Microscope

Microscope

1. MRS. MERLIN DAYANA. E BSC (N ) NURSING TUTOR, GANGA COLLEGE OF NURSING COIMBATORE MRS. MERLIN DAYANA. E BSC (N ) NURSING TUTOR, GANGA COLLEGE OF NURSING COIMBATORE

2. OUT LINE Introduction Microscopy History of Microscopy Properties of Microscopy Types of Microscopy Handlig and care of Microscopy

3. INTRODUCTION Microscope,instrument that produces enlarged images of small objects, allowing the observer an exceedingly close view of minute structures at a scale convenient for examination and analysis. A. The microscope may provide a dynamic image.

4. MICROSCOPY Definition : A microscope ( Greek: micron= small and scopos = to look) Microscope: Is an interment for viewing objects that are too small to be seen by the naked or unaided eye. Microscopy: The science of investigating small object using such an instruments is called microscopy.

5. HISTORY OF MICROSCOPY Hans & Zachary's janssen In 1590 , both are dutch eyeglass makers inventors. The first compound microscopes are invented by Hans & zacharis janssen. That is tube with lences at each end. HANS & ZACHARIS JANSSEN In 1590 , both are Dutch eyeglass makers inventors. The first compound microscopes are invented by Hans & Zacharias janssen. That is tube with lenses at each end.

6. HISTORY OF MICROSCOPY Anton van leeuwenhook Is generaly credited with bringing the microscopeto attention of biologists. In 1661 he discoverd bacteria,free living & parasitic microscopic protists,sprem cells.blood cells. He is the father of microbiology. Anton van Leeuwenhoek Is generally credited with bringing the microscope to attention of biologists. In 1661 he discovered bacteria, free living & parasitic microscopic protists,sprem cells. blood cells. He is the father of microbiology .

7. HISTORY OF MICROSCOPY Robert hook In 1635 - 1703, English chemist , mathematician , physicists & inventor. He improved the compound microscope.

8. PROPERTIES OF MICROSCOPY Good magnification A good microscope should have at least three properties Good resolution Good contrast

9. PROPERTIES OF MICROSCOPY Good resolution:- Resolution power refers to the ability to produce separate images of closely placed objectives. That they can be distinguished as two separate entities. The resolution power of:- Unaided human eye is about 0.2mm (200μm) Light microscope is about 0.2μm Electron microscope is about 0.5nm.

10. PROPERTIES OF MICROSCOPY Good contrast :- Contrast is improved by staining the specimen. When the stain binds to the cells, the contrast is increased.

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

12. Compound microscopy Simple microscopy Interference microscopy Bright field or light microscopy Dark field microscopy Phase contrast microscopy Fluorescence microscopy Electron microscopy TYPES OF MICROSCOPE

13. SIMPLE MICROSCOPE: Simple microscope: contains only one lenses. Ex. Magnifying glass.

14. COMPOUND MICROSCOPY Compound microscope: A system of two lens that works together Compound microscope: A system of two lens that works together.

15. BRIGHT FIELD OR LIGHT MICROSCOPE Light microscope forms a dark image against a brighter background, hence the name Bright field M echanical part M agnifying part Illuminating part

16. BRIGHT FIELD OR LIGHT MICROSCOPE

17. MECHANICAL PART base : It holds various part of microscope, such as the light source , the fine and coarse adjustment . c- shaped arm : It holds 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.

18. MAGNIFYING PART ocular lens : the arm contains an eye piece that bears an ocular lens of 10X magnification power. Microscope with two eye piece are called as binocular microscopes. ocular lens : The arm contains an eye piece that bears an ocular lens of 10X magnification power. Microscope with two eye piece are called as binocular microscopes. Objective lens: The arm also contains a revolving nose piece that bear three to five objectives with lenses of different magnifying power (4X,10X,40X,and 100X).

19. ILLUMINATING PART condenser :It is mounted beneath the stage which focuses a cone of light on the slide. iris diaphragm : I t 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.

20. PRINCIPLE 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.

21. DARK FIELD MICROSCOPE In dark field microscope, the object appears bright against a dark background. It is made possible by special dark condenser. Hence the name dark field microscope.

22. DARK FIELD MICROSCOPE

23. DARK FIELD MICROSCOPE

24. PRINCIPLE DARK FIELD MICROSCOPE 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. Only the which is reflected by the specimen enters the objective lens where as the un reflected light dose not enter the object. As a result, the specimen is brightly illuminated; but the background appears dark.

25. Application: Dark field microscope is used to identify the living, unstained cells and thin bacteria like spirochetes which is cannot visualized by light microscope. Application of dark filed microscopy

26. 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 microbes, the properties of living cells cannot be studied.

27. PHASE CONTRAST MICROSCOPE

28. PRINCIPLES The condenser is similar to that of dark field microscope, consist of an opaque central area with a thin transparent ring, which procuces a hollow cone of light. As this cone of light passes through a cells, some light rays are bent due to variations in density and refractive index within the specimen and are retracted by about one fourth of a wave length. The un deviated light rays strike a phase plate, 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 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 un deviated light, is bright, while the unstained object appears dark and well defined.

29. APPLICATION To study unstained living cells. Detailed examination of internal structures in living microorganisms . To study flagellar movements and motility of bacteria and protozoan’s To study intestinal and other live protozoa such as amoebae . To examine fungi grown in culture.

30. FLUORESCENCE MICROSCOPY Refers to any microscope that uses Fluorescence property to generate an image.

31. When Fluorescence dyes are exposed to ultraviolet rays, they become excited and are said to Fluorescence .i.e. they covert this invisible, short wavelength rays into light of longer wavelength. 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. PRINCIPLES

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

33. APPLICATION: EpiFluorescence microscope: It is the simplest form of Fluorescence microscope, which has the following applications. Auto Fluorescence microscope: Some microbes directly fluoresce when UV lamp e.g. cyclospora. 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. Aura mine phenol: is used for the detection of tubercle bacilli. Immunofluorescence: It uses florescent dye tagged immunoglobulin to detect cell surface antigen or antibodies bound to cell surface antigens.

34. ELECTRON MICROSCOPY An electron microscope uses accelerated e 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 if a cell.

35. Electron microscopes are of two types: A Transmission Electron microscope B scanning Electron microscope ELECTRON MICROSCOPY

36. 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 collision with air molecules.

37. TRANSMISSION ELECTRON MICROSCOPE

38. TRANSMISSION ELECTRON MICROSCOPE

39. ELECTRON PATHWAY Electron pathway: Electron pass through a magnatic condenser and then bombardon thin sliced 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 .

40. MEASURES TO INCREASE CONTRAST OF EM – STAINING: In electron microscope the stain is used are solution of heavy metal salts like lead citrate and uranyl acetate. Negative staining: The specimen is spread out in a thin film with heavy metal like phosphotungstic acid and uranyl acetate. Shadowing: The technique is particularly useful in studying virus morphology, bacterial flagella, and plasmids.

41. SCANNING ELECTRON MICROSCOPY scanning Electron microscope has been used to examine the surface of microorganisms in great detail. It has a resolution of7nm or less. The SEM differs from TEM, in producing an image from electrons emitted by an objects surface rather than from transmitted electrons.

42. SCANNING ELECTRON MICROSCOPY

43. INTERFERENCE MICROSCOPE Uses a differential - I Interference contrast (DIC) microscope. Allows for detailed view of live, unstained specimens. Includes two prisms that add contrasting colors to the image. The image is colorful and three- dimensional.

44. INTERFERENCE MICROSCOPE

45. RULES OF USING A MICROSCOPY Always carry with 2 hands. Only use lens paper for cleaning . Do not force knobs . Always store covered Be careful of the cords.