1. MICROSCOPY A Brief Overview Dr Saleh M.Y. Practical Lab Experiment -1- MBBS-Phase II 19/10/2010

7. Microscope used by Anton von Leeuwenhoek An old pocket Microscope

8. VARIABLES USED IN MICROSCOPY

11. LR = 0.61 x W W = Wavelength NA = Num aperture NA Types of microscope Resolving power Compound Microscope 200 nanometers Scanning Electron Microscope 10 nanometers Transmission Electron Microscope 0.2 nanometers

17. COMPOUND MICROSCOPE Compound microscope made by John Cuff 1750

22. What’s the power of this lens? To calculate the power of magnification, multiply the power of the ocular lens by the power of the objective , e..g.: 10x40=400 times What are the powers of magnification for each of the objectives we have on our microscopes? Fill in the table on your worksheet.

24. Comparing Powers of Magnification We can see better details with higher the powers of magnification, but we cannot see as much of the image. 10x 40x Which of these images would be viewed at a higher power of magnification?

25. Let’s give it a try ... 1 – Turn on the microscope and then rotate the nosepiece to click the red-banded objective into place. 2 – Place a slide on the stage and secure it using the stage clips. Use the coarse adjustment knob (large knob) to get it the image into view and then use the fine adjustment knob (small knob) to make it clearer. 4 – When you are done, turn off the microscope and put up the slides you used. 3 – Once you have the image in view, rotate the nosepiece to view it under different powers. Draw what you see on your worksheet! Be careful with the largest objective! Sometimes there is not enough room and you will not be able to use it!

26. How to make a wet-mount slide … 1 – Get a clean slide and coverslip from your teacher. 2 – Place ONE drop of water in the middle of the slide. Don’t use too much or the water will run off the edge and make a mess! 3 – Place the edge of the cover slip on one side of the water drop. You do not need to use the stage clips when viewing wet-mount slides! 5 – Place the slide on the stage and view it first with the red-banded objective. Once you see the image, you can rotate the nosepiece to view the slide with the different objectives. 4 - Slowly lower the cover slip on top of the drop. Cover Slip Lower slowly

27. ILLUMINATION - Lamp, sunlight, battery operated lamp, 60 W bulb, Quartz halogen light. FILTERS - Blue, Green, Heat absorbing filters, Barrier filters.

28. 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

29. The Parts of a Microscope

30. The Parts of a Microscope Body Tube Objective Lenses Stage Clips Diaphragm Light Source Ocular Lens Arm Stage Coarse Adj Fine Adjustment Base Nose Piece

45. HOW A MICROSCOPE WORKS ?

46. OPTICAL PATH IN COMPOUND MICROSCOPE

47. Method of using Compound Microscope

52. How to observe a slide ?

54. PHASE CONTRAST MICROSCOPE

60. Condenser annulus

62. Phase plate

63. Images of Phase Contrast Microscpy Clostridium botulinum Spirilium volutans

64. Comparision of Images of Bright Field and Phase Contrast Microscopy

67. Optical path in Dark Ground Microscopy

69. Uses of Dark Ground Microscopy Treponema pallidum Useful in demonstrating - Treponema pallidum - Leptospira - Campylobacter jejuni - Endospore

73. Electron Microscope

78. - 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

79. Working of SEM

80. SEM IMAGES Vibrio cholerae with polar flagella Treponema pallidum

84. Comparison of Depth of Light Collection and Image clarity Light Microscope Confocal Scanning Laser Microscope

85. PRINCIPLE OF CONFOCAL MICROSCOPY