification ranges from 40X to 100X based on the magnification power of the lenses.
Magnification is calculated as follows: Objective lens magnification divided by eyepiece lens magnification
The objective lens is crucial for both enlargement and resolution, which is the ability to make a picture clear enough to see. Prescott defined resolution as a lens’s capacity to differentiate or identify minute items that are closely connected to one another.
Although the eyepiece magnifies the final picture, its magnification capacity is less 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.
Bright field light microscope applications (Compound light microscope)
The microscope, which is widely employed in microbiology, is used to study both static and live specimens coloured by simple colors. This offers contrast for simple visibility under a microscope. Therefore, it may be used to differentiate between cells of basic bacteria and parasitic protozoans such as Paramecium.
Phase Contrast Microscope
- With this particular kind of optical microscope, light entering the unstained material causes minute light aberrations known as phase shifts.These phase changes create an illuminated (bright) image in the background when light passes through an opaque object. This is how phase shifts are turned into images.
- When employing a transparent specimen, the phase-contrast microscope gives high-contrast pictures, particularly of microorganism cultures, thin tissue fragments, cell tissues, and subcellular particles.
- The optical technique used to convert a specimen into an amplitude picture, which is visible via the microscope’s eyepiece, provides the basis for the operation of the phase-contrast microscope.
- Unstained cells, sometimes referred to as phase objects, may be seen with the PCM. Consequently, the morphology of the cell is conserved and the cells may also be seen in their original state, with strong contrast and excellent clarity. This is due to the fact that staining and fixing the specimens kills the majority of the cells, a trait that is only reversed using a brightfield light microscope.
- The changes that occur with light penetration are transformed to amplitude changes, which result in the contrast in the picture.
- They create better visuals of the specimens’ pictures when combined with contrast-enhancing components like fluorescence.
Parts of the Phase Contrast Microscope
The Phase Contrast Microscope’s equipment depends on the light paths it uses to get from the source of light to the picture it sees.
Its components are thus listed in order:
- Light source (Mercury arc lamp)
- Collective lens
- Condenser annular
- Phase plate
- Deflected light
- phase ring
The functioning of the Phase Contrast microscope
- With a specific wavelength, both deviated scattered (deflected) light and undeviated light, which penetrates the specimen and is absorbed, generate colour, which is what causes the change. Amplitude fluctuations refer to the difference in the amplitudes of the absorbed and dispersed light. These amplitude fluctuations are sensitive enough for photography tools like the phase contrast microscope to be able to capture them, making them visible to the human eye.
- The condenser of a phase-contrast microscope is a clear circle which generates a cone of light as it travels via the specimen and an opaque disc known as an annular ring. Due to changes in light density, some light bends at the specimen and causes image formation at the objective lens. The phase ring on the phase plate will be struck by the undeviated light. While the deviating light would skip the phase ring and pass directly through the phase plate, it will create an image.
When used by contrast enhancement techniques like fluorescence, the objective lenses of the phase-contrast microscope are capable of performing a variety of tasks. The objective lenses were positioned inside the phase plate, which produces a broad spectrum of contrast between the specimen and the backdrop by variable light uptake and phase shift, or undiffraction.
Applications of the Phase-Contrast Microscope
- Identify the morphologies of living cells, including those of plants and animals.
- investigating the mechanisms of microbial locomotion
- to find specific microbiological components, such as bacterial endospores.
Dark-Field Light Microscope
It is a specialised kind of bright-field light microscope which resembles the phase-contrast microscope in a number of ways. A dark field microscope is created by placing a darkfield stop underneath and a condenser lens that forms a hollow cone ray of light which only reaches the objective from the specimen (Prescott, pg 22).
Visualizing live, unstained cells is accomplished with this method. This is impacted by how the specimen is illuminated, as when a hollow cone beam of light is transmitted to the specimen, the undeviated (reflected/refracted) light travels through the objectives to the specimen, creating an image, while the deviated (unreflected/unrefracted) light does not.
As a result, the specimen will look bright and its surroundings will appear dark. The gloomy backdrop that gives dark-field microscopy its name makes this possible.
Applications of the Dark Field Microscope
Larger cells, like eukaryotic cells’ interior organs, may be seen using it.
Recognising bacterial cells having characteristic morphologies, such as Treponema pallidum, the syphilis-causing pathogen,
The Fluorescent Microscope
Normally, pictures from the aforementioned microscopes appear after light has penetrated and gone through the specimen.
The specimen under the fluorescent microscope emits light. How? By incorporating a dye molecule into the sample, When a dye molecule absorbs light energy, it typically becomes agitated and releases any stored energy as light. Compared to its radiating light, the excited molecule’s light energy has a large wavelength. The dye molecule is often a fluorochrome, which fluoresces when introduced to a particular wavelength of light. The light that was emitted is converted into an image that has been fluorochrome-labeled.
The fluorescent microscope’s working mechanism is based on the idea that by exposing the specimen to ultra-violet, blue, or ultraviolet light, the fluorescent light creates a picture of the specimen. They feature an exciter filter that allows a strong beam of light produced by a mercury vapour arc lamp to pass through. The exciter filter transmits a certain wavelength to the specimen that has been stained with fluorochrome, creating the picture at the objective that has been labelled with fluorochrome.
Following the focus, there is a buffer filter whose primary function is to filter out unwanted ultraviolet radiation that might damage the viewer’s vision and reduce the image’s contrast.
Applications of the Fluorescent Microscope
It is used to display bacterial entities like Mycobacterium tuberculosis.
used to detect particular antibodies generated in immunofluorescence methods against bacterial antigens or pathogens are labelled with fluorochromes.
Fluorochrome-labeled microorganisms are used in ecological research to locate and study them.
By observing the colour that bacteria release after being exposed to certain stains, it is also possible to distinguish between dead and living bacteria.
In addition to the previously mentioned microscopes, there is another uncommon microscope called differential interference contrast microscopy. The pictures are created from differences in the light, either deviated or undeviated, much like in a phase-contrast microscope. The distinction is that two light beams are directed at the specimen and focused by a prism in this instance. While one beam travels through the prism to the specimen, the other travels through the transparent portion of the glass slide without the specimen. The result of the two beams combining and interfering with one another is a picture. For unstained specimens, it can be utilised to view cell components like endospores, bacterial cell walls, nuclei, and granules.
- Microbiology by Laning M. Prescott, 5th Edition
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