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)