Post on 28-Jan-2016
transcript
IVIV
Practical Aspects of Lens DesignPractical Aspects of Lens Design
October 2008October 2008
Rudi Rottenfusser – Carl Zeiss MicroImaging
The Objective
The Most Important Microscope Component
Lenses
Glass Parameters (excerpt)
• Refractive Index• Dispersion• Thermal Expansion Coeff.• Spectral Transmission• No Autofluorescence• No Schlieren, bubbles, inclusions• Reflectivity• Film Adhesion (AR coatings)• Chemical Resistance • Resistance to Humidity• Availability
Topics
• Airy Disk / Point Spread Function• Resolution Criteria – Rayleigh, Sparrow, etc.• Definition: Depth of field / focus
• Aberrations
• The Objective – What do the markings mean– What to consider when selecting an objective
• Website - References
What happens to the image of the object when it travels through the various microscope components?
1) No Lens Aberrations (“perfect lens”)
On Axis image
Wave fronts
In phase ½λ Out of phase
NA: 0.4 0.8 1.3
Relative sizes of Airy disk (D) as a function of Numerical Aperture
DD
D
Airy Disk
D = Diameter of Airy disk in image
plane ObjectiveNAD
22.1
D
Resolution in z as defined by the “Airy Body” is
ObjectiveNAD
22.1
2NA
nz
Airy Disk
ObjectiveNAd
61.0
Rayleigh Limit of Lateral Resolution
d = ½ D
D
• Minimum distance dmin is reached, when the principal maximum of object 1 (center of Airy Disk) coincides with first minimum of object 2
• Intensity of maxima =
20% higher than intensity of “dip” between maxima
Intensity
Xdmin
Two points at minimum distance to be “resolved”
ObjectiveNAd
61.0Rayleigh Limit of
lateral resolutiond = ½ D (radius)
Airy Disks of 2 clearly imaged separate points:
Rayleigh Criterion for resolution
Objectives - Definitions: Depth of Field / Focus
Example: C-Apochromat 40x/1,2W 1 Rayleigh unit = 0,42 µm in object plane
= 0,668 mm in image plane
eNAM
n
NA
nT
2
T = Depth of field (µm)
λ = Wavelength (µm)n = Refractive IndexM = Magnification (Image Ratio)e = diffraction-limited resolution
d in image plane (µm)From Shinya Inoué / Kenneth R. Spring book: “Video Microscopy Fundamentals - 2nd edition”Chapter 2.4.6
Different formulas (e.g. Berek 1927, Shillaber 1944, Françon 1961, Martin 1966, Michel 1981, Piller 1977)
In general: At high NA the depth of field is small and the depth of focus at the image side is large. This reverses at low magnifications!
What happens to the image of the object when it travels through the various microscope components?
2) Considering Aberrations
• Spherical Aberration
• Chromatic Aberration (axial)
• Chromatic Aberration (lateral or radial)
• Curvature of Field
• Astigmatism
• Distortion
• Internal Reflexes
Aberrations
Plan-Apochromat 40x/0.95 corr.
Correction Collar set at 0.21mm
Spherical Aberration
Plan-Apochromat 40x/0.95 corr.
Correction Collar set at 0.17mm
Spherical Aberration
Spherical Aberration
Infinite number of prisms with different angles
and, therefore, different refractive
powers
Due to the spherical character of the lens, rays do not cross over at the same Focal
Point
Spherical Aberration
Spherical Aberration is reduced by smaller aperture
Less confused “Zone of Confusion”
Fixing Spherical Aberration
Multiple Elements
Aspheric Lens
Exaggerated
Reducing Spherical Aberration
How to generate Spherical Aberration:
Incorrect Cover Glass
Maxim
um
Inte
nsi
ty in a
n
image o
f a p
oin
t obje
ct
How to generate Spherical Aberration:
Incorrect Cover Glass
Reso
luti
on [
µm
]
(Fu
ll W
idth
, H
alf
Max)
Use 0.170 mm thick cover slips !
Types and Thickness Ranges
No.0 ......... 0.08 - 0.12 mm
No.1 ......... 0.13 - 0.17 mm
No.1.5....... 0.16 - 0.19 mm
No.2 ......... 0.19 - 0.23 mm
No.3 ......... 0.28 - 0.32 mm
No.4 ......... 0.38 - 0.42 mm
No.5 ......... 0.50 - 0.60 mm
Choose the right cover glass!
No.1.5....... 0.16 - 0.19 mm
40x/1.3 Oil immersion objective – Energy at different depths of penetration z in water
How to generate Spherical Aberration:
Focusing deeper into the sample
Aquaeus Medium
Water Immersion
Benefit of Water Immersion
Objectives (with cover slip correction)
Cover Slip n=1.52
n~1.3
n~1.3
Chromatic Aberration (Axial)
Remember “Dispersion” of Light!
Fixing Chromatic Aberration
The classic “Achromat” (Doublet)
n ≈ 1.55 n ≈ 1.65
Objectives - Definitions:
Corrected Wavelength (nm):
UV VIS IR
Plan Neofluar - - (435) 480 546 - 644 - -
Plan Apochromat - - 435 480 546 - 644 - -
C-Apochromat 365 405 435 480 546 608 644 - -
IR C-Apochromat - - 435 480 546 608 644 8001064
Objectives - Best Focus
1 RU
480 nm546 nm
644 nm
RU = Rayleigh Unit
Image
Lateral Chromatic Aberration (LCA)
(Chromatic Magnification Difference)
Lateral Chromatic Aberration (LCA)
Lateral Chromatic Aberration (LCA)
Different manufacturers correct for LCA in different ways:
Leica:The tube lens corrects for a fixed amount of LCA
Nikon:The objectives themselves are fully corrected
Olympus:The objectives themselves are fully corrected
Zeiss:The tube lens corrects for objectives with different LCA’s
Sagittal
Tan
gen
tial
Astigmatism
Spherical Aberration
Astigmatism
Coma
Intensity Distribution
in Airy Disk
Curvature of field: Flat object does not project a flat image
(Problem: Camera Sensors are flat)
f1
image
object
f2
Objectives - Definitions: Flatness
Flatness at 435nm:
SF 18 SF 25
Plan Neofluar 1 R
Plan Apochromat < 0,5 R
C-Apochromat 0,6 - 1 R 1 - 2R
Distortion
Pincushion
Barrel
Internal Straylight
% R
[nm]400 700
4
2Anti Reflection (AR) Coating
uncoated
Single layer
Multi layer
Reflexes (unwanted reflections)
~ 1%
~ 0.1%
Internal Straylight
How does it work?
Anti-Reflex (AR) Coating
/4
Questions?
Objective Markings
Thread Diameter0.8”x1/36” (RMS) 27mm, 25mm
Mounting Distance (Specimen to Flange):22, 45, 60, 75mm, others?
..1.2589254.101010R
“Standard” Sequence
Magnification
1.251
1.252
1.253
1.254
1.255
1.256
1.257
1.258
1.259
1.2510
1.2511
=
=
=
=
=
=
=
=
=
=
=
1.25x
1.6x
2x
2.5x
3.2x
4x
5x
6.3x
8x
10x
12.5x…
Why these strange numbers?
What to consider when selecting an objective:
1. Magnification
2. Working Distance
3. Numerical Aperture (NA) – Resolution / Depth of Field
4. Image Quality – minimized Aberrations (spherical, chromatic, flatness of field, astigmatism, coma, distortion)
5. Adaptation to specific Applications (Contrasting Techniques, Cover Slips, Chambers, Shape of front lens for Access)
6. Spectral Transmission (Visible,IR,UV?)
7. No Autofluorescence
8. No Strain (Pol)
9. Temperature Tolerance
10. Temperature Isolation (heating!)
11. Chemical Resistance
12. Electrical Shielding
13. Minimal Path Gradient (2-photon)
14. Perfect Parfocality and Parcentricity
15. Compact yet durable
16. Inexpensive
For a comprehensive lookup of objectives, consult Websites!(Example for Zeiss: www.zeiss.com/campus)
Description of Classes of Objectives
Example (Screenprint)
Please refer to appropriate web sites from
Leica
Nikon
Olympus
Thank you, and do enjoy your microscopes !
Rudi Rottenfusser
Office: 508/289-7541
Cell: 508/878-4784
Email: rrotte@mbl.edu
Microscopy Support: 800/233-2343
Imaging Support: 800/509-3905
Website: www.zeiss.com/micro
Educational Site: www.zeiss.com/campus