Compound microscope

compound microscope
florescent microscope
DGI
Phase contrast
Electron microscope and its types
TEM
SEM

1. COMPOUND MICROSCOPE OR LIGHT MICROSCOPE PRESENTED BY MEGHA SHRIDHAR

2. INTRODUCTION The term “compound” in compound microscopes refers to the microscope having more than one lens. Devised with a system of combination of lenses, a compound microscope consists of two optical parts, namely the objective lens and the ocular lens.

3. PRINCIPLE A beam of visible light from the base is focused by a condenser lens onto the specimen. The objective lens picks up the light transmitted by the specimen and creates a magnified image of the specimen called the primary image inside the body tube.

4. HOW IT WORKS or PROCEDURE The specimen or object, to be examined is usually mounted on a transparent glass slide and positioned on the specimen stage between the condenser lens and objective lens. A beam of visible light from the base is focused by a condenser lens onto the specimen. The objective lens picks up the light transmitted by the specimen and creates a magnified image of the specimen called the primary image inside the body tube. This image is again magnified by the ocular lens or eyepiece. When higher magnification is required, the nose piece is rotated after low power focusing to bring the objective of a higher power (generally 45X) in line with the illuminated part of the slide. Occasionally very high magnification it required (e.g. for observing bacterial cell). In that case, an oil immersion objective lens (usually 100X) is employed .

5. Magnification of compound microscope In order to ascertain the total magnification when viewing an image with a compound light microscope, take the power of the objective lens which is at 4x, 10x or 40x and multiply it by the power of the eyepiece which is typically 10x. Therefore , a 10x eyepiece used with a 40X objective lens will produce a magnification of 400X. The naked eye can now view the specimen at magnification 400 times greater and so microscopic details are revealed.

6. COMPOUND MICROSCOPE

7. Parts of a Compound Microscope Eyepiece And Body Tube. The eyepiece is the lens through which the viewer looks to see the specimen. It usually contains a 10X or 15X power lens. The body tube connects the eyepiece to the objective lenses . Objectives and Stage Clips Objective Lenses are one of the most important parts of a Compound Microscope. They are the closest to the specimen. A standard Microscope has three to four Objective Lenses which range from 4X to 100X. Stage Clips are metal clips that held the slide in a place.

8. Arm and Base The Arm connects the Body Tube to the base of the Microscope. The Base supports the Microscope and its where Illuminator. Illuminator and Stage The illuminator is the light source for a microscope. A compound light microscope mostly uses a low voltage bulb as an illuminator. The stage is the flat platform where the slide is placed.

9. Nosepiece and Aperture Nosepiece is a rotating turret that holds the objective lenses. The viewer spins the nosepiece to select different objective lenses. The aperture is the middle of the stage that allows light from the illuminator to reach the specimen . Condenser, Iris diaphragm, and Diaphragm A condenser gathers and focuses light from the illuminator onto the specimen being viewed. Iris diaphragm adjusts the amount of light that reaches the specimen. The diaphragm is a five holed disk placed under the stage. Each hole is of a different diameter. By turning it, you can vary the amount of light passing through the stage opening

10. Applications A compound microscope is of great use in pathology labs so as to identify diseases. Various crime cases are detected and solved by drawing out human cells and examining them under the microscope in forensic laboratories. The presence or absence of minerals and the presence of metals can be identified using compound microscopes. Students in schools and colleges are benefited by the use of a microscope for conducting their academic experiments. It helps to see and understand the microbial world of bacteria and viruses, which is otherwise invisible to the naked eye. Plant cells are examined and the microorganisms thriving on it can be ascertained with the help of a compound microscope. Thereby , a compound microscope has proved to be crucial to biologists

11. Advantages Simplicity and its convenience. A compound light microscope is relatively small, therefore it’s easy to use and simple to store, and it comes with its own light source. Because of their multiple lenses, compound light microscopes are able to reveal a great amount of detail in samples.

12. B) DGI MICROSCOPE or DARK GROUND ILLUMINATION MICROSCOPE or DARK FIELD MICROSCOPE

13. INTRODUCTION This is similar to the ordinary light microscope; however, the condenser system is modified so that the specimen is not illuminated directly. The condenser directs the light obliquely so that the light is deflected or scattered from the specimen, which then appears bright against a dark background. Living specimens may be observed more readily with dark field than with bright field microscopy .

14. PRINCIPLE The dark-ground microscopy makes use of the dark-ground microscope, a special type of compound light microscope. The dark-field condenser with a central circular stop, which illuminates the object with a cone of light, is the most essential part of the dark-ground microscope. This microscope uses reflected light instead of transmitted light used in the ordinary light microscope. It prevents light from falling directly on the objective lens. Light rays falling on the object are reflected or scattered onto the objective lens with the result that the microorganisms appear brightly stained against a dark background.

15. Uses of Dark-field Microscope The dark ground microscopy has the following uses: It is useful for the demonstration of very thin bacteria not visible under ordinary illumination since the reflection of the light makes them appear larger. This is a frequently used method for rapid demonstration of Treponema pallidum in clinical specimens. It is also useful for the demonstration of the motility of flagellated bacteria and protozoa. Darkfield is used to study marine organisms such as algae, plankton, diatoms, insects, fibers, hairs, yeast and protozoa as well as some minerals and crystals, thin polymers and some ceramics. Darkfield is used to study mounted cells and tissues. It is more useful in examining external details, such as outlines, edges, grain boundaries and surface defects than internal structure.

16. Advantages of Darkfield Microscope Dark-field microscopy is a very simple yet effective technique. It is well suited for uses involving live and unstained biological samples, such as a smear from a tissue culture or individual, water-borne, single-celled organisms. Considering the simplicity of the setup, the quality of images obtained from this technique is impressive .

17. Limitations of Darkfield Microscope The main limitation of dark-field microscopy is the low light levels seen in the final image. The sample must be very strongly illuminated, which can cause damage to the sample.

18. Definition A fluorescence microscope is an optical microscope that uses fluorescence and phosphorescence instead of, or in addition to, reflection and absorption to study properties of organic or inorganic substances. Fluorescence is the emission of light by a substance that has absorbed light or other electromagnetic radiation while phosphorescence is a specific type of photoluminescence related to fluorescence .

19. Principle Light of the excitation wavelength is focused on the specimen through the objective lens. The fluorescence emitted by the specimen is focused on the detector by the objective. Since most of the excitation light is transmitted through the specimen, only reflected excitatory light reaches the objective together with the emitted light.

20. Parts of Fluorescence Microscope 1) A light source : Lasers are mostly used for complex fluorescence microscopy techniques, while xenon lamps, and mercury lamps, and LEDs with a dichroic excitation filter are commonly used. 2) The excitation filter: The exciter is typically a band pass filter that passes only the wavelengths absorbed by the fluorophore

21. 3) The dichroic mirror: A dichroic filter or thin-film filter is a very accurate color filter used to selectively pass light of a small range of colors while reflecting other colors . 4) The emission filter: The emitter is typically a band pass filter that passes only the wavelengths emitted by the fluorophore and blocks all undesired light outside this band – especially the excitation light .

22. Advantages of Fluorescence Microscope Fluorescence microscopy is the most popular method for studying the dynamic behavior exhibited in live-cell imaging. This stems from its ability to isolate individual proteins with a high degree of specificity amidst non-fluorescing material. The sensitivity is high enough to detect as few as 50 molecules per cubic micrometer. Different molecules can now be stained with different colors, allowing multiple types of the molecule to be tracked simultaneously.

23. Limitations of Fluorescence Microscope Fluorophores lose their ability to fluoresce as they are illuminated in a process called photo-bleaching . Photo-bleaching occurs as the fluorescent molecules accumulate chemical damage from the electrons excited during fluorescence. Cells are susceptible to photo toxicity , particularly with short-wavelength light.

24. Phase contrast microscopy

25. Principle When light passes through cells, small phase shifts occur, which are invisible to the human eye. In a phase-contrast microscope, these phase shifts are converted into changes in amplitude, which can be observed as differences in image contrast.

26. Parts of Phase Contrast Microscope Phase-contrast microscopy is basically a specially designed light microscope with all the basic parts in addition to which an annular phase plate and annular diaphragm are fitted . The annular diaphragm: It is situated below the condenser. It is made up of a circular disc having a circular annular groove. The light rays are allowed to pass through the annular groove. Through the annular groove of the annular diaphragm, the light rays fall on the specimen or object to be studied. At the back focal plane of the objective develops an image.

27. Applications of Phase contrast Microscopy To produce high-contrast images of transparent specimens, such as living cells (usually in culture), microorganisms, thin tissue slices, lithographic patterns, fibers, latex dispersions, glass fragments, and subcellular particles (including nuclei and other organelles).

28. Advantages Living cells can be observed in their natural state without previous fixation or labeling. It makes a highly transparent object more visible. No special preparation of fixation or staining etc. is needed to study an object under a phase-contrast microscope which saves a lot of time. Examining intracellular components of living cells at relatively high resolution. eg : The dynamic motility of mitochondria, mitotic chromosomes & vacuoles. It made it possible for biologists to study living cells and how they proliferate through cell division.

29. Limitations Phase-contrast condensers and objective lenses add considerable cost to a microscope, and so phase contrast is often not used in teaching labs except perhaps in classes in the health professions.

30. Definition An electron 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

31. Working Principle of Electron microscope Electron microscopes use signals arising from the interaction of an electron beam with the sample to obtain information about structure, morphology, and composition. Working The electron gun generates electrons. Two sets of condenser lenses focus the electron beam on the specimen and then into a thin tight beam. To move electrons down the column, an accelerating voltage (mostly between 100 kV-1000 kV) is applied between tungsten filament and anode. 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 .

32. 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

33. Types of Electron microscope There are two types of electron microscopes, with different operating styles: The transmission electron microscope (TEM ) The scanning electron microscope (SEM)

34. The transmission electron microscope (TEM) The transmission electron microscope is used to view thin specimens through which electrons can pass generating a projection image . TEM is used, among other things, to image the interior of cells (in thin sections ), the structure of protein molecules the organization of molecules in viruses and cytoskeletal filaments, the arrangement of protein molecules in cell membranes

35. TEM

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37. The scanning electron microscope (SEM) Conventional scanning electron microscopy depends on the emission of secondary electrons from the surface of a specimen. 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.

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39. Parts of Electron microscope Electron gun The electron gun is a heated tungsten filament, which generates electrons . Electromagnetic lenses 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 .

40. Specimen Holder The specimen holder is an extremely thin film of carbon held by a metal grid. 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.

41. Applications 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. Science of microbiology owes its development to the electron microscope. Study of microorganisms like bacteria, virus and other pathogens have made the treatment of diseases very effective.

42. Advantages Very high magnification Incredibly high resolution Material rarely distorted by preparation It is possible to investigate a greater depth of field Diverse applications

43. Limitations The live specimen cannot be observed. As the penetration power of the electron beam is very low, the object should be ultra-thin. For this, the specimen is dried and cut into ultra-thin sections before observation. As the EM works in a vacuum, the specimen should be completely dry. Expensive to build and maintain Requiring researcher training Image artifacts resulting from specimen preparation. This type of microscope is a large, cumbersome extremely sensitive to vibration and external magnetic fields.