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Microscopy department of botany sagar m.pPPT.pptx
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- 2. MICROSCOPY: OVERVIEW, PRINCIPLESAND ITS TYPES PRINCIPLES OF MICROSCOPY • Biochemical analysis is generally go along with microscopic investigation of cell, tissue or organelle preparations. Such type of investigation are used in various applications, for e.g. for the biochemical characterization or to examine the integrity of samples during the experiment. • Microscopy is a technique use for making very tiny things to visible to the naked eyes and the instrument used to make things visible to the unaided or naked eye is known as Microscope. • The microscope is the instrument most frequently characteristics of microbiology laboratory. • The magnification provides by the microscope enables us to see microorganisms and their structures otherwise invisible to the naked eye. • There are two fundamental types of microscope: • Light microscope • Electron microscope
- 4. Light microscope • Lightmicroscope involves the use of series of glass lenses to focus light in order to create an image whereas, electron microscope uses electromagnetic lenses to focus beam of electrons. • Light microscopes have ability to magnify objects up to a maximum of approximately 1500 times whereas electron microscopes are able to magnify the objects maximum of approximately 200,000 times. • Magnification is not the best measure of microscope, however. Rather, resolution, that is the ability to distinguish between two closely spaced objects in a specimen which is the more reliable estimate of a microscope’s utility. • Resolution limit of standard light microscope is about 0.5 micrometers but in contrast lateral resolution limit of electron microscope is up to 1 nanometer (nm). • Light microscopes are used to view both living and dead specimens and often in real color, but in electron microscopes only dead specimens are viewed and never in real color.
- 7. LIGHT MICROSCOPE • Itis a type of microscopy in which magnifications is obtained by a system of optical lenses using light waves. • Light microscope abide of a single lens mounted in a metal frame is the simple form of microscope- a magnifying lens. • Light microscope usually uses sun or ambient indoor light as a source of illuminations. • Because of the travelling of light through the specimens, this instrument is also called as transmission light microscope. • The light microscope forms a magnified image of a specimen which is based on the principles of absorption, transmission, diffraction and refraction of light waves. • All modern light microscopes are usually made up of more than one glass in combinations in which the major components are the eyepiece lens or ocular lens, the objective lens, and the condenser lens, and instruments of such combinations are therefore called compound microscopes.
- 8. TYPES OF LIGHTMICROSCOPES 1.Bright field microscope: In this type of microscopy, the area observed or the microscopic field is appears brightly lighted whereas the microorganisms appears dark because they absorb some of the light. 2.Dark filed microscope: In this, effects produced by the dark field techniques is that of a dark background against which objects are brightly illuminated. 3.Phase contrast microscope: In principle, this technique is based on the fact that light passing through one material and into another material of a slightly different refractive index and/or thickness will undergo in change in phase. 4.Fluorescence microscope: In this the specimens itself acts as a light source, the specimens used to study are either fluorescent materials or stained with fluorescent dye.
- 9. Brightfield Light Microscope(Compound light microscope) • This is the most basic optical Microscope used in microbiology laboratories which produces a dark image against a bright background. Made up of two lenses, it is widely used to view plant and animal cell organelles including some parasites such as Paramecium after staining with basic stains. Its functionality is based on being able to provide a high-resolution image, which highly depends on the proper use of the microscope. This means that an adequate amount of light will enable sufficient focusing of the image, to produce a quality image. It is also known as a compound light microscope.
- 11. • It iscomposed of: • Two lenses which include the objective lens and the eyepiece or ocular lens. • Objective lens is made up of six or more glasses, which make the image clear from the object • The condenser is mounted below the stage which focuses a beam of light onto the specimen. It can be fixed or movable, to adjust the quality of light, but this entirely depends on the microscope. • They are held together by a sturdy metallic curved back used as an arm and a stand at the bottom, known as the base, of the microscope. The arm and the base hold all the parts of the microscope. • The stage where the specimen is placed, allowing movement of the specimen around for better viewing with the flexible knobs and it is where the light is focused on. • Two focusing knobs i.e the fine adjustment knob and the coarse adjustment knob, found on the microscopes’ arm, which can move the stage or the nosepiece to focus on the image. the sharpen the image clarity. • It has a light illuminator or a mirror found at the base or on the microbes of the nosepiece. • The nosepiece has about three to five objective lenses with different magnifying power. It can move round to any position depending on the objective lens to focus on the image. • An aperture diaphragm also is known as the contrast, which controls the diameter of the beam of light that passes through the condenser, in that, when the condenser is almost closed, the light comes through to the center of the condenser creating high contrast. But when the condenser is widely open, the image is very bright with very low contrast
- 12. Applications of theBright Field Light Microscope (Compound light microscope) • Vastly used in Microbiology, this microscope is used to view fixed and live specimens, that have been stained with basic stains. This gives contrast for easy visibility under the microscope. Therefore it can be used to identify basic bacteria cells and parasitic protozoans such as Paramecium.
- 13. Phase Contrast Microscope •This is a type of optical microscope whereby small light deviations known as phase shifts occur during light penetration into the unstained specimen. These phase shifts are converted into the image to mean, when light passes through the opaque specimen, the phase shifts brighten the specimen forming an illuminated (bright) image in the background. • The phase-contrast microscope produces high contrast images when using a transparent specimen more so those of microbial cultures, thin tissue fragments, cell tissues, and subcellular particles. • The principle behind the working of the phase-contrast microscope is the use of an optical method to transform a specimen into an amplitude image, that’s viewed by the eyepiece of the microscope. • The PCM can be used to view unstained cells also known as the phase objects, which means that the morphology of the cell is maintained and the cells can be observed in their natural state, in high contrast and efficient clarity. This is because if the specimens are stained and fixed, they kill most cells, a characteristic that is uniquely undone by the brightfield light microscope.
- 15. Applications of Phase-Contrast Microscope •Determine morphologies of living cells such as plant and animal cells • Studying microbial motility and structures of locomotion • To detect certain microbial elements such as the bacterial endospores
- 16. Dark Field Microscope •This is a specialized type of bright field light microscope that has several similarities to the Phase-Contrast Microscope. To make a dark field Microscope, place a darkfield stop underneath and a condenser lens which produces a hollow cone beam of light that enters the objective only, from the specimen. • This technique is used to visualize living unstained cells. This is affected by the way illumination is done on the specimen in that, when a hollow cone beam of light is transmitted to the specimen, deviated light (unreflected/unrefracted) rays do not pass through the objectives but the undeviated (reflected/refracted) light passes through the objectives to the specimen forming an image. • This makes the surrounding field of the specimen appear black while the specimen will appear illuminated. This is enabled by the dark background this the name, dark-field Microscopy.
- 18. Applications • It isused to visualize the internal organs of larger cells such as the eukaryotic cells • Identification of bacterial cells with distinctive shapes such as Treponema pallidum, a causative agent of syphilis.
- 20. Fluorescent Microscope • Inthe case of the fluorescent Microscope, the specimen emits light. How? By adding a dye molecule to the specimen. This dye molecule will normally become excited when it absorbs light energy, hence it releases any trapped energy as light. The light energy that is released by the excited molecule has a long wavelength compared to its radiating light. The dye molecule is normally a fluorochrome, that fluoresces when exposed to the light of a certain specific wavelength. The image formed is a fluorochrome-labeled image from the emitted light • The principle behind this working mechanism is that the fluorescent microscope will expose the specimen to ultra or violet or blue light, which forms an image of the specimen that is emanated by the fluorescent light. They have a mercury vapor arc lamp that produces an intense beam of light that passes through an exciter filter. The exciter filter functions to transmit a specific wavelength to the fluorochrome stained specimen, producing the fluorochrome-labeled image, at the objective. • After the objective, there is a barrier filter that functions primarily to remove any ultraviolet radiation that may be harmful to the viewer’s light, thus reducing the contrast of the image.
- 22. Applications of theFluorescent Microscope • Used in the visualization of bacterial agents such as Mycobacterium tuberculosis. • Used to identify specific antibodies produced against bacterial antigens/pathogens in immunofluorescence techniques by labeling the antibodies with fluorochromes. • Used in ecological studies to identify and observe microorganisms labeled by the fluorochromes • It can also be used to differentiate between dead and live bacteria by the color they emit when treated with special stains
- 23. ELECTRON MICROSCOPE • Anelectron microscope is a microscope that uses a beam of accelerated electrons as a source of illumination. It is a special type of microscope having a high resolution of images, able to magnify objects in nanometres, which are formed by controlled use of electrons in a vacuum captured on a phosphorescent screen. • Electron microscope is used when the greatest resolution is required, and when the living state can be ignored. • The image produced in an electron microscope reveals the ultra structure of cells. • The source of light in an electron microscope is the electron gun or electron beam. • A tungsten filament emits electrons, when a high voltage of between 40 000 and 100 000 volts (the accelerating voltage) is passed between the cathode and the anode.
- 24. TYPES OF ELECTRONMICROSCOPE
- 25. Working Principle ofElectron Microscope 1.The electron gun generates electrons. 2.Two sets of condenser lenses focus the electron beam on the specimen and then into a thin tight beam. 3.To move electrons down the column, an accelerating voltage (mostly between 100 kV- 1000 kV) is applied between the tungsten filament and anode. 4.The specimen to be examined is made extremely thin, at least 200 times thinner than those used in the optical microscope. Ultra-thin sections of 20-100 nm are cut which is already placed on the specimen holder. 5.The electronic beam passes through the specimen and electrons are scattered depending upon the thickness or refractive index of different parts of the specimen. 6.The denser regions in the specimen scatter more electrons and therefore appear darker in the image since fewer electrons strike that area of the screen. In contrast, transparent regions are brighter. 7.The electron beam coming out of the specimen passes to the objective lens, which has high power and forms the intermediate magnified image. 8.The ocular lenses then produce the final further magnified image.
- 26. • Transmission electronmicroscope (TEM): In the TEM, electrons that passes through the specimen are imaged. • Scanning electron microscope (SEM): In the SEM electrons that are reflected back from the specimen (secondary electrons) are collected, and the surfaces of specimens are imaged.
- 27. Parts of ElectronMicroscope • 1.Electron gun • The electron gun is a heated tungsten filament, which generates electrons. • 2. Electromagnetic lenses • The condenser lens focuses the electron beam on the specimen. A second condenser lens forms the electrons into a thin tight beam. • The electron beam coming out of the specimen passes down the second of magnetic coils called the objective lens, which has high power and forms the intermediate magnified image. • The third set of magnetic lenses called projector (ocular) lenses produce the final further magnified image. • Each of these lenses acts as an image magnifier all the while maintaining an incredible level of detail and resolution. • 3. Specimen Holder • The specimen holder is an extremely thin film of carbon or collodion held by a metal grid. • 4. Image viewing and Recording System • The final image is projected on a fluorescent screen. • Below the fluorescent screen is a camera for recording the image.
- 29. • The transmissionelectron microscope is used to view thin specimens through which electrons can pass generating a projection image. • The TEM is analogous in many ways to the conventional (compound) light microscope. • TEM is used, among other things, to image the interior of cells (in thin sections), the structure of protein molecules (contrasted by metal shadowing), the organization of molecules in viruses and cytoskeletal filaments (prepared by the negative staining technique), and the arrangement of protein molecules in cell membranes (by freeze-fracture).
- 30. SEM • Conventional scanningelectron microscopy depends on the emission of secondary electrons from the surface of a specimen. • Because of its great depth of focus, a scanning electron microscope is the EM analog of a stereo light microscope. • It provides detailed images of the surfaces of cells and whole organisms that are not possible by TEM. It can also be used for particle counting and size determination, and for process control. • It is termed a scanning electron microscope because the image is formed by scanning a focused electron beam onto the surface of the specimen in a raster pattern.
- 32. Applications of Electronmicroscope • Electron microscopes are used to investigate the ultrastructure of a wide range of biological and inorganic specimens including microorganisms, cells, large molecules, biopsy samples, metals, and crystals. • Industrially, electron microscopes are often used for quality control and failure analysis. • Modern electron microscopes produce electron micrographs using specialized digital cameras and frame grabbers to capture the images. • The science of microbiology owes its development to the electron microscope. The study of microorganisms like bacteria, virus, and other pathogens have made the treatment of diseases very effective.