1. Tutorial on Microscopy September 15, 2007

2. Why the need to study microscopy Why the need to study microscopy? It is a tool complementary to molecular biology It has become an indispensable tool for many biologists and pathologists to check sterility of cultures to study the histology of biopsies to study the developmental program of organ to follow the movement of a protein from the cytoplasm to the chloroplast

3. Students need to understand the microscope to get a sense of size, to get the best image possible, to learn how to enjoy using it, to use it for cool and fun purposes, to keep it in good shape, to be able to share the instrument with others.

4. MOTIC ZEISS

5. Eye-piece or ocular Revolving nose with objectives

6. Eye-piece or ocular Revolving nose with objectives Stage and its controls Focussing knob

7. Eye-piece or ocular Revolving nose with objectives Stage Condenser Light source Focussing knob and its controls

8. Light and lenses are the two pieces of equipment which are used to manipulate the light. Both are inherent to the microscope you used; they cannot be changed.

9. The lenses: three types in your microscope The condenser The objective The eye-piece or ocular

10. The lenses: three types in your microscope The condenser The objective The eye-piece or ocular

11. http://micro.magnet.fsu.edu/primer/anatomy/condensers.html The condenser: a combination of lenses

12. http://micro.magnet.fsu.edu/primer/anatomy/condensers.html The simplest condenser Role: to condense and focus light onto the specimen Allows more or less light to enter the condenser The condenser: a combination of lenses Also called an iris diaphragm

13. The condenser: a combination of lenses Will possess specific characteristics (correction, numerical aperture and others) depending on manufacturer specifications http://www.zeiss.com/C1256B5E0047FF3F?Open

14. The lenses: three types in your microscope The condenser The objective The eye-piece or ocular

15. http://micro.magnet.fsu.edu/primer/ The objective: a combination of lenses

16. http://micro.magnet.fsu.edu/primer/ The objective: a combination of lenses MoticZeiss

17. http://micro.magnet.fsu.edu/primer/ The objective: a combination of lenses MoticZeiss PlanCP-Achromat

18. Achromat: Good color correction – exactly for two wavelengths. Field flatness in the image center, refocusing also covers the peripheral areas. Designed for fields of view up to 18 mm diameter. Versions for phase contrast. Budget-priced objectives. Names: CP-Achromat (CP: Clinical Plan) and Achrostigmatism. http://www.zeiss.com/C1256B5E0047FF3F?Open

19. The effect of chromatic aberration Rays of longer λ focus further away that those of shorter λ. http://micro.magnet.fsu.edu/primer/

20. The effect of chromatic aberration Rays of longer λ focus further away that those of shorter λ. http://micro.magnet.fsu.edu/primer/ Rays of longer λ focus further away that those of shorter λ. http://micro.magnet.fsu.edu/primer/

21. Achromat: Good color correction – exactly for two wavelengths. Field flatness in the image center, refocusing also covers the peripheral areas. Designed for fields of view up to 18 mm diameter. Versions for phase contrast. Budget-priced objectives. Names: CP-Achromat (CP: Clinical Plan) and Achrostigmatism. http://www.zeiss.com/C1256B5E0047FF3F?Open Plan and Epiplan: Improved Achromat objectives with good image flatness for fields of view with dia. 20 or even 23 mm. Therefore ideal for photomicrography. Zeiss Motic

22. Plant microtechnique and microscopy. E. Ruzin Field curvature: the sharpest focus of a lens is on a curved surface rather than on a flat plane.

23. http://micro.magnet.fsu.edu/primer/java/aberrations/curvatureoffield

25. http://micro.magnet.fsu.edu/primer/ The objective: a combination of lenses MoticZeiss PlanCP-Achromat 4x / 0.10 10x / 0.25 40x / 0.65 Magnification / Numerical Aperture

26. http://micro.magnet.fsu.edu/primer/ The objective: a combination of lenses MoticZeiss PlanCP-Achromat 4x / 0.10 10x / 0.25 40x / 0.65 5x / 0.12 10x / 0.25 40x / 0.65 100x / 1.25 oil Magnification / Numerical Aperture

27. http://micro.magnet.fsu.edu/primer/ The objective: a combination of lenses MoticZeiss PlanCP-Achromat 4x / 0.10 10x / 0.25 40x / 0.65 5x / 0.12 10x / 0.25 40x / 0.65 100x / 1.25 oil

28. http://micro.magnet.fsu.edu/primer/ The objective: a combination of lenses MoticZeiss PlanCP-Achromat 4x / 0.10 10x / 0.25 40x / 0.65 5x / 0.12 10x / 0.25 40x / 0.65 100x / 1.25 oil ∞ / 0.17 ∞ / - ∞ / 0.17

29. All the objectives mentioned here are members of the family of ICS-Optics (ICS: Infinity Color-corrected System). These objectives project their images to “infinity” first. Only the tube lens produces an intermediate image – to be more precise, at a distance of approx. 164.5 mm behind the tube lens. This distance was chosen to comply with the classical tube length.

30. http://micro.magnet.fsu.edu/primer/ The objective: a combination of lenses MoticZeiss PlanCP-Achromat 4x / 0.10 10x / 0.25 40x / 0.65 5x / 0.12 10x / 0.25 40x / 0.65 100x / 1.25 oil ∞ / 0.17 ∞ / - ∞ / 0.17

31. A coverslip is part of the image-forming system, a lens element, its power has been taken into account by the manufacturer, its thickness and the making of the glass will affect the deviation of the light.

32. The thickness of the coverslip and the refractive index of the glass will have an effect of the light path 1 thickness= 0.13 to 0.17 mm Satisfactory for NA ≤ 0.4. Klosevych, 1989

33. http://micro.magnet.fsu.edu/primer/ The objective: a combination of lenses MoticZeiss PlanCP-Achromat 4x / 0.10 10x / 0.25 40x / 0.65 5x / 0.12 10x / 0.25 40x / 0.65 100x / 1.25 oil ∞ / 0.17 ∞ / - ∞ / 0.17 Any questions?

34. The lenses: three types in your microscope The condenser The objective The eye-piece or ocular

35. http://micro.magnet.fsu.edu/primer/anatomy/oculars.html Eyepieces are not just simple lenses, but are corrected optical systems consisting of several lenses.

36. Eyepieces: magnifiers to view the intermediate Image produced by the objective and the tube lens. http://www.zeiss.com/C1256B5E0047FF3F?Open 1. Position of the intermediate image (also for the reticle) 2. Limit of the field of view (black edge of image) 3. Eye-piece optics (Ramsden ocular) 4. Position of the eyepiece pupil (pupil of the observer’s eye) 5. Focusing ring for the diopter compensation

37. The lenses: three types in your microscope The condenser The objective The eye-piece or ocular There will be specifications on these lenses Motic WFPL 10x / 20 Glasses symbol WF for Wide Field of view PL to match the objective correction Magnification 10X Field number which refers to the diameter (in mm) of the fixed diaphragm in the eyepiece.

38. The lenses: three types in your microscope The condenser The objective The eye-piece or ocular There will be specifications on these lenses Zeiss PL 10x / 18 Glasses symbol Designed for eyeglass wearers. The exit pupil is at a considerable distance from the eyepiece.

39. Specimen Intermediate image Final image

40. What is the main purpose of the microscope? The condenser The objective The eye-piece or ocular

41. The condenser The objective The eye-piece or ocular To condense and focus light onto the specimen What is the main purpose of the microscope?

42. The condenser The objective The eye-piece or ocular To condense and focus light onto the specimen To form a clear intermediate image What is the main purpose of the microscope?

43. The condenser The objective The eye-piece or ocular To condense and focus light onto the specimen To form an intermediate image To form the final image What is the main purpose of the microscope?

44. Specimen Intermediate image Final image Any questions?

45. Magnification: apparent increase in size Resolution: the minimum distance between 2 dots that can be discerned What is the main purpose of the microscope?

46. http://micro.magnet.fsu.edu/primer/lightandcolor Airy disc: defined as the region enclosed by the first minimum of the Airy pattern and contains 84 % of the luminous energy.

47. http://micro.magnet.fsu.edu/primer/ Resolution: the minimum distance between 2 dots that can be discerned

48. The smaller the diameter of the Airy disc produced by a lens, the higher is the resolving power of that lens, the better you can separate two distinct points. http://micro.magnet.fsu.edu/primer/

49. The larger the numerical aperture of a lens, The smaller the Airy disc, The smaller and better the resolution. http://micro.magnet.fsu.edu/primer/ Resolution = (0.61 λ) / Numerical Aperture

50. n: refractive index of the medium in the object space Θ: angular aperture Resolution = (0.61 λ) / (n x sin θ) λ: wavelength of light Airy disk sizes vary with changes in objective numerical aperture and illumination wavelength.

51. Resolution = (0.61 λ) / Numerical Aperture n: refractive index of the medium in the object space Θ: angular aperture. Resolution = (0.61 λ) / (n x sin θ) λ: wavelength of light

52. Angular aperture is a measure of the number of the highly diffracted image-forming light rays captured by the objective expressed as the angle between the microscope optical axis and the direction of the most oblique light rays captured by the objective. = θ http://micro.magnet.fsu.edu/primer/ Specimen Objective

53. In theory, θ cannot be superior to 90 o sin(θ) cannot be superior to 1. In the best microscopes, θ is about 70 o with a sine of 0.94. Resolution = (0.61 λ) / (n x sin θ) The other limitation for the objective is the refractive index of the medium; generally, it is air with n=1.

54. Resolution = (0.61 λ) / (n x sin θ) where 0.61 represents the degree to which image points can overlap and still be recognized by an observer as separate points the lower the wavelength, the lower and better the resolution, If in air and maximum θ, then r = (0.61 λ) / 0.94

55. Resolution = (0.61 λ) / (n x sin θ) If in air and maximum θ, then r = (0.61 λ) / 0.94 Monochromatic light better Possibility to use coloured filters If blue light, λ = 450 nm, r = 274.5 / 0.94 = 292 nm If green light, λ = 550 nm, r = 335.5 / 0.94 = 356 nm If UV light, λ = 250 nm, r = 152.5 / 0.94 = 162 nm Because the λ of an e - is much shorter than a that of a photon, resolution is much greater in an e - microscope (0.2 nm). The shorter the λ, The better the resolution

56. Resolution = (0.61 λ) / (n x sin θ) To further improve resolution, to visualize minute specimens, one could use a medium with a n higher than that of air.

57. Use of immersion lenses allows more light to be collected More points to form an image, There is no diffraction of light because of the homogeneity of the refractive indices. Better resolution http://micro.magnet.fsu.edu/primer/

59. Resolution = (0.61 λ) / (n x sin θ) If maximum θ and blue light, then r = 274 / (n x 0.94) If air (n = 1), r = 274 / 0.94 = 292 nm If 50% glycerol, r = 274 / (1.4 x 0.94) = 208 nm If generic imm. oil, r = 274 / ( 1.515 x 0.94) = 192 nm If permount, r = 274 / (1.525 x 0.94) = 191 nm If Canada balsam, r = 274 / (1.545 x 0.94) = 189 nm The larger the refractive index of the medium, The better the resolution.

60. angular aperture The larger the angular aperture, The higher the numerical aperture, The more light the lens can capture, The more information the lens can transmit. NA = n sin(θ) The larger the numerical aperture of a lens, The smaller the Airy disc, The better the resolution. Any questions?

61. http://micro.magnet.fsu.edu/primer/ The objective: a combination of lenses MoticZeiss PlanCP-Achromat 4x / 0.10 10x / 0.25 40x / 0.65 5x / 0.12 10x / 0.25 40x / 0.65 100x / 1.25 oil ∞ / 0.17 ∞ / - ∞ / 0.17 r = (0.61 λ) / NA

62. It would be a pity if the intermediate image produced with such sophisticated optics were to be impaired just before it reaches the eye because of poor handling of the microscope. Koehler illumination

63. Koehler or Köhler illumination Switch on the microscope Check that the light is on http://www.zeiss.com

64. Koehler or Köhler illumination Open wide the field diaphragm: the spot of light should be at its maximum diameter http://www.zeiss.com

65. Koehler or Köhler illumination http://www.zeiss.com Open wide the iris diaphragm of the condenser. The small light spot shows its maximum brightness

66. Koehler or Köhler illumination http://www.zeiss.com Place slide on stage Reduce brightness of light if need be Position the oculars so that you are comfortable when looking through them Adjust your view by focussing the diopter compensation ring

67. First, look into the distance with your eyes relaxed and then into the eyepieces – without changing the setting of your eyes. Only then should you set the inter-pupillary distance of the eyepieces via the folding bridge until you see only one circle instead of two. Remember to use both your eyes for viewing. Relaxed viewing is important

68. Keep your distance Microscopes for teaching labs are usually designed for eyeglass wearers. Therefore, the exit pupil of the eyepiece is at a considerable distance from the eyepiece. Users who do not wear eyeglasses should also keep this distance to permit the entire light from the microscope to find its way to the iris of the eye. If you slowly move your head to and fro in front of the eyepieces, you will soon find the optimum, relaxed posture allowing you to see the entire circle of the field of view.

69. Koehler or Köhler illumination http://www.zeiss.com Focus grossly on the specimen

70. Koehler or Köhler illumination http://www.zeiss.com Close the field diaphragm Focus the condenser by using the focussing knob on the left of the microscope

71. Koehler or Köhler illumination http://www.zeiss.com When the condenser is focussed, you will see the sharp edges of the field diaphragm

72. Koehler or Köhler illumination http://www.zeiss.com Center the light with the centering knobs placed below the stage You should now see a centered image of the specimen surrounded by black

73. Koehler or Köhler illumination Open now the field diaphragm Do open just enough to fill the field of view Do not touch again the condenser knob, because your condenser is now focussed

74. I was taught to set up Koehler illumination with a focussed slide on the microscope stage but with the specimen out of the field-of-view so that its staining could not interfere with the light source. I was also taught to use whenever possible a blue filter so that a monochromatic light with a low λ is obtained.

75. Koehler or Köhler illumination http://www.zeiss.com Remove gently one of the eye-pieces (us. the left). Look down the tube to see the back focal plane of the objective

76. Koehler or Köhler illumination http://www.zeiss.com

77. Koehler or Köhler illumination http://www.zeiss.com Three elements essential for optimal results: circular outline of the objective aperture, iris opening of the aperture diaphragm of the condenser, image of the light source.

78. Koehler or Köhler illumination http://www.zeiss.com Adjust the size of the light disc by swinging the iris diaphragm of the condenser Three elements essential for optimal results: circular outline of the objective aperture, iris opening of the aperture diaphragm of the condenser, image of the light source.

79. Koehler or Köhler illumination A compromise between resolution and contrast 9/10: best resolution 2/3: best contrast

80. For research purposes, you should set-up Koehler each time you change objectives.

81. 9/10 position of aperture diaphragm Maximum resolution 2/3 position of aperture diaphragm Maximum contrast Maximum depth of detail 1/2 position of aperture diaphragm Creation of artefacts Klosevych, 1989

82. 2 3 1: Aperture diaphragm = NA obj lack of contrast and low visibility 2: Aperture diaphragm at 90% better contrast & excellent resolution 3: Aperture diaphragm at 50% excessive diffraction & creation of artefacts 1 Microscopy and photomicrography. R.F. Smith, CRC Press

83. Tips given by Zeiss: Relaxed viewing is important Keep your distance Exclusively for eyeglass wearers: a little test Avoid the use of force Protect your investment: the dust cover Please avoid “do-it-yourself” work on the microscope

84. http://micro.magnet.fsu.edu/primer Two invaluable web-sites to visit when you want to learn more about microscopy: http://www.zeiss.com (Microscopy from the beginning)