Post on 01-Jun-2020
www.iap.uni-jena.de
Medical Photonics Lecture 1.2
Optical Engineering
Lecture 9: Instruments II
2019-06-12
Michael Kempe
Summer term 2019
2
Contents
No Subject Ref Date Detailed Content
1 Introduction Zhong 10.04.Materials, dispersion, ray picture, geometrical approach, paraxial approximation
2 Geometrical optics Zhong 17.04.Ray tracing, matrix approach, aberrations, imaging, Lagrange invariant
3 Diffraction Zhong 24.04.Basic phenomena, wave optics, interference, diffraction calculation, point spread function, transfer function
4 Components Kempe 08.05. Lenses, micro-optics, mirrors, prisms, gratings
5 Optical systems Zhong 15.05.Field, aperture, pupil, magnification, infinity cases, lens makers formula, etendue, vignetting
6 Aberrations Zhong 22.05. Introduction, primary aberrations, miscellaneous7 Image quality Zhong 29.05. Spot, ray aberration curves, PSF and MTF, criteria8 Instruments I Kempe 05.06. Human eye, loupe, eyepieces, photographic lenses, scan lenses
9 Instruments II Kempe 12.06.Microscopic systems, micro objectives, illumination, scanning microscopes, contrasts
10 Instruments III Kempe 19.06.Medical optical systems, endoscopes, ophthalmic devices, surgical microscopes, zoom lenses
11 Photometry Zhong 26.06.Notations, fundamental laws, Lambert source, radiative transfer, photometry of optical systems, color theory
12 Illumination systems Gross 03.07.Light sources, basic systems, quality criteria, nonsequential raytrace
13 Metrology Gross 10.07. Measurement of basic parameters, quality measurements
3
s = 250 mmo
flens
y
y'
lens
eye Lnom
f
mmm
250
Objective
exit pupil
intermediate
focus
Eyepiece
Eye pupil
tube length
eyepiece
eye
f
mmm
250
US 7643225L = 4.2 mm , F'=2.8 , f = 3.67 mm , 2w=2x34°
US 6844989L = 6.0 mm , F'=2.8 , f = 4.0 mm , 2w=2x31°
EP 1357414L = 5.37 mm , F'=2.88 , f = 3.32 mm , 2w=2x33.9°
Olympus 2L = 7.5 mm , F'=2.8 , f = 4.57 mm , 2w=2x33°
Eye
Loupe
Eyepiece
Photographic lens
Resolution:
1’ corresponds to
35µm at 250mm
Lecture Instruments I
f=6mm (fisheye)
to 1200mm (telephoto)
F# from 1 to 11
Historical Development of Optical Microscopes
1670 Hooke
1632 Leeuwenhoek
1870 Zeiss
today
Study of Life: Driver of Microscope Development
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
Microscopic Magnification
Basic geometrical consideration
objective lens
object
focal length of
objective lens
focal length of
eyepiece
eye lens
eyepiece
real intermediate
image
image
virtual
image
Image Planes and Pupils
Principal setup of a classical compound optical microscope
upper row : image planes, lower row : pupil planes
Köhler illumination (object in pupil plane of illumination setup)
source
collector condenser objective eyepiece eyetube lens
eye
pupil
exit pupil
objective
aperture
stopfield
stop
object intermediate image image
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
tubeocobjmicro
f
mm
f
fmmm
250
obj
tubeobj
f
fm
obj
tubeobjExP
m
NAfNAfD
22
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
ftube
Microscope Resolution
Typically, microscope optical systems are corrected to be 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
objunx
sin
61.0
objill ununx
sinsin
22.1
sinn
kx
Resolution and Magnification
The useful magnification should enable a detection of the smallest resolvable features
of the object
For observation with the eye the smallest features should appear under nor more than 2’ (otherwise: empty magnification)
(1’ = 0.017°=1/3438 rad)
The magnification must be large enough to avoid overfilling the eye pupil
For camera detection the smallest features should be detected by no more than 2 pixels
𝑚𝑚𝑖𝑐𝑟𝑜
∆𝑥
250𝑚𝑚≤
2
3438
𝑚𝑚𝑖𝑐𝑟𝑜 ≤𝑝
𝜆∙ 𝑁𝐴
𝑚𝑚𝑖𝑐𝑟𝑜 ≤0.15𝑚𝑚
∆𝑥≈ 600 𝑁𝐴
𝐷𝐸𝑥𝑃
𝐷𝑒𝑦𝑒=
𝑓𝑡𝑢𝑏𝑒𝑓𝑒𝑦𝑒
𝑚𝑚𝑖𝑐𝑟𝑜 ≥250𝑚𝑚
𝐷𝑒𝑦𝑒2𝑁𝐴 ≈ 200 𝑁𝐴
𝐷𝑒𝑦𝑒 ≈ 2…2.5 𝑚𝑚
Abbe
Abbe
𝑚𝑚𝑖𝑐𝑟𝑜 = 𝑚𝑜𝑏𝑗 ∙ 𝑚𝑝ℎ𝑜𝑡𝑜𝑒𝑦𝑒 ≤2𝑝
∆𝑥
Increased magnification does not necessarily generate more details
Increasing resolution is required
12
Magnification and Resolution
x2
x4x8
x16
x32
resolved
magnification
not
resolved
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
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,...)
Standard specifications depend on vendor / system
Exit pupil: in general inside, diameter and z-position
depend on aperture / correction
Correction for chromatic difference of magnification
either built into the objectives themselves
(Olympus and Nikon) or corrected in the tube lens
(Leica and ZEISS).
DIC slider
position
Rear stop
Exit pupil
Pupil
Object
plane Pupil manipulations
Ph: internal phase ring near back focal plane,
diameters fit to position of 1. diffraction order
DIC: manipulations outside (DIC-slider)
with negligible field dependence
(low field angle at slider position and high depth of
focus for pupil)
Objective Lenses: Conventions
15
Parfocal
distance
Working
distance
Source:
www.microscopyu.com
Objective Lens: Performance Classes
Classification:
1. performance in color 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
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
Microscope Objective Lens Types
Medium magnification system
40x/0.65
High NA system 100x/0.9
without field flattening
High NA system 100x/0.9
with flat field
Large-working distance
objective lens 40x/0.65
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
Microscope Objective Lens: Cover glass
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
Microscope objective lens : Index mismatch
Objective lens with immersion
3 materials : Immersion (I), cover glass (C) and sample (S)
Refraction law :
Problems by index mismatches with sample points deep inside
Strong sphericalaberrations for high-NA
Standard immersion(index of refraction at 546,1 nm)
Water (𝑛𝐼 = 1.33)
Glycerol (𝑛𝐼 = 1.47)
Oil (𝑛𝐼 = 1.518)
for comparison: cover glass 𝑛𝐶𝐺 = 1.5255
first lens
immersion cover
glassprobe
mediumenlarged picture of
the ray caustic
paraxial
focus
marginal
focus
nCG
nM
SSCCII nnnNA coscoscos
Tube Optical System: Tube Lens
Simple tube lens
Magnification
On axis : diffraction limited
Dominant residual aberration:
lateral color (corrected together with objective lens)
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
Tube Optical System: Prisms
Tube prism systems to generate two binocular 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
23
Stereo Microscopes
Greenough Type• Well-corrected objective
lenses
• Inclined image planes
CMO Type (Common Main Objective)
• Main objective used off-
axis
• Varying aberrations on
both channels (globe
effect)
Illumination Optics: Overview
Four possibilities for practical needs
Epi vs. trans-illumination
Bright vs. dark field illumination
Comparison of light cones for
imaging and illumination parts
axis
observation
epi-dark
field
trans-bright
field
trans-dark
field
epi-bright
field
object
plane
objective
condenser
Illumination Optics: Overview
Instrumental realizations
a) incident illumination
bright fieldb) incident illumination
dark fieldc) transmitted illumination
bright field
d) transmitted illumination
dark field
ring
mirrorobservation
illumination
object
plane
ring
mirror
objective
lens
object
plane
observation
illumination
observation
ring
condenser
object
plane
illumination
condenser
object
plane
observation
illumination
Köhler Illumination Real Setups
Additional relay lenses, space for switchable components
Aberrations of field stop imaging clearly visible
For high resolution high NA condensor necessary
Modifications at aperture stop for special illumination settings / contrast methods
Köhler Illumination Real setups
aperture
stop
field
stop
condenser
objectplane
aperturestop
field stop
filter
collector
source
26
Ref: B. Böhme
Contrasts in Microscopy
• Biomedical specimen exhibit weak natural contrast in transillumination
or brightfield imaging
Source: zeiss-campus.magnet.fsu.edu
Phase Contrast Imaging
• Pure phase objects are not visible in brightfield
imaging
• Zernike phase contrast:
access of diffracted (𝑟𝑑) and undiffracted (𝑟𝑢)
light by ring illumination
phase shift and attenuation of undiffracted
light
Phase Contrast Imaging
𝐼𝑖𝑚 = 𝑟2 = 𝑟𝑑2 + 𝑟𝑢
′ 2 − 2𝑟𝑑𝑟𝑢′ cos
𝜙
2
= 𝑡2 + 2 1 − 𝑐𝑜𝑠𝜙 − 2𝑡 2 1 − 𝑐𝑜𝑠𝜙 cos𝜙
2≈ (𝜙 − 𝑡)²
𝑟𝑑 ² = 𝑟𝑑² = 2 (1 − 𝑐𝑜𝑠𝜙
𝑟𝑢 ² = 1 𝑟𝑢′ ² = 𝑡²
• Example: pure phase object
• Undiffracted light before and
behind phase ring
• Resulting image and contrast
with strong nonlinear
dependence on object phase
not suited for quantitative
imaging 𝐶 =𝐼𝑖𝑚 − 𝐼𝑏𝑔𝑟
𝐼𝑖𝑚 + 𝐼𝑏𝑔𝑟≈
𝜙 − 𝑡 2 − 𝑡2
𝜙 − 𝑡 2 + 𝑡2=
𝜙² − 2𝑡𝜙
𝜙² − 2𝑡(𝜙 − 𝑡
0 40 80 120 160 200 240 280 320 360
-1,0
-0,5
0,0
0,5
1,0
co
ntr
ast C
object phase (degrees)
t=0.10
t=0.25
t=1.0
Differential Interference Contrast (DIC)
• Contrast of phase objects
can also be obtained by
interference of sheared
beams
• In DIC the beams (of
orthogonal polarization)
are separated and
combined by Wollaston
prisms
• Interference of the beams
with displacement 𝛿𝑥 by
analyzer
phase gradient imaging
• Without the prisms
polarization contrast can
be realized (typ. polarizer
and analyzer with
orthogonal orientation)
2
,,, yxxryxryxI yxirr ,exp
x
rxyxryxxr
,,
2
22,
xxryxI
Differential Interference Contrast
• The image depends on the orientation of the beam separation and the
bias phase (introduced by translation of the prism)
Fluorescence Microscopy
Fluorescence microscopy is the most frequently employed mode of light microscopy
used in biomedical research today
Setup:
Necessary components:
Dicroitic beam splitter, excitation filter with
sharp edge
UV
source
objectobjective
lens
image
plane
illumination
at 365 nm
fluorescence
red or
infrared
dicroitic
beam splitter
excitation
filter
UV bloc
filter
emission filter
𝐼 𝑟 = 𝑃𝑆𝐹 𝑟 ⊗ 𝑂 𝑟
For shift-invariant PSF
Source: zeiss-campus.magnet.fsu.edu
Light Sources for Fluorescence Microscopy
• Many fluorophores require UV light for excitation – mercury lamps (e.g. HBO 100,
a 100-watt high-pressure mercury plasma arc-discharge lamp) provide the
sufficient light power from the UV to the yellow/red spectral range
• LEDs are more stable and efficient with
intensities ranging from 5-25 mW/cm²
Laser scan microscope
Depth resolution (sectioning) with
confocal pinhole
Transverse scan on field of view
Digital image
Only light comming out of the
conjugate plane is detected
Perfect system: scan mirrors
conjugate to pupil location
System needs a good correction
of the objective lens,
symmetric 3D distribution of
intensity
http://zeiss-campus.magnet.fsu.edu/tutorials/opticalsectioning/confocalwidefield/indexflash.html
Confocal Microscope
'
objective
lens
pinhole
lens pinhole CCD
in focus
out of focus
laser
illumination
𝐼 𝑟 = 𝑃𝑆𝐹𝑐𝑜𝑛 𝑟 ⊗ 𝑂 𝑟
For shift-invariant PSF
𝑃𝑆𝐹𝑐𝑜𝑛 𝑟 = 𝑃𝑆𝐹² 𝑟
detector
Confocal Microscopy
35
Confocal
Wide FieldWide Field
(laser)
Confocal
high z-resolution
3D via sectioning
(haze suppressed)
limited z-resolution
thick sections
(off-focus haze)“snapshot”
scanning
z
z
Excitation
Plan-APOCHROMAT
40x /1,3 Oil
Emission
Plan-APOCHROMAT
40x /1,3 Oil
Excitation
Plan-APOCHROMAT
40x /1,3 Oil
Emission
Plan-APOCHROMAT
40x /1,3 Oil
Source: Carl Zeiss Microscopy GmbH
Scan Systems
Source:
http://www.zamisel.com/SSpostavka2.htm
l
Wide Field
Objective
exit pupil
intermediate
focus
Eyepiece
Eye pupil
tube length
Beam
deflecting
elementScan lens
• Beam deflecting element conjugate to
pupil location
• Often scanning mirrors for two
orthogonal scan directions used –
pupil plane between two scanners
Scan Systems: Introduction
Scan resolution:
Number of resolvable points in the field of view
corresponds to angle resolution
Information capacity:
1. Resolvable points
2. Speed of scanning
Etendue: product of scan range andscanner area
max2 ExP
Airy
D
D
LN
log
log v
angle
resolution
scan speed
growing scan
capacity
acoustic optical modulator
polygon
mirror
galvo
scanner
holographic
scanner
electro
optical
modulator
resonant
galvo
scanner
max ExPMirMir DD
Deflecting Components
Different types of deflecting elements
Scanning
Non-Mechanical
Deflection
Electro-optic EOD
Acousto-optic AOD
Mechanical
Oszillation
Galvanometric Galvoscanner
HolographicHolographic
Scanner
Electrostatic MEMS Scanner
Rotation
PolygonPolygon-scanner
Rotating Prisms
Dove Prism
TranslationLenses and lens arrays
Galvanometer and Electrostatic Scanner
Galvo scanner MEMS-Scanner
Source: scanlab.de Source: researchgate.net
Scanner Lenses
Ideal scanner lens (F- lens): h = f
Flat-field corrected lens: h = f tan
nonlinear displacement: distortion correction needed
• In addition telecentricity
ensures minimum beam
distortions
40
Source: thorlabs.com
Confocal Signal with Spherical Aberration
S(u)
u-30 -20 -10 0 10 20 30
0
1
2
3
4
5
6
7
8
9
10
relative pinhole size:a = 3a = 2a = 1a = 0.5
spherical aberration 2
Spherical aberration:
- PSF broadened
- PSF no longer symmetrical around image plane during defocus
Confocal signal:
- loss in contrast
- decreased resolution
Multispectral Detection
• Simultaneous imaging of many
colors by spectrally resolved
detection enables the imaging of
multi-label samples
• Particularly relevant for fluorophores
with overlapping spectra (e.g.
fluorescent proteins)
Source: Carl Zeiss Microscopy GmbH
grating
32-Channel PMT
Multispectral Detection
CFP, CGFP, GFP and YFPCultured cells expressing 4 FPs in ER, nuclei, plasma membranes and mitochondria, repectively
Sample: Drs. Miyawaki, Hirano, RIKEN, Wako, Japan
CFP CGFP
GFP YFP
Source: Carl Zeiss Microscopy GmbH