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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
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 theposition 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
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
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___
Homework 8
Work through the Microscope Illumination Section under Microscope Anatomy at: http://micro.magnet.fsu.edu/primer/index.html
Objective Specifications
Why can a high resolution objective cost $4000?: Correction of Geometrical
Aberrations
• Monochromatic: Spherical, Coma, Astigmatism, Distortion, Curvature of Field
• Chromatic: Longitudinal, Lateral
Spherical Aberration
Coma
B'
B
a. negative
b. positive, (Pluta)
B'
B
Astigmatism
Distortion
Distortion
(Pluta)
Curvature of Field
(Pluta)
Curvature of Field
Chromatic AberrationChromatic Aberration
a. Longitudinal
b. Latteral
4 3 2 1 0 V B G R
RB
Chromatic (and Spherical) Aberrations Corrected by the Achromatic Doublet
Chester More Hall Makes the Discovery in 1730, diddles, andJohn Dolland Learns the Secret,and Patents it in about 1759.
The 3 Classes of Objectives
Chromatic and Mono-Chromatic Corrections
Chromatic Correction
Plan Objectives
Apochromat Objectives
Mechanical LengthsStandard Mechanical Connecting Lengths
45 (60) mmPar-FocalDistance
WorkingDistance
Slide
Coverslip Shoulder ofObjective
Primary ImagePlane
Shoulder ofOcular
10 mm
Objective Specifications
Parfocal Distance and Turret Mount
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
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
Importance of Objective NA
• Light Collection: I ~ NAobj2/Mtot
2
• Lateral Resolution:
-Fluorescence: r = 0.61/NAobj
-Trans-Illumination: r = /(NAobj + NAcond)
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)
Objective Special Use
• Phase Contrast Ph1, Ph2, Ph3
• Polarized Light Pol, DIC
• UV fluorescence U-, U340/380
• Darkfield Iris in BFP
Dry Objectives must correct for refractions at air/coverslip interface; Oil immersion
Increases NA
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
Correction Collars for Spherical Aberration
Muli-Immersion and Variable Coverslip Thickness Objectives
Front Element Design in Oil Immersion Objectives
Why Use A Water Immersion Objective
Anti-Reflection Coatings Reduce Scattered Light
Anti-Reflection Coatings Reduce Scattered Light
5
4
3
2
1
0 400 500 600 700 800 nm
Uncoated glass n= 1.52
Single layer coating
Multilayer coating
Relative Transmission of Objectives (%)
• Name 320 350 400 500600nm
• 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
Abbe Condenser
Achromatic Condenser
Aplanatic Condenser
Swing-Out Top Lens Condenser
Ocular or Eyepiece
Ocular Designs
Stage and Eyepiece Micrometers for Microscope Distance Measurements
100m
Scale under coverslip
a. Stage Micrometer:
b. Ocular Micrometer:
1 2 3 4 5 6 mm
Place on the rim at the frontfocal plane of the ocular.
Projection Oculars
Elements of a Simple Stage
Higher Quality Specimen Stage
Circular stage
FRAP Scope with Cooled CCD Camera
Inverted Microscope Stage
Inverted Microscopes and Micromanipulation
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)