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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. And therewithal, whenever I found out anything remarkable, I have thought it my duty to put down my discovery on paper, so that all ingenious people might be informed thereof.  Antony van Leeuwenhoek. Letter of June 12, 1716
MICROSCOPY A Brief Overview Dr SANTOSH KARADE
[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]
INTRODUCTION TO MICROSCOPY ,[object Object],[object Object],[object Object]
DEFINITION ,[object Object],[object Object],[object Object]
HISTORICAL BACKGROUND ,[object Object],[object Object],[object Object]
Anton van Leeuwenhoek (1632-1723)   ,[object Object],[object Object],[object Object]
Microscope used by Anton von Leeuwenhoek An old pocket Microscope
VARIABLES USED IN MICROSCOPY
[object Object],[object Object],[object Object],[object Object],[object Object]
[object Object],[object Object]
[object Object],[object Object],NA Types of microscope Resolving power Compound Microscope  200 nanometers  Scanning Electron Microscope  10 nanometers  Transmission Electron Microscope  0.2 nanometers
NUMERICAL APERTURE(NA) ,[object Object],[object Object],[object Object],[object Object],θ /2 A B D C ,[object Object],[object Object]
DEFINITION ,[object Object],[object Object]
ABERRATION ,[object Object],[object Object],Blue focus Red focus Incident light
[object Object],Focus of marginal rays Focus of axial rays
TYPES OF MICROSCOPE ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]
COMPOUND MICROSCOPE Compound microscope made by John Cuff 1750
PARTS OF COMPOUND MICROSCOPE ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]
OBJECTIVE LENS ,[object Object],[object Object],[object Object],[object Object],Focal Length ,[object Object],[object Object],[object Object],[object Object],TYPES
OIL IMMERSION OBJECTIVE ,[object Object],[object Object],GLASS OIL A B C D E G F FBEG - OIL ABCD - AIR
[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]
PARTS OF MICROSCOPE (Cont) Iris Diaphragm Condenser Field Diaphragm Diaphragm control ring Focusing knob illumination source
I LLUMINATION  - Lamp, sunlight, battery operated lamp, 60 W bulb, Quartz halogen light. FILTERS  -  Blue, Green, Heat absorbing filters, Barrier filters.
Multiple step operation employed to attain optimal illumination: 1. Remove any diffusing filter.  2. Put a slide on the stage and focus.  3. Completely close the field diaphragm .  4. Move the condenser until the border or the iris hexagon is neat and clear.  5. Center if necessary.  6. Open the field diaphragm until the tip of the hexagon touches the field limit KOHLER’S  ILLUMINATION
HOW A MICROSCOPE WORKS ?
OPTICAL PATH IN COMPOUND MICROSCOPE
METHOD OF USING COMPOUND  MICROSCOPE
[object Object],[object Object],[object Object],[object Object],arm arm
[object Object],[object Object],[object Object],[object Object]
[object Object],10. Looking through the eyepiece, very slowly move the coarse adjustment knob until the specimen comes into focus.  11. Adjust distance between eye piece.
[object Object],[object Object],[object Object],[object Object]
HOW TO OBSERVE A SLIDE ?
CAUSES OF ERROR IN FOCUSING  ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]
PHASE CONTRAST MICROSCOPE
PHASE CONTRAST MICROSCOPE ,[object Object],[object Object],[object Object]
PRINCIPLE OF PHASE CONTRAST MICROSCOPY ,[object Object],[object Object],[object Object]
[object Object],[object Object],[object Object]
CONDENSER ANNULUS ,[object Object],[object Object]
Condenser annulus
PHASE PLATE ,[object Object],[object Object],[object Object],[object Object]
Phase plate
IMAGES OF PHASE CONTRAST MICROSCPY Clostridium botulinum Spirilium volutans
COMPARISION OF IMAGES OF BRIGHT FIELD AND PHASE CONTRAST MICROSCOPY
USES OF PHASE CONTRAST MICROSCOPY ,[object Object],[object Object],[object Object]
DARK GROUND MICROSCOPE ,[object Object],[object Object],[object Object]
OPTICAL PATH IN DARK GROUND MICROSCOPY
REQUISITES FOR DARK GROUND MICROSCOPY ,[object Object],[object Object],[object Object]
USES OF DARK GROUND MICROSCOPY Treponema pallidum ,[object Object],[object Object],[object Object],[object Object],[object Object]
FLUORESCENCE MICROSCOPY ,[object Object],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
[object Object],[object Object],[object Object]
USE OF FLUORESCENCE MICROSCOPY ,[object Object],[object Object],[object Object],[object Object]
ELECTRON MICROSCOPE
ELECTRON  MICROSCOPE ,[object Object],[object Object],[object Object],[object Object],[object Object]
TYPES OF EM ,[object Object],[object Object]
TRANSMISSION ELECTRON MICROSCOPE (TEM) ,[object Object],[object Object],[object Object],[object Object]
TEM (Cont) ,[object Object],[object Object],[object Object],[object Object]
[object Object],[object Object],[object Object],SCANNING ELECTRON MICROSCOPE
WORKING OF SEM
SEM IMAGES Vibrio cholerae  with polar flagella Treponema pallidum
INVERTED MICROSCOPE ,[object Object],[object Object],[object Object],[object Object],[object Object],OTHER  MICROSCOPES
STEREO MICROSCOPE ,[object Object],[object Object],POLARIZING MICROSCOPE ,[object Object],[object Object],[object Object],[object Object]
[object Object],[object Object],CONFOCAL SCANNING LASER MICROSCOPE
Comparison of Depth of Light Collection and Image clarity Light Microscope Confocal Scanning Laser Microscope
PRINCIPLE OF CONFOCAL MICROSCOPY
USES OF CONFOCAL MICROSCOPE ,[object Object],[object Object],[object Object]
What is the advantage of using a confocal microscope? ,[object Object],[object Object]
NEWER MICROSCOPE ,[object Object],[object Object],[object Object]
CARE OF THE MICROSCOPE ,[object Object],[object Object],[object Object],[object Object],[object Object]
THANK YOU

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Microscopy

  • 1. 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. And therewithal, whenever I found out anything remarkable, I have thought it my duty to put down my discovery on paper, so that all ingenious people might be informed thereof. Antony van Leeuwenhoek. Letter of June 12, 1716
  • 2. MICROSCOPY A Brief Overview Dr SANTOSH KARADE
  • 3.
  • 4.
  • 5.
  • 6.
  • 7.
  • 8. Microscope used by Anton von Leeuwenhoek An old pocket Microscope
  • 9. VARIABLES USED IN MICROSCOPY
  • 10.
  • 11.
  • 12.
  • 13.
  • 14.
  • 15.
  • 16.
  • 17.
  • 18. COMPOUND MICROSCOPE Compound microscope made by John Cuff 1750
  • 19.
  • 20.
  • 21.
  • 22.
  • 23.
  • 24. PARTS OF MICROSCOPE (Cont) Iris Diaphragm Condenser Field Diaphragm Diaphragm control ring Focusing knob illumination source
  • 25. I LLUMINATION - Lamp, sunlight, battery operated lamp, 60 W bulb, Quartz halogen light. FILTERS - Blue, Green, Heat absorbing filters, Barrier filters.
  • 26. Multiple step operation employed to attain optimal illumination: 1. Remove any diffusing filter. 2. Put a slide on the stage and focus. 3. Completely close the field diaphragm . 4. Move the condenser until the border or the iris hexagon is neat and clear. 5. Center if necessary. 6. Open the field diaphragm until the tip of the hexagon touches the field limit KOHLER’S ILLUMINATION
  • 27. HOW A MICROSCOPE WORKS ?
  • 28. OPTICAL PATH IN COMPOUND MICROSCOPE
  • 29. METHOD OF USING COMPOUND MICROSCOPE
  • 30.
  • 31.
  • 32.
  • 33.
  • 34. HOW TO OBSERVE A SLIDE ?
  • 35.
  • 37.
  • 38.
  • 39.
  • 40.
  • 41.
  • 43.
  • 45. IMAGES OF PHASE CONTRAST MICROSCPY Clostridium botulinum Spirilium volutans
  • 46. COMPARISION OF IMAGES OF BRIGHT FIELD AND PHASE CONTRAST MICROSCOPY
  • 47.
  • 48.
  • 49. OPTICAL PATH IN DARK GROUND MICROSCOPY
  • 50.
  • 51.
  • 52.
  • 53.
  • 54.
  • 56.
  • 57.
  • 58.
  • 59.
  • 60.
  • 62. SEM IMAGES Vibrio cholerae with polar flagella Treponema pallidum
  • 63.
  • 64.
  • 65.
  • 66. Comparison of Depth of Light Collection and Image clarity Light Microscope Confocal Scanning Laser Microscope
  • 67. PRINCIPLE OF CONFOCAL MICROSCOPY
  • 68.
  • 69.
  • 70.
  • 71.