www.iap.uni-jena.de Microscopy Lecture 1: Optical System of the Microscopy I 2012-10-15 Herbert Gross Winter term 2012
www.iap.uni-jena.de
Microscopy
Lecture 1: Optical System of the Microscopy I
2012-10-15
Herbert Gross
Winter term 2012
Lectures:
Alexander Heisterkamp / IAO
Rainer Heintzmann / IPHT
Kai Wicker / IPHT
Herbert Gross / IAP
Lecture:
dates: Monday, 14.00 – 15.30
Location: HS 2, Fröbelstieg 1, Abbeanum
Seminar:
starts at xxx
bi-weekly
location: xxx
Web page on IAP homepage under ‚learning‘ provides slides and exercises
2 1 Optical System of the Microscopy I
Lecture data
3 1 Optical System of the Microscopy I
Preliminary time schedule
No Date Main subject Detailed topics Lecturer
1 15.10. Optical system of a microscope I overview, general setup, binoculars, objective lenses, performance and types of lenses, tube optics Gross
2 22.10. Optical system of a microscope II Etendue, pupil, telecentricity, confocal systems, illumination setups, Köhler principle, fluorescence systems and TIRF, adjustment of objective lenses Gross
3 29.10. Physical optics of widefield microscopes
Point spread function, high-NA-effects, apodization, defocussing, index mismatch, coherence, partial coherent imaging Gross
4 05.11. Performance assessment
Wave aberrations and Zernikes, Strehl ratio, point resolution, sine condition, optical transfer function, conoscopic observation, isoplantism, straylight and ghost images, thermal degradation, measuring of system quality
Gross
5 12.11. Fourier optical description basic concepts, 2-point-resolution (Rayleigh, Sparrow), Frequency-based resolution (Abbe), CTF and Born Approximation Heintzmann
6 19.11. Methods, DIC Rytov approximation, a comment on holography, Ptychography, DIC Heintzmann
7 26.11. Imaging of scatter
Multibeam illumination, Cofocal coherent, Incoherent processes (Fluorescence, Raman), OTF for incoherent light, Missing cone problem, imaging of a fluorescent plane, incoherent confocal OTF/PSF
Heintzmann
8 03.12. Incoherent emission to improve resolution Fluorescence, Structured illumination, Image based identification of experimental parameters, image reconstruction Heintzmann
9 10.12. The quantum world in microscopy Photons, Poisson distribution, squeezed light, antibunching, Ghost imaging Wicker
10 17.12. Deconvolution Building a forward model and inverting it based on statistics Wicker
11 07.01. Nonlinear sample response STED, NLSIM, Rabi the information view Wicker
12 14.01. Nonlinear microscopy two-photon cross sections, pulsed excitation, propagation of ultrashort pulses, (image formation in 3D), nonlinear scattering, SHG/THG - symmetry properties Heisterkamp
13 21.01. Raman-CARS microscopy principle, origin of CARS signale, four wave mixing, phase matching conditions, epi/forward CARS, SRS. Heisterkamp
14 28.01 Tissue optics and imaging Tissue optics, scattering&aberrations, optical clearing,Optical tomography, light-sheet/ultramicroscopy Heisterkamp
15 04.02. Optical coherence tomography principle, interferometry, time-domain, frequency domain. Heisterkamp
1. Seward, Optical design of microscopes, SPIE Press, 2010
2. H. Gross / F. Blechinger / B. Achtner, Survey of optical instruments, Wiley 2007
3. W.T. Welford, Aberrations of optical systems, Hilger 1986
4. V. Mahajan, Optical Imaging and aberrations, part I: Ray geometrical, SPIE Press, 1998
5. V. Mahajan, Optical Imaging and aberrations, part II: Wave diffraction, SPIE Press, 2001
6. D. Murphy, Fundamentals of light microscopy, Wiley, 2001
4 1 Optical System of the Microscopy I
Literature for Part 1
1. Introduction
2. General optical setup of microscopes
3. Binoculars
4. Objective lenses
5. Performance and types of lenses
6. Tube optics
5 1 Optical System of the Microscopy I
Contents of 1st Lecture
1 Optical System of the Microscopy I
History of Microscopy
Ernst Abbe:
Beginning of scientific microscopy
Theory of diffraction imaging
Systematic development and correction of
objective lenses
Major landmark in understanding
microscopic resolution
Ernst Abbe memorial in Jena
Ref: W. Osten
1 Optical System of the Microscopy I
Data Sheet of Abbe
Scientific calculation of lens
systems for microscopes
Systematic development of
new materials together with
Otto Schott
First apochromate
1 Optical System of the Microscopy I
Application Fields of Microscopy
Ref: M. Kempe
Cell biology
biological development
toxicology,...
Biomedical basic
research
Material
research
Research
Medical
routine
Pharmacy
semiconductor inspection
semiconductor manufacturing
Industrial
routine
Routine
applications
Microscopy
Micro system technology
geology
polymer chemistry
Pathology
clinical routine
forensic,...
Microscopic surgery
ophthalmology
1 Optical System of the Microscopy I
Image Planes and Pupils
Principal setup of a classical optical microscope
Upper row : image planes
Lower row : pupil planes
Köhler setup
source
collector condenser objective eyepiece eyetube lens
eye
pupil
exit pupil
objective
aperture
stopfield
stop
object intermediate image image
1 Optical System of the Microscopy I
Microscopic Image Formation
Basic microscopic system with finite image location
The objective lens forms an intermediate image, this is observed by the eyepiece
Magnification of the objective lens
Magnification of the 2-stage imaging setup:
eyepiece
chief ray
marginal ray w'
intermediate
imageobjective
lens
object
eye
stube length t
1feyep
h'
h
fObj
w
pupil
obj
imaobj
m
DD
eyeobj
microf
mm
f
tm
250
1 Optical System of the Microscopy I
Microscope with Infinite Image Setup
Basic microscopic system with infinite image location and tube lens
Magnification of the first stage:
Magnification of the complete setup
Exit pupil size
eyeobj
tubemicro
f
mm
f
fm
250
obj
tubeobj
f
fm
obj
obj
objExPm
NAfNAfD
2'2
marginal
ray
eyepiece
chief ray
w'
intermediate
imageobjective
lens
object
eye
tube length t
h'
h
fobj
w
pupil tube lens
s1
feye
eye
pupil
1 Optical System of the Microscopy I
Microscope resolution
Typically, microscope optical systems are corrected diffraction limited
The resolution therefore follows the Abbe formula
Self-luminous object
Pupil is filled
Non-self-luminous object
The relative pupil filling determines
the degree of partial coherence and
the resolution
If the magnification exceeds the resolution of the eye of the human observer:
empty resolution
Typical: 500 NA .... 1000 NA
objunx
sin
61.0
objill ununx
sinsin
22.1
1 Optical System of the Microscopy I
Microscope magnification
Magnification :
objective and
eyepiece
12.5
16
20
25
32
40
50
63
80
100
125
160
200
250
320
400
500
630
800
1000
1250
1600
2000
2500
320032x
meye
eyepieces25x
20x
16x
12.5x
10x
8x
6.3x
5x
100 / 1.25
63 / 0.90
40 / 0.65
25 / 0.45
16 / 0.35
10 / 0.22
6.3 / 0.16
2.5 / 0.08
objective lenses :
magnification mobj
/ NA
m = 1000 NA
m = 500 NA
magnification
mmicro
4 / 0.10
eyeobjmicro mm
1 Optical System of the Microscopy I
Setup of the Microscope
Standards in microscopic setups
Terms and normalized lengths
Differences in the numbers depending on the vendor are possible
3.5 mm
92.5 mm
w
object
planemarginal
ray
chief ray
back focal,
plane
exit pupil
F'
matching
length 45 mm
tube
lens
optical tube length t
reference plane
objective shoulder
rear stop
intermediate
image
focal length tube lens
ftube
= 165 mm
eyepiece
matching
length
10 mm
Dinter
field
size
Dobj
objective lens tube system
mechanical tube length
1 Optical System of the Microscopy I
Upright-Microscope
Sub-systems:
1. Detection / Imaging path
1.1 objective lens
1.2 tube with tube lens and
binocular beam splitter
1.3 eyepieces
1.4 optional equipment
for photo-detection
2. Illumination
2.1 lamps with collector and filters
2.2 field aperture
2.3 condenser with aperture stop
eyepiece
photo
camera
tube lens
objective
lens
lamp
lamp
collector
collector
condensor
intermediate
image
binocular
beamsplitter
object
film plane
1 Optical System of the Microscopy I
Inverse Microscope
Additional relay-system in
tube
Applications:
1. liquid covered probe
2. sample observed through
coverglas
display
tube lens
objective
lens
lamp
lamp
collector
collector
condensor
relay
optic
binocular
beamsplitter
object
film
plane
eyepiece
Stereo microscopes Upright microscopes Inverse microscopes
Routine microscopes
From M. Kempe
1 Optical System of the Microscopy I
Microscope Stands
1 Optical System of the Microscopy I
Basic Setup
Eyepieces images a finite image of an instrument to infinity
Viewing with a relaxed eye
Magnification
Problem: - Location of the eye pupil inside
- Pupil of the eyepiece outside : large height of chief ray
Aperture : usually small
Objective
exit pupil
intermediate
focus
Eyepiece
Eye pupil
tube length
eyepiecef
mm250
instrument
pupil
x'
z'
e x
s'
F'
f1
f2
field
lens
intermediate
image
stopeye
lens
eye
pupil
1 Optical System of the Microscopy I
Eyepiece: Notations
Field lens reduces chief ray
height
Eye lens adapts pupil diameter
Matching of
1. Field of view
2. Pupil diameter
3. Pupil location
Eye relief :
- distance between last lens surface and
eye cornea
- required : 15 mm
- with eyeglasses : 20 mm
Pupil size: 2-8 mm
1 Optical System of the Microscopy I
Evolution of Eyepiece Designs
Monocentric
Plössl
Erfle
Von-Hofe
Erfle diffractive
Wild
Erfle type(Zeiss)
BerteleScidmore
Loupe
Erfletype
Bertele
Kellner
Ramsden
Huygens
Kerber
König
Nagler 1
Nagler 2
Bertele
Aspheric
Dilworth
1 Optical System of the Microscopy I
Kellner Eyepiece
Classical setup
Corresponds to Ramsden type
Field lens moved
Eye lens achromatized
-1.000 0.000 1.000
0.250
0.500
0.750
1.000
LONGITUDINALSPHERICAl ABER.
DIOPTER
-3.000 0.000 3.000
2.625
5.250
7.875
10.500
tan sag
ASTIGMATICFIELD CURVES
DIOPTER
-20.00 0.00 20.00
2.625
5.250
7.875
10.500
DISTORTION
Distortion (%)
0°
10°
20°
24°
20
arc
min
1 Optical System of the Microscopy I
Bertele Eyepiece
High performance system
Enlarged eye relief
Larger field of view: 2 x 33°
Larger diameter necessary
More lenses necessary for correction
-1.000 0.000 1.000
0.250
0.500
0.750
1.000
LONGITUDINALSPHERICAl ABER.
DIOPTER
-3.000 0.000 3.000
3.750
7.500
11.250
15.000
tan sag
ASTIGMATICFIELD CURVES
DIOPTER
-20.00 0.00 20.00
3.750
7.500
11.250
15.000
DISTORTION
Distortion (%)
0°
10°
20°
33°
20
arc
min
1 Optical System of the Microscopy I
Microscope Objective Lens
Focal length as function of magnification
for f=165 mm - tubelens
Large angles in object space
Colour coding of magnification
0 20 40 60 80 1000
5
10
15
20
25
fobj
[mm]
mmicro
Colour Magnification
black 1x
grey 2x
red 4x
yellow 10x
green 20x
bright blue 40x
blau 50x
dark blue 60x
white 100x
NA = 0.95 / 72°
NA = 0.85 / 58°
NA = 0.60 / 37°
NA = 0.30 / 17°
axis
object
planeaperture angles
1 Optical System of the Microscopy I
Microscopic Objective Lens: Legend
Legend of data, type
and features
immersion
contrast
magnification
oil
water
glycerin
all
magnification
numerical aperture
additional data:
- immersion
- cover glass
correction
- contrast method
mechanical adjustment
for
1. cover slide
2. immersion type
3. temperature
4. iris diaphragm
tube length
thickness of cover glass
0 without cover glass
- insensitive
type of lens
special features
(long distance,...)
1 Optical System of the Microscopy I
Microscope Objective Lens
Typical ray paths and outfits
1 Optical System of the Microscopy I
Microscopic Objective Lens: Housing
Mechanical
setup
1. thread
2. interface plane
3. spring for damage protection
4. -7. middle lens groups
8. correction ring
9. front lens group
10. socket of front lens
1 Optical System of the Microscopy I
Microscope Objective Lens: Performance Classes
Classification:
1. performance in colour correction
2. correction in field flattening
Division is rough
Notation of quality classes depends on vendors
(Neofluar, achro-plane, semi-apochromate,...)
improved
field
flatness
improved colour correction
Achromate
Plan-
Apochromat
Fluorite Apochromatno
PlanPlan-
achromat
Plan-
Fluorite
1 Optical System of the Microscopy I
Microscope Objective Lens: Performance Classes
Quality
classes of
the manufac-
turers
Objective class
Leica Nikon Olympus Zeiss
Name Feature Name Feature Name Feature Name Feature
Plan LD
Ph
Pol
Plan
Plan-Epi
DIC
Epi
Pol
Epiplan LD
Achromat Achromat DIC Achromat Ph
Plan-Achromat
DIC Plan Achroplan Wd
LD
Fluorite / Semi-Apochromat
Fluotar Fluor UV
Wd
Fluor Fluar UV
Plan-Fuorit Plan-Fluotar
Pol
Epi
DIC
Ph
Plan-Fluor HT
LD
W
Plan-Fluor Pol
Wd
Epi
DIC
LD
Ph
Plan-Fluar
Plan-Neofluar
Epiplan-Neofluar
Pol
HT
TIRF
Epi
Apochromat Apochromat UV
Wd
Apochromat W
UV
TIRF
Apochromat Epi
Plan-Apochromat
Plan-Apochromat
W
UV
HT
Ph
C
Plan-Apochromat
HT
C
W
Plan-Apochromat
HT
W
TIRF
Epi
C
Plan-
Apochromat
C-Apochromat
HT
W
C
1 Optical System of the Microscopy I
Microscopic Objective Lens: Performance Classes
Different color correction types
Classes of lenses with flattened field:
Chromatical correction:
1. Achromate
2. Fluorite
3. Apochromate
Increasing etendue
Increasing NA / resolution
0
0.5
1
1.5
0 20 40 60 80 100
Plan Apochromate
Plan Fluorite
Plan Achromat
magnifi-
cation
NA
dry
immersion
1 Optical System of the Microscopy I
Microscope Objective Lens: Performance Classes
Quality classes a...d / color and field performance
Diffraction limited on axis in the green guaranteed
Usual degradation for outer wavelengths and far off-axis field positions
1
Wrms
0.3
0
diffraction limit
0
rel.
field
480 nm
546 nm
644 nm
poly
Wrms
0.3
00
Wrms
0.3
00
Wrms
0.3
00
a b
c d
1
rel.
field 1
rel.
field
1
rel.
field
1 Optical System of the Microscopy I
Microscope Objective Lens: Field Flattness
Three different classes:
1. No effort
2. Semi-flat
3. Completely flat D
S
rel.
field0 0.5 0.707 1
1
0.8
0.6
0.4
0.2
0
diffraction
limit
plane
semi
plane
not
plane
1 Optical System of the Microscopy I
Microscope Objective Lens: Structure
Typical parts of lens structure for high NA-objective lenses
Separation of the lens setup in 3 major sections
afront part :
1. spherical aberration : only small
2. coma : only small
3. astigmatism : only small
4. curvature : only small
b
middle part :
1. spherical aberration : correction
2. color : correction
3. coma : correction
rear part :
1. curvature : correction
2. astigmatism : correction
3. color : correction
c
1 Optical System of the Microscopy I
Microscope Objective Lens: Simple Lenses
Simple type for low aperture and
small magnifications
Improved system for
higher apertures by an
aplanatic-concentric
lens
Diffraction limited performance
up to NA = 0.65
field
Ds
1
0.8
0.6
0.4
0.2
0
0 10 mm5 mm
diffraction limit546 nm
644 nm
480 nm
1 Optical System of the Microscopy I
Microscope Objective Lens: Simple Lenses
Improved design:
cemented front lens
Plane front surface due to practical reasons
Colour correction significantly better
object
Wrms
in
field
9 mm
diffraction limit
546 nm
644 nm
480 nm
4.5 mm00
1
2
poly
1 Optical System of the Microscopy I
Microscope Objective Lens: High NA 100x/0.93 oil
Two lenses in the front group
Colour correction uniformity good
Field curvature bad:
best focal plane depends on field
height
0y' [m]0
0.2
0.4
0.6
0.8
1
Wrms
[]
40 8020 60
diffraction limit
480 nm
546 nm
644 nm
poly
2 40-2-4
diffraction limit
0
0.2
0.4
0.6
0.8
1
Wrms
[]
axis20 m
40 m
z [m]
60 m
70 m
80 m
1 Optical System of the Microscopy I
Microscope Objective Lens: High NA 100x/0.93
Point spread function
Diffraction limit: 80% Strehl ratio
Typical: performance in the blue critical
644 nm
0 1.5 m0 1.5 m 0 1.5 m
546 nm480 nm
-1.5 m
diffraction
limit
1 Optical System of the Microscopy I
Microscope Objective Lens: High NA 100x/0.93
Objective lens 100x/0.9
No effort for field flattening
Small range of diffraction limit depending on
wavelength and field size
0y [mm]0
0.5
Wrms
[]
4 82 6
480 nm
546 nm
644 nm
poly0.4
0.3
0.2
0.1
diffraction limit
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
0.48 0.5 0.52 0.54 0.56 0.58 0.6 0.62 0.64
y'
[m]
diffraction
limit0.08
0.16
0.24
0.32
0.4
0.48
0.56
0.64
0.0714
1 Optical System of the Microscopy I
Microscope Objective Lens: High NA 100x/0.93
Seidel surface contributions
for 100x/0.90
No field flattening group
Lateral color in tube lens corrected
Distortion plays minor role in bio-med
applications in microscopy
5 10
-0.5
0
0.5
-0.02
0
0.02
-4
-2
0
2
4
-5
0
5
-2
0
2
-0.02
0
0.02
-1
0
1
spherical
coma
astigmatism
curvature
distortion
axial
chromatic
lateral
chromatic
1
518
11
13
sum
1 Optical System of the Microscopy I
Microscope Objective Lens: Cover glass
Enhancement of numerical
aperture
Standard data: K5, d=0.17 mm
Effect on spherical
correction for NA > 0.6 air uim
immersion
cover
glass
objective
lens
uair
a) b)
0.2 0.4 0.6 0.8 1 1.2 1.4 1.60.6
0.7
0.8
0.9
1
1.05
DS
NA
d=0.22 mm
d=0.17 mm
1 Optical System of the Microscopy I
Microscope objective lens: Free Working Distance
Free working distance decreases
with increasing magnification m
Large distance enlarges the lens
diameter for large NA
0 20 40 60 80 1000
0.5
1
1.5
2
2.5
m
dfr
[mm]
oil
dsmall
dlarge
Dlarge
Dsmall
1 Optical System of the Microscopy I
Microscope Objective Lens Types
Medium magnification system
40x0.65
High NA system 100x0.9
without field flattening
High NA system 100x0.9
with flat field
Large-working distance
objective lens 40x0.65
1 Optical System of the Microscopy I
Microscope Objective Lens: Correcting lens
Floating element to
adjust and correct
spherical aberration
Applications:
1. different thickness values
of cover glass
2. index mismatch at
the sample
floating
element
+1.80 mm
+1.63 mm
0.7 mm
cover
slide
air distance
1.2 mm
1.7 mm
0 mm
4.46
mm
3.99
mm
3.66
mm
3.32
mm
+2.04 mm
1 Optical System of the Microscopy I
Microscope Objective Lens: Catadioptric Lenses
Catadioptric lenses:
1. Schwarzschild design: first large mirror
2. Newton design: first small mirror
Advantageous:
1. Large working distance
2. Field flattening
3. Colour correction
Drawback:
central obscuration reduces
contrast / resolution
a) Schwarzschild b) Newton
1 Optical System of the Microscopy I
Microscope Objective Lens: Catadioptric Lenses
More complicated setups
1. four surfaces, two refractive
monolithic
2. Catadioptric,
cemented monolithic
image
object
r2
r4
r3
r1
1 Optical System of the Microscopy I
Lateral Chromatical Aberration
Transverse chromatical aberration of microscopic lenses:
hard to correct with the other components
Possible Solutions:
1. Compensating ocular, correction in the eyepiece (Abbe)
Intermediate image not corrected
2. Correcting with the tube lens
In infinity color corrected (ICS) optics
3. Correcting in the back part of the objective lens
Using inverted achromates with positive flint lens
1 Optical System of the Microscopy I
Combined colour correction
Problem : Lateral colour
correction is critical
Different solutions:
a) Compensation with
eyepiece
b) Corrector-null-power-
component
c) Compensation with tube
lens
Disadvantage:
- Intermediate image non
corrected
- no modularity
objective lens
y'LCh
= + 1.4 %
a) conventional, objective with finite image
eyepiece
- 1.4 %
eyepiece
0 %
intermediate image
corrected
objective lens
y'LCh
= + 1.2 %corrector lens
- 1.2 %tube lens
0 %
b) infinite objective lens with corrector
objective lens
y'LCh
= + 1.5 %tube lens
- 1.50 %
c) infinite objective lens without corrector
intermediate image
correctedeyepiece
0 %
intermediate image
not corrected
1 Optical System of the Microscopy I
Tube Optical System: Tube Lens
Simple tube lens
Magnification
On axis : diffraction limited
Dominant residual aberration:
lateral colour
objective
exit pupil
d = 100 mmf'
TL = 164 mm
tube
lens
yTL
DFV
= 25 mm
intermediate
image
DExP
480 nm
0
8.8 mm
12.5 mm
546 nm 644 nm
obj
tubeobj
f
fm
1 Optical System of the Microscopy I
Tube Optical System: Improved Systems
Simple tube lens for field position:
strong astigmatism
More complicated tube lenses:
1. Chromatical correction
2. Magnification factor modified
3. Adjust pupil location
cj []
sph coma ast tre 4 5-0.1
-0.08
-0.06
-0.04
-0.02
0
0.02
0.04
0.06
mTL
= 1 mTL
= 0.8mTL
= 1 mTL
= 1.25
1 Optical System of the Microscopy I
Tube Optical System: Prisms
Tube prism systems to generate two bincular channels
Adjustable pupillary distance required
Two versions: shift / tilt movement
a) shift version tube prims set
left
right
dIPD
= 65 mm
D = 28 mm
D = 28 mm
left
right
dIPD
= 65 mm
D = 28 mm
D = 28 mm
shift x
b) tilt version tube prims set
shift x
tilt axis