Microscope

overview of different types of microscope.

1. MICROSCOPE

2. What is microscope? A  microscope   is an instrument  used to see objects that are too small for the naked eye.

3. Properties of a microscope A good microscope should have at least three properties: Good Resolution Good Contrast Good Magnification

4. Good Resolution :- Resolution power refers to the ability to produce separate images of closely placed objects. So that they can be distinguished as two separate entities. The resolution power of- Unaided human eye is about 0.2mm (200 µm) Light microscope is about 0.2 µm. Electron microscope is about 0.5 nm. I mmersion Oil has a higher refractive index than air, hence, use of oil enhances the resolution power of a microscope.

5. Good Contrast: Contrast is improved by staining the specimen. When the stain bind to the cells, the contrast is increased .

6. Good Magnification: Ocular lens with a magnification power of 10X. Objective lens- Scanning (4X) Low power (10X) High power (40X) Oil immersion (100X)

7. Total magnification of a field is the product of the magnification of objective and ocular lens: Scanning field (40X) Low power field (100X) High power field (400X) Oil immersion field (1000X)

8. TYPES OF MICROSCOPE The following types of microscopes are in use now- Bright field or light microscope Dark field microscope Phase contrast microscope Fluorescence microscope Electron microscope

9. Bright field or light microscope Light microscope forms a dark image against a brighter background, hence the name bright field. Structure: The parts of light microscope is divided into three groups- Mechanical part Magnifying part Illuminating part

10. Mechanical part: Base: It holds various part of microscope, such as the light source, the fine and coarse adjustment knobs. C- shaped arm: It hold the microscope, and it connects the ocular lens to the objective lens. Mechanical stage: The arm bears a stage with stage clips to hold the slides and the stage control knobs to move the slide during viewing. It has an aperture at the center that permit light to reach the object from the bottom.

11. Magnifying part: Ocular lens: The arm contains an eye piece that bears an ocular lens of 10X magnification power. Microscope with two eye pieces are called as binocular microscopes. Objective lens: The arm also contains a revolving nose piece that bears three to five objectives with lenses of differing magnifying power (4X,10X,40X, and 100X).

12. Illuminating parts: Condenser: It is mounted beneath the stage which focuses a cone of light on the slide. Iris diaphragm: it control the light pass through the condenser. Light source: it may be a mirror or an electric bulb. Fine and coarse adjustment knob: They sharpen the image .

13. MICROSCOPE

14. Principle : The rays emitted from the light source pass through the iris diaphragm and fall on the specimen. The rays passing through the specimen is gathered by the objective and a magnified image is formed. This image is further magnified by the ocular lens to produce the final magnified virtual image.

15. Compound microscope Dark-field microscope

16. Dark field microscope: In dark field microscope, the object appears bright against a dark background. It is made possible by special dark field condenser. Hence the name dark field microscope. Principle: The dark field condenser has a central opaque area that blocks light from entering the object lens directly and has a peripheral annular hollow area which allows the light to pass through and focus on the specimen obliquely.

17. Only the which is reflected by the specimen enters the objective lens whereas the unreflected light does not enter the objective. As a result, the specimen is brightly illuminated; but the background appears dark. Application: Dark field microscope is used to identify the living, unstained cells and thin bacteria like spirochetes which is can not visualised by light microscope

18. Phase contrast microscope: As per name, in phase contrast microscope the contrast is enhanced . This microscope visualizes the unstained living cells by creating difference in contrast between the cells and water. It converts slight differences in refractive index and cell density into easily detectable variations in light intensity. Contrast can be enhanced by staining of the specimens, but as staining kills the microbes, the properties of living cells cannot be studied.

19. Principle: The condenser is similar to that of dark field microscope, consist of an opaque central area with a thin transparent ring, which produces a hollow cone of light. As this cone of light 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 one fourth of a wave length. The undeviated light rays strike a phase plate,(a special disk located in the objective) while the deviated rays miss the ring and pass through the rest of the plate.

20. The phase ring is constructed in such a way that the undeviated light passing through it is advanced by one-fourth of a wavelength out of the phase and will be cancel each other when they come together to form an image. The background, formed by undeviated light ,is bright ,while the unstained object appears dark and well defined. Summary of application- The light rays go through condenser specimen phase ring objective lens ocular lens

21. Ray diagram of Phase contrast microscope

22. Application: To study unstained living cells Detailed examination of internal structures in living microorganisms. To study flagellar movements and motility of bacteria and protozoans. To study intestinal and other live protozoa such as amoebae and Trichomonas. To examine fungi grown in culture .

23. Fluorescence microscope: Refers to any microscope that uses fluorescence property to generate an image. Principle: When fluorescent dyes are exposed to ultraviolet rays, they become excited and are said to fluoresce, i.e. they convert this invisible, short wavelength rays into light of longer wavelengths (i.e. visible light).

24. Ray diagram of Fluorescent Microscope

25. The source of light may be a mercury lamp which emits rays that pass through an excitation filter . The excitation filter is so designed that it allows only short wavelength UV light (about 400nm, called as the exciting wavelength of light) to pass through; blocking all other long wavelength rays .

26. The exciting rays then get reflected by a dichromatic mirror in such a way that they fall on the specimen which is priorly stained with fluorescent dye. The fluorescent dye absorb the the exciting rays of short wavelength, gets activated and it turns emits fluorescent rays of higher wavelength.

27. The barrier filter positioned after the objective lenses removes any remaining UV light, which could damage the viewer eyes, or blue and violet light, which would reduce the image contrast. Applications: Epifluorescence microscope: It is the simplest form of fluorescence microscope, which has the following applications. Auto fluorescence: Some microbes directly fluoresce when placed under UV lamp e.g. Cyclospora .

28. Microbe coated with fluorescent dyes: Certain microbes fluoresce when they are stained with fluorochrome dyes. e.g. Acridine orange dye is used in QBC examination for the detection of malarial parasites. Auramine phenol is used for the detection of tubercle bacilli. Immunofluorescece : It uses florescent dye tagged immunoglobulin to detect cell surface antigen or antibodies bound to cell surface antigens.

29. Electron Microscopy : An Electron microscope uses accelerated electrons as a source of illumination. Because the wave length of electrons can be up to 100,000 time shorter than that of visible light photons. The electron microscope has a much better resolving power than a light microscope. Hence, it can reveal the details of flagella, fimbriae and intracellular structures of a cell.

30. It was invented by G erman Physicist Ernst and Ruska in 1931. Difference between light microscope and electron microscope

31. Electron microscope are of two types: Transmission electron microscope Scanning electron microscope Transmission electron microscope Electrons are generated by electron gun, which travel in high speed. The medium of travel in EM should be a fully vacuum path because in air path, electrons can get deflected by collisions with air molecules.

32. Electron pathway: Electron pass through a magnetic condenser and then bombardon thin sliced specimen mounted on the copper slide. The specimen scatters electrons passing through it, and then the electron beam is focused by magnetic lenses to form an enlarged, visible image of the specimen on a fluorescent screen. Measures to increase contrast of EM – Staining: In electron microscope the s tain is used are solution of heavy metal salts like lead citrate and uranyl acetate.

33. Negative staining: The specimen is spread out in a thin film with heavy metals like phosphotungstic acid or uranyl acetate. Shadowing: This technique is particularly useful in studying virus morphology, bacterial flegella , and plasmids.

34. Scanning electron microscope Scanning electron microscope has been used to examine the surface of microorganisns in great detail. It has a resolution of 7nm or less. The SEM differs from TEM, in producing an image from electrons emitted by an object’s surface rather than from transmitted electrons

35. Rules of using a microscope Always carry with 2 hands Only use lens paper for cleaning Do not force knobs Always store covered Be careful of the cords