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Practical Aspects

Practical Aspects. See: 1) http://micro.magnet.fsu.edu/primer/anatomy/anatomy.html 2)http://micro.magnet.fsu.edu/primer/anatomy/specifications.html 3) Murphy: Pgs 50-60 E. D. Salmon University of North Carolina at Chapel Hill. Homework Problem 5.

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Practical Aspects

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  1. Practical Aspects See: 1)http://micro.magnet.fsu.edu/primer/anatomy/anatomy.html 2)http://micro.magnet.fsu.edu/primer/anatomy/specifications.html 3) Murphy: Pgs 50-60 E. D. Salmon University of North Carolina at Chapel Hill

  2. Homework Problem 5 The light source is a 3-mm square tungsten filament. The design of the illumination system requires that (1) the filament be 300 mm away from the condenser diaphragm, (2) the image of the filament must be in focus at the condenser diaphragm and (3) the filament must be 15-mm square to fill the condenser aperture with light. Assuming the lamp collector lens is an ideal thin lens, determine the focal length, and the position of the collector lens between the lamp filament and the condenser diaphragm. Ans: Eqn 1: M = i/o = 15/3 = 5; i = 5o Eqn 2: i+ o = 300; 5o +o = 300; o = 300/6 = 50; i = 250 Eqn 3: 1/i +1/o = 1/f; 1/250 +1/50 = 1/f; f = 41.67 mm

  3. Homework Problem 6 A field diaphragm or iris is placed in front of the collector lens as shown for the Koehler illumination system. The field iris is used to control the illuminated area of the specimen. The condenser lens is translated back and forth along the central axis until an image of the field diaphragm is in sharp focus on the specimen. When the opening of the field diaphragm is 20 mm, the image on the specimen must be 2 mm in diameter. In addition, the field diaphragm is placed 160 mm away from the condenser lens. What is the focal length of the condenser needed to meet these requirements? Answer: Eqn. 1): 1/o +1/i = 1/f, or 1/160 +1/i = 1/f and Eqn. 2): M = i/o = .1, so i = .1 *160 = 16 mm Solving Eqn 1 1/160 +1/16 = 1/f; f = 14.5 mm

  4. Homework Problem 7 Indicate “In-focus” or “out-of-focus”for: Field Diaphragm Light Source at: Field Diaphragm ____In_______ ______Out___ Condenser Diaphragm ____Out______ ______In____ Specimen ____In______ ______Out___ Objective BFP ____Out______ ______In____ Ocular FFP ____In_______ ______Out___ Ocular BFP (Ramdens Disk) ____Out______ ______In____ Retina (or camera detector) ____In_______ ______Out___

  5. Homework 8 Work through the Microscope Illumination Section under Microscope Anatomy at: http://micro.magnet.fsu.edu/primer/index.html

  6. Objective Specifications

  7. Why can a high resolution objective cost $4000?: Correction of Geometrical Aberrations • Monochromatic: Spherical, Coma, Astigmatism, Distortion, Curvature of Field • Chromatic: Longitudinal, Lateral

  8. Spherical Aberration

  9. Coma

  10. Astigmatism

  11. Distortion

  12. Curvature of Field

  13. Chromatic Aberration

  14. Chromatic (and Spherical) Aberrations Corrected by the Achromatic Doublet Chester More Hall Makes the Discovery in 1730, diddles, and John Dolland Learns the Secret, and Patents it in about 1759.

  15. The 3 Classes of Objectives Chromatic and Mono-Chromatic Corrections

  16. Chromatic Correction

  17. Plan Objectives

  18. Apochromat Objectives

  19. Mechanical Lengths

  20. Objective Specifications

  21. Parfocal Distance and Turret Mount

  22. Tube lens and Chromatic Correction:Leica-200mm, in tube lens;Zeiss-160 mm, in tube lens;Olympus-180 mm, in tube lensNikon-200 mm, in objective

  23. Working Distance of Some Objectives (mm) • Zeiss PlanApo100X/1.4 oil……..0.1 • Olympus “ “ “ “ …….0.2 • Nikon PlanApo 60X/1.4 oil……..1.1 • Zeiss PlanApo 40X/1.2 water…..0.22 • Olympus “ 60X “ “ …..0.22 • Zeiss Plan Acro 100X/1 water…..1.00 • Nikon Fl 40X/.75 air…………….0.51 • Nikon Fl 40X/.7 LWD air……….2.? • Nikon Fl 10X/.30 air…………….10

  24. Importance of Objective NA • Light Collection: I ~ NAobj2/Mtot2 • Lateral Resolution: -Fluorescence: r = 0.61l/NAobj -Trans-Illumination: r = l/(NAobj + NAcond)

  25. Objective Immersion Type • Dry (no marking) • Water (direct) W.WI • Water (coverglass) W Korr • Glycerol G, Gly • Oil Oil, Oel • Multi-immersion Imm (Water, glycerol, oil)

  26. Objective Special Use • Phase Contrast Ph1, Ph2, Ph3 • Polarized Light Pol, DIC • UV fluorescence U-, U340/380 • Darkfield Iris in BFP

  27. Dry Objectives must correct for refractions at air/coverslip interface; Oil immersion Increases NA

  28. Cover Slip (see below) and Slide Thickness: Slide is 1 mm thick; both have n= 1.52 crown glass • # 0: 0.1-0.13 mm • # 1: 0.13-0.17 mm • # 1.5: 0.15-0.20 mm; 0.17 mm for Dry Obj. • # 2: 0.17-0.25 mm • # 3: 0.25-0.5 mm

  29. Correction Collars for Spherical Aberration

  30. Muli-Immersion and Variable Coverslip Thickness Objectives

  31. Front Element Design in Oil Immersion Objectives

  32. Why Use A Water Immersion Objective

  33. Anti-Reflection Coatings Reduce Scattered Light

  34. Anti-Reflection Coatings Reduce Scattered Light 5 4 3 2 1 0 Uncoated glass n= 1.52 Single layer coating Multilayer coating 400 500 600 700 800 nm

  35. Relative Transmission of Objectives (%) • Name 320 350 400 500 600nm • Fluor 40X/1.3 16 66 80 90 91 • “ “ “ 29 79 88 95 99 • 40X/0.9 water 56 88 • Planapo 40X/1.2 water 20 54 86 89 92

  36. Abbe Condenser

  37. Achromatic Condenser

  38. Aplanatic Condenser

  39. Swing-Out Top Lens Condenser

  40. Ocular or Eyepiece

  41. Ocular Designs

  42. Stage and Eyepiece Micrometers for Microscope Distance Measurements

  43. Projection Oculars

  44. Elements of a Simple Stage

  45. Higher Quality Specimen Stage

  46. Circular stage

  47. FRAP Scope with Cooled CCD Camera

  48. Inverted Microscope Stage

  49. Inverted Microscopes and Micromanipulation

  50. Modern Upright Research Light Microscope (1995) *Bright, High Contrast Optics *Epi-Fluorescence *Phase-Contrast *Polarization *DIC *Diffraction Limited Resolution *Multiple Ports *Auto. Photography *Electronic Imaging- (Video---CCD)

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