Objectives

1
Objectives
2
Parameters of Objectives
3
Plan-Apochromate 63x, N.A. 1.4
Inoué, p.59
4
Optical Magnification

• Optical magnification w/ eyepieces
  Magnification of Lens 63x x Magnification of
  tube lens (e.g. Optovar) 1x x Magnification of
  eyepiece 10x Total optical magnification 630x

5
Magnification of Camera System

• Optical magnification w/ camera Magnification
  of Lens 63x x Magnification of tube lens (e.g.
  Optovar) 1x x Magnification of camera
  adapter 0.63x Total optical magnification 40x
• Electronic magnification Diagonal of
  monitor 17 inch / Diagonal CCD chip 2/3 11mm
  0.43 inch Total electronic magnification 40x
• Magnification of camera system Optical
  magnification 40x x Electronic
  magnification 40x Total optical
  magnification 1600x

6
Useful Magnification

• The resolution is limited by the Numerical
  Aperture, not the magnification
• Eyepiece
• The overall magnification of a microscope should
  be higher than 500 times, but less than 1000
  times the objective aperture.
• Camera or scanning microscope
• Use Nyquist criterion

7
Field of View

• Eyepieces

FOV field number diameter of intermediate
image plane (e.g. 23mm)

• Camera

ChipSize diagonal dimension of CCD chip (e.g.
11mm for 2/3 Axiocam chip)
8
Depth of Field

• Area above and below focal plane still perceived
  as sharp

9
Magnification, Field of View and Depth of Field
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Tube Lens and Magnification
Zeiss ICS Microscope ftube 164.5 mm Olympus ?-Optics Microscope ftube 180 mm Leica, Nikon ?-Optics Microscope ftube 200 mm
Zeiss 100x/1.4 Objective fObjective 1.65 mm fObjective 1.65 mm fObjective 1.65 mm
Actual Magnification 100x 109x 122x
Brightness comparisons 1 0.84 0.68
Olympus 100x/1.4 Objective fObjective 1.8 mm fObjective 1.8 mm fObjective 1.8 mm
Actual Magnification 91x 100x 111x
Brightness comparisons 1.2 1 0.8
Nikon or Leica 100x/1.4 Objective fObjective 2 mm fObjective 2 mm fObjective 2 mm
Actual Magnification 82x 90x 100x
Brightness comparisons 1.5 1.2 1
Zeiss 63x/1.4 Objective fObjective 2.6 mm fObjective 2.6 mm fObjective 2.6 mm
Actual Magnification 63x 69x 77x
Brightness comparisons 2.5 2.1 1.7
11
Numerical Aperture Determines Resolution
Large Angle
Small Angle
Low NA
High NA
Large Details
Small Details
12
Higher N.A. Results in Brighter Images
Alpha Fluar 100x/1.45Oil
PlanNeo100x/1.3Oil
PlanApo 100x/1.4Oil
NoteThe transmission in the blue in the Alpha is
5 times higher as with the PlanApo! NotePinhole
and collimator settings were not changed between
Alpha Fluar and the other objectives.
13
Higher N.A. Results in Brighter Images
Transmitted light
10x 0.5 NA is 3 times brighter than 10x 0.3NA
Epifluorescence
10x 0.5 NA is 8 times brighter than 10x 0.3NA
14
Transmittance of Objectives
C-Apochromat 40x/1.20 W Corr UV-VIS-IR (Carl
Zeiss) C-Apochromat 63x/1.20 W Corr UV-VIS-IR
(Carl Zeiss)
Brightness (transmission) ? NA2/Magnification2
15
Transmittance of Objectives
Achroplan IR 40x/0.8 W (Carl Zeiss) Ultrafluar
40x/0.6 Glyc (Carl Zeiss)
Brightness (transmission) ? NA2/Magnification2
16
Optical Aberrations Imperfections in optical
systems

• Chromatic aberration
• Caused by variations in refractive index for
  different wavelengths
• Spherical or geometrical aberration
• Related to spherical nature of lens and
  approximations used to obtain Gaussian lens
  equation
• Field Curvature
• Caused by lenses with curved surfaces

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Field Curvature
Center in Focus
Edges in Focus
Entire Field in Focus
18
Spherical Aberration
19
Chromatic Aberration
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Chromatic Aberration Dispersion Depends On
Wavelength
Material Blue (486nm) Yellow (589nm)
Red (656nm) Crown Glass 1.524 1.517
1.515 Flint Glass 1.639 1.627 1.622
Water 1.337 1.333 1.331 Cargille
Oil 1.530 1.520 1.516
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Objective Correction for Optical Aberration
ObjectiveType SphericalAberration ChromaticAberration FieldCurvature
Achromat 1 Color 2 Colors No
Plan Achromat 1 Color 2 Colors Yes
Fluorite 2-3 Colors 2-3 Colors No
Plan Fluorite 3-4 Colors 2-4 Colors Yes
Plan Apochromat 3-4 Colors 4-5 Colors Yes



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Correction of Infinity Optics is Manufacturer
Dependent
Manufacturer Tube Lens Focal Length mm Parfocal Distance mm Thread Type Correction for Lateral Chromatic Aberation
Leica 200 45 M25 Fixed amount in tube lens
Nikon 200 60 M25 Objective
Olympus 180 45 RMS Objective
Zeiss 160 45 RMS M27 Automatic in tube lens
23
Match Refractive Index of Sample and Immersion
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Refractive Index

• Air 1
• Water 1.3381
• Glycerin 1.455
• Immersion Oil 1.5180
• Cover slip 1.525
• Gel/Mount (Biomeda Inc) 1.3641
• Methyl Salicylate (Sigma) 1.5409
• Dimethyl Sulfoxide (Sigma) 1.4836
• VectaShield (Vector Labs) 1.4577
• DPX (Fluke) 1.5251

Source Confocal Listserver contribution ofY.S.
Prakash 4-176 Anes. Res., SMH Mayo Clinic
Rochester, MN 55905 www.zeiss.de/micro J.P.Pawley
(Edt.) Handbook of Biological Confocal
Microscopy p. 320
25
PSF Along Z With Refractive Index Mismatch
z0µm
z2µm
z4µm

• Medium n1.33
• Oil n1.51
• Z distance sample-cover slip

z10µm
3.20µm
z20µm
z60µm
Inoué, p.54
26
Mixing Oil Immersion with Sample in Water Medium
Medium Oil
Medium Water
Focus set 5 µm
Focus set 20 µm
Source Hell Stelzer Lens Aberrations in
Confocal Fluorescence Microscopy In Pawley
(Edt.) Handbook of Biological Confocal Microscopy
27
CTF Water Immersion Objective w/ Sample in Water
Inoué, p.54
28
CTF Oil Immersion Objective w/ Sample in Water
Inoué, p.54
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Use 1.5 (0.170 mm) coverslips

• Standard thicknessNo.0 ......... 0.08 - 0.12
  mmNo.1 ......... 0.13 - 0.17 mmNo.1.5........
  0.16 - 0.19 mmNo.2 ......... 0.19 - 0.23 mmNo.3
  ......... 0.28 - 0.32 mmNo.4 ......... 0.38 -
  0.42 mmNo.5 ......... 0.50 - 0.60 mm

Acceptable deviations 0,01 mm for N.A. gt
0.7 0,03 mm for 0.3 lt N.A. lt 0.7
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The Effect of Coverslips
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Effect of Error in Cover Glass Thickness
Inoué, p.54
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Diffraction Pattern w/ Spherical Aberration
Inoué, p.63
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Z-Scan of Interface Slide - Embedding Medium
Schwertner et al., 2004
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Optimization Procedure for Correction Collar

• Z-scan at interface slide-embedding medium
• Measure Intensity I for different settings R of
  collar
• Calculate C(R)
• Maximize C(R)

Schwertner et al., 2004
35
Objectives for NLO applications
Objective lens Working Distance mm Dispersion Parameter fsec2 Dispersion Parameter fsec2 Pulse broadening factor of 100 fsec pulse Max. PTD fsec ()
Objective lens Working Distance mm On axis Variation Pulse broadening factor of 100 fsec pulse Max. PTD fsec ()
IR-Achroplan 40x/0.8 W 3.61 1714 ? 20 1.14 -3
IR-Achroplan 63x/0.9 W 2.00 1494 ? 15 1.11 -9
Plan Neofluar 40x/1.3 oil 0.20 2328 ? 30 1.23 9
Plan Apochromat 20x/0.75 0.61 1531 ? 10 1.12 10
Table 1 Summary of specific parameters for Zeiss
microscope objective lenses recommended for
2-Photon applications. All data were measured at
800 nm. () Propagation time difference (PTD) is
calculated for the whole optical setup including
the LSM 510 NLO and the microscope stand.
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Transmission for Achroplans