Microscopy
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The document provides an overview of microscopy, detailing its history, components, types, and usage. It outlines the development of various microscopes, including light microscopes (such as brightfield, darkfield, phase-contrast, and fluorescence) and electron microscopes (transmission and scanning). Key care instructions for microscopes are also highlighted, ensuring proper maintenance and optimal functionality.
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Microscopy
- 1. BARKATULLAH UNIVERSITY, BHOPAL MICROSCOPY Presented by: Navjot Singh M.Sc. 1st semester Application no. – B208140024
- 2. Introduction A microscope is an instrument used to view objects that are not visible to the naked eye. The microscope magnifies the image of the object being viewed through it. The magnification of the object is produced by the combined action of two lenses, the objective lens near the object, and the eye piece lens near the viewer’s eye.
- 3. History Zacharias Janssen and his son Hans made the early microscope in 1590. Galileo Galilei develops a compound microscope in 1609.
- 4. Robert Hooke publishes Micrographia, includes drawings of the honeycomb structure of cork in 1665. Antonie van Leeuwenhoek described cells and bacteria in 1676.
- 5. Ernst Ruska and Max Knoll from the ideas of Leo Szilard designe and built the Transmission electron microscope in 1931. Frits Zernike developed the phase contrast microscope in 1932. Ernst Ruska developed the Scanning electron microscope in 1942.
- 6. Components of a microscope The tube The body The arm The stage The substage The base Coarse adjustments Fine adjustments Condenser adjustments The lens systems
- 7. Uses and care of a bright field microscope Always lift and carry the microscope with well supported hands. Protect the microscope from dust, moisture and direct sunlight. Place the microscope on a firm surface so that it does not vibrate. If the objective lens is dirty, it should be cleaned with a clean piece of soft linen or lens tissue. Little xylene may be used if the dirt is difficult to remove. Never use alcohol. The underside of a glass slide should be completely dry before it is placed onto the stage. At the end of the working day, all the objective lenses must be wiped clean and the microscope should be covered with its protective cover.
- 8. Types of microscopes Light Microscope Light microscopes include Brightfield Microscopes, Darkfield Microscopes, Phase-contrast Microscopes, Fluorescence Microscopes. Electron microscope Electron microscopes include Transmission Electron Microscope (TEM) and Scanning Electron Microscope (SEM).
- 9. Brightfield microscope The brightfield microscope is a compound microscope with two or more lenses. It produce a dark image on a bright background. Eyepiece contains a lens called an ocular lens. The ocular lenses typically magnify images 10 times (10x). The magnification of objective lenses typically ranges from 4x to 100x. The total magnification is Ocular magnification x objective magnification For example, if a 40x objective lens is selected and the ocular lens is 10x, the total magnification would be (40x)(10x) = 400x.
- 10. Darkfield microscope Living, unstained cells and organisms can be observed by simply changing the way in which they are illuminated. A hollow cone of light is focused on the specimen in such a way that unreflected and unrefracted rays do not enter the objective. Only light that has been reflected or refracted by the specimen forms an image. The field surrounding a specimen appears black, while the object itself is brightly illuminated. Fig.: Treponema pallidum under darkfield microscope
- 11. Phase-contrast Microscope The condenser of a phase-contrast microscope has an annular stop, an opaque disk with a thin transparent ring, which produces a hollow cone of light. As this cone passes through a cell, some light rays are bent due to variations in density and refractive index within the specimen and are retarded by about 1/4 wavelength. The deviated light is focused to form an image of the object. Undeviated light rays strike a phase ring in the phase plate, a special optical disk located in the objective, while the deviated rays miss the ring and pass through the rest of the plate. The phase ring is constructed in such a way that the undeviated light passing through it is advanced by 1/4 wavelength, the deviated and undeviated waves will be about 1/2 wavelength out of phase and will cancel each other when they come together to form an image.
- 12. Phase-contrast Microscope The background, formed by undeviated light, is bright, while the unstained object appears dark and well-defined. Fig.: Paramecium under phase-contrast microscope
- 13. Fluorescence Microscope The fluorescence microscope exposes a specimen to ultraviolet, violet, or blue light and forms an image of the object with the resulting fluorescent light. A mercury vapor arc lamp or other source produces an intense beam, and heat transfer is limited by a special infrared filter. The light passes through an exciter filter that transmits only the desired wavelength. A darkfield condenser provides a black background against which the fluorescent objects glow. Usually the specimens have been stained with dye molecules, called fluorochromes, which fluoresce brightly upon exposure to light of a specific wavelength.
- 14. Fluorescence Microscope A barrier filter positioned after the objective lenses removes any remaining ultraviolet light. Fig.: Escherichia coli under Fluorescent microscope
- 15. Transmission Electron Microscope (TEM) In TEM, a heated tungsten filament in the electron gun generates a beam of electrons that is then focused on the specimen by the condenser. Since electrons cannot pass through a glass lens, doughnut-shaped electromagnets called magnetic lenses are used to focus the beam. The column containing the lenses and specimen must be under high vacuum to obtain a clear image because electrons are deflected by collisions with air molecules. The specimen scatters electrons passing through it, and the beam is focused by magnetic lenses to form an enlarged, visible image of the specimen on a fluorescent screen. A denser region in the specimen scatters more electrons and therefore appears darker in the image since fewer electrons strike that area of the screen. In contrast, electron-transparent regions are brighter.
- 16. Transmission Electron Microscope (TEM) The screen can also be moved aside and the image captured on photographic film as a permanent record. Fig.: Fimbria and Flagellum under TEM
- 17. Scanning Electron Microscope (SEM) The SEM scans a narrow, tapered electron beam back and forth over the specimen. When the beam strikes a particular area, surface atoms discharge a tiny shower of electrons called secondary electrons, and these are trapped by a special detector. Secondary electrons entering the detector strike a scintillator causing it to emit light flashes that a photomultiplier converts to an electrical current and amplifies. The signal is sent to a cathode-ray tube and produces an image like a television picture, which can be viewed or photographed.
- 18. Scanning Electron Microscope (SEM) Fig.: Staphylococcus aureus under SEM
- 19. References Ochei, J. and Kolhatkar A., (2015), Medical Laboratory Science, Theory and Practices, Tata McGraw-Hill. Lansing M. Prescott, John P. Harley, Donald A. Klein, (2002), Microbiology, Tata McGraw-Hill. https://www.microscopemaster.com/microscope-timeline.html https://www.sciencelearn.org.nz/resources/1692-history-of-microscopy- timeline
- 20. Thank You