Microscopy and Types of Microscopes
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The document provides a comprehensive overview of microscopy, detailing its history, types of microscopes, their functions, and proper usage. It highlights important figures like Anton van Leeuwenhoek and discusses methods to enhance observation and care for microscopes. Various techniques, including electron microscopy and phase contrast microscopy, are explored for their applications in biological studies.
Microscopy and Types of Microscopes
- 1. Dr. Roma Goyal Assistant Professor, Department of Microbiology MICROSCOPES
- 2. My work, which I've done for a long time, was not pursued in order to gain the praise I now enjoy, but chiefly from a craving after knowledge, which I notice resides in me more than in most other men. Whenever I found out anything remarkable, I have thought it my duty to put down my discovery on paper, so that all people might be informed thereof. Antony van Leeuwenhoek. Letter of June 12, 1716
- 3. • INTRODUCTION • DEFINITION • HISTORICAL BACKGROUND • VARIABLES USED IN MICROSCOPY • VARIOUS TYPES OF MICROSCOPES • COMPOUND MICROSCOPE - Structure and Function • USE OF MICROSCOPE • CARE OF MICROSCOPE MICROSCOPY
- 4. 4 INTRODUCTION TO MICROSCOPY • Understanding the optical principles and construction of microscopes • Role of microscopy • Microscopic techniques and application
- 6. 6 DEFINITION • A microscope (Greek: micron = small and scopos = aim) • MICROSCOPE - An instrument for viewing objects that are too small to be seen by the naked or unaided eye • MICROSCOPY - The science of investigating small objects using such an instrument is called microscopy
- 7. 7 HISTORICAL BACKGROUND • 1590 - Hans Janssen and his son Zacharias Janssen, developed first microscope. • 1609 - Galileo Galilei - occhiolino or compound microscope. • 1620 - Christian Huygens, another Dutchman, developed a simple 2-lens ocular system that was chromatically corrected.
- 8. 8 Anton van Leeuwenhoek (1632-1723) • Anton van Leeuwenhoek is generally credited with bringing the microscope to the attention of biologists. • A tradesman of Delft, Holland. 1661 - He discovered bacteria, free-living and parasitic microscopic protists, sperm cells, blood cells, microscopic nematodes etc.
- 9. 9 Microscope used by Anton von Leeuwenhoek An old pocket Microscope
- 10. USES OF MICROSCOPE • Magnification of an object. • Maximization of resolution • To give contrast between structures, organisms and background.
- 12. 12 MAGNIFICATION • Degree of enlargement • No of times the length, breadth or diameter, of an object is multiplied.
- 13. 13 Types of microscope Resolving power Compound Microscope 200 nanometers Scanning Electron Microscope 10 nanometers Transmission Electron Microscope 0.2 nanometers RESOLUTION Ability to reveal closely adjacent structural details as separate and distinct
- 14. RESOLVING POWER Optical Instrument Resolving Power R.P. in Angstroms Human eye 0.2 mm 2,000,000 Å Light microscope 0.2 m 2000 Å Scanning electron microscope 5-10 nm 50-100 Å Transmission electron microscope 0.5 nm 5 Å
- 15. 15 TYPES OF MICROSCOPE • Simple microscope • Compound microscope • Phase Contrast Microscope • Dark Ground Microscope • Fluorescent Microscope • Electron Microscope • Others
- 16. 16 COMPOUND MICROSCOPE Compound microscope made by John Cuff 1750
- 17. PARTS OF COMPOUND MICROSCOPE
- 18. 18 EYE PIECE • Forms magnified virtual & erect image • TYPES (a) Monocular (b) Binocular (c) Trinocular
- 19. 19 OBJECTIVE LENS • It forms magnified real image. Mounted on Nose piece Scan - 4X Low Power - 10X High Power - 40X Oil immersion - 100X TYPES
- 20. 20
- 21. Light Path • Light enters objective lens • Collect light from specimen • Forms a magnified inverted image • Image magnified by ocular lens & passed to eye • Total magnification (40x X 10x = 400x) • Parfocal
- 22. Oil Immersion Lens • Light bends (refracts) as it passes from glass into air • Some light does not enter this smaller objective lens • Use oil between slide and 100x lens • Displaces air between lens and specimen • Glass and oil have same RI so less bending • Oil becomes part of the optics of glass • Increases resolution
- 23. 23 ILLUMINATION - Lamp, sunlight, battery operated lamp, 60 W bulb, Quartz halogen light. FILTERS - Blue, Green, Heat absorbing filters, Barrier filters.
- 24. 24 HOW A MICROSCOPE WORKS ?
- 25. 25 METHOD OF USING COMPOUND MICROSCOPE
- 26. 26 1. Grasp the microscopes arm with one hand and place your other hand under the base. 2. Place the microscope on a bench. Adjust seat 3. Clean Lenses. 4. Turn the coarse adjustment knob to raise the body tube arm arm
- 27. 27 5. Revolve the nose piece to set low- power objective lens. 6. Adjust the Condenser lenses and diaphragm . 7. Place a slide on the stage and secure with stage clips. 8. Switch on the light at low intensity and then increase intensity.
- 28. 28 9. Turn the coarse adjustment knob to lower the body tube until the low power objective reaches its lowest point. 10. Looking through the eyepiece, very slowly move the coarse adjustment knob until the specimen comes into focus. 11. Adjust distance between eye piece.
- 29. 29 12. Switch to the high power objective lens only after adjusting condenser and iris diaphragm. 13. Place a drop of oil over specimen before using oil immersion objective. 14. Lower the objective until oil makes contact with objective. 15. Looking through the eyepiece, very slowly focus the objective away from the slide i.e by raising the objective lens.
- 30. 30 HOW TO OBSERVE A SLIDE ?
- 31. 31 CAUSES OF ERROR IN FOCUSING • Revolving Nose Piece is off centre • Preparation is upside down • Thick cover slip • Dirt or Dried oil over Lens • Air bubble in immersion oil • Poor illumination – Condenser not fully racked up
- 32. Smear of gram positive bacteria with cocci and bacilli shape, under 100X light microscope.
- 33. DARK FIELD MICROSCOPY • special condenser diaphragm • occludes direct light, passes wide angle light • angle too wide to enter objective
- 35. 35 PHASE CONTRAST MICROSCOPE • First described in 1934 by Dutch physicist Frits Zernike • Produces high-contrast images of transparent specimens • Advantage - Living cells can be examined in their natural state
- 36. 36 USES OF PHASE CONTRAST MICROSCOPY • Phase contrast enables visualization of internal cellular components. • Diagnosis of tumor cells . • Examination of growth, dynamics, and behavior of a wide variety of living cells in cell culture
- 37. 37 DARK GROUND MICROSCOPE • Optical system to enhance the contrast of unstained bodies . • Specimen appears gleaming bright against dark background PRINCIPLE OF DGI
- 38. 38 USES OF DARK GROUND MICROSCOPY Treponema pallidum Useful in demonstrating -Treponema pallidum - Leptospira - Campylobacter jejuni - Endospore
- 39. 39 FLUORESCENCE MICROSCOPY • PRINCIPLE UV light Fluorochrome Visible radiation FITC EX - 495 nm EM - 520nm TRITC EX – 540 nm EM – 590 nm Texas Red Ex – 600 nm EM – 615 nm
- 40. 40 • UV rays passes through exciter filter • Dark ground condenser • Micro organisms stained with fluorescent dye, when examined under microscope with ultraviolet light are seen as bright object against dark background
- 41. 41 USE OF FLUORESCENCE MICROSCOPY • Auramine Rhodamine – Yellow Fl - Tubercle bacilli • Acridine Orange R - gives orange red Fl with RNA and yellow green Fl with DNA • QBC • IMMUNOFLUORESCENCE
- 43. 43 ELECTRON MICROSCOPE • Electron Microscopes uses a beam of highly energetic electrons to examine objects on a very fine scale. This examination can yield the info about • Topography • Morphology • Composition • Crystallographic structure
- 44. 44 TYPES OF EM • Transmission Electron Microscope (TEM) • Scanning Electron Microscope (SEM)
- 45. 45 TRANSMISSION ELECTRON MICROSCOPE (TEM) • Stream of electrons is formed • Accelerated using a positive electrical potential • Focused by metallic aperture and Electro magnets • Interactions occur inside the irradiated sample which are detected and transformed into an image .
- 46. 46 TEM (Cont) • Projector Lens forms image on Fluorescent viewing screen • 2D Image • Magnification 10,000 X to 100,000 X
- 47. 47 Scan a gold-plated specimen to give a 3-D view of the surface of an object which is black and white. Used to study surface features of cells and viruses. Scanning Electron microscope has resolution 1000 times better than Light microscope . SCANNING ELECTRON MICROSCOPE
- 48. 48 SEM IMAGES Vibrio cholerae with polar flagella Treponema pallidum
- 50. 50 INVERTED MICROSCOPE • Examination of cultures in flat bottom dishes • Micro dissection • Examination of parasites • Observation of agglutination in serology OTHER MICROSCOPES
- 51. 51 CARE OF THE MICROSCOPE • Handling • Proper storage • Care of Lenses • Care of oil emersion objective • Care of lamp
- 53. THANKS