microscopy - uni-jena.de · 2012. 10. 16. · lecture 2: optical system of the microscopy ii...
Post on 09-Nov-2020
5 Views
Preview:
TRANSCRIPT
www.iap.uni-jena.de
Microscopy
Lecture 2: Optical System of the Microscopy II
2012-10-22
Herbert Gross
Winter term 2012
2 2 Optical System of the Microscope II
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. Etendue
2. Pupil
3. Telecentricity
4. Confocal systems
5. Illumination setups
6. Köhler principle
7. Fluorescence systems and TIRF
8. Objective adjustment
3 2 Optical System of the Microscope II
Contents of 2nd Lecture
2 Optical System of the Microscope II
Microscope Objective Lens
No rigid relationship between
magnification and aperture
Product of field size and NA fixes the
overall capacity
Variation of image-sided numerical
aperture
NA'
mobj
0.03
1000 20 40 8060
0.02
0.01
NA
1.5
1
0.5
00 100
mobj
80604020
immersion
2 Optical System of the Microscope II
Etendue of Microscope Objective Lenses
Information capacity,
etendue, space-bandwidth product,
number of PSF‘s resolved in the field
Non-rigid correlation between
magnification and etendue
Largest etendue for medium
magnifications
Immersion systems have larger
etendue
Dfield
in [mm]10-1
100 1
10-2
10-1
100
G [mm2]
air
oil
water
m150 100
10
63 50 40 20 10 5 4 2.5 1.25
2
4NADG field
Relevance of the system pupil :
Brightness of the image
Transfer of energy
Resolution of details,
Information transfer, location of Fourier spectrum
Image quality
Aberrations due to aperture
Image perspective
Perception of depth
Compound systems:
matching of pupils is necessary, location and size
2 Optical System of the Microscope II
Properties of the Pupil
2 Optical System of the Microscope II
Entrance and Exit Pupil
exit
pupil
upper
marginal ray
chief
ray
lower coma
raystop
field point
of image
UU'
W
lower marginal
ray
upper coma
ray
on axis
point of
image
outer field
point of
object
object
point
on axis
entrance
pupil
2 Optical System of the Microscope II
Microscope Objective Lens: Pupil
Object space telecentric
Real rear stop is not
defining the pupil
Collimated outgoing
beam
Exit pupil usually
not accessible
object
plane
objective
lens
rear stop
exit pupil
marginal ray
chief ray
telecentric,
entrance pupil infinity
y'p
u
f'f'
chief
ray
exit
pupil
rear
stopobject
plane
pupil
2 Optical System of the Microscope II
Microscope Objective Lens: Pupil
Diameter of pupil only weak correlated with magnification
Pupil distorsion:
Pupil distortion small
(sine condition corrected)
DExP
2
3
4
5
6
7
8
9
10
11
m0 10 20 30 40 50 60 70 80 90 100 110
1sin'
'
uf
yD
p
distExP
a) Lister
0 0.2 0.4 0.6 0.8 10
0.005
0.01
0.015
0.02
0.025
0.03
0.035
yp y
p
b) 100x0.93 oil
c) Laikin
yp
0 0.2 0.4 0.6 0.8 10
0.002
0.004
0.006
0.008
0.01
0.012
d) Schwarzschild
yp0 0.2 0.4 0.6 0.8 1
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
x 10-3
0 0.2 0.4 0.6 0.8 10
0.5
1
1.5
2
2.5
3
3.5
4
4.5
5
x 10-3
Ddist ExP D
dist ExP
Ddist ExP
Ddist ExP
2 Optical System of the Microscope II
Microscope Objective Lens: Pupil
Imaging of the pupil is important
Residual aberrations:
sharp edge of aperture is
desired
Real systems : smoothed
illumination profile
I(r)
1
0.5
00 1 2
r
[mm]
ideal
edge
lens
pupil
stop
v
2 Optical System of the Microscope II
Microscope Objective Lens: Pupil
Example of larger residual aberrations
of pupil image :
1. axial shift of pupil with field size
2. no sharp imaging of aperture stop
3. coloured edge of stop boundary
480 nm
0
11.6°
546 nm 644 nm
16.3°
20.0°
23.1°
30
15
0
diffraction limit
-0.5 0.50
Dspot
[m]
z
[mm]
axis
11.6°
16.3°
20°
23.1°
2 Optical System of the Microscope II
Microscope Objective Lens: Pupil
Vignetting of the pupil :
1. truncation of the bundle for finite field sizes
2. chief ray not identical with centroid
3. perturbation of telecentricity
4. in microscopic systems mostly at the
front lens
chief ray
telecentric
chief ray not
telecentric
full aperture
vignetted
aperture
centre of
gravity
shifted
2 Optical System of the Microscope II
Vignetting
Truncation at front lens
Pupil vignetting:
crescent shape of
light cone
Illumination fall-off towards
field boundary
cover
glass oil
front
lens
truncating
edge
axis
zone
field
1
0.675
pupil shape
I(r)/Io
1
0.5
00 10.5
y/ymax
2 Optical System of the Microscope II
Vignetting
Truncation of regions with large
aberrations as correction method
Improved performance
Psf elliptical, anisotropiuc resolution
Energy reduced
DS
0.5
1
0.8
00 10.5
y'/ymax
480 nm
546 nm
644 nm
diffraction limit
solid line : vignetted
dashed line : without vignetting
axis field zone field edge
yxyxx
Special stop positions:
1. stop in back focal plane: object sided telecentricity
2. stop in front focal plane: image sided telecentricity
3. stop in intermediate focal plane: both-sided telecentricity
Telecentricity:
1. pupil in infinity
2. chief ray parallel to the optical axis
2 Optical System of the Microscope II
Telecentricity
telecentric
stop
object image
chief rays
parallel to axis
in image spacef f'
imageobject telecentric
stop
chief rays
parallel to axis
in object space
a) image sided telecentric b) object sided telecentric
Design problems with telecentricity:
Usual telecentricity only fulfilled for one wavelength
Telecentricity is related to the centroid ray.
Therefore the telecentricity is disturbed by
vignetting effects
2 Optical System of the Microscope II
Telecentricity
wtele
[°]
y'/y'max
0 0.2 0.4 0.6 0.8 1-0.2
-0.15
-0.1
-0.05
0
0.05
480 nm
587 nm
656 nm
0 0.2 0.4 0.6 0.8 1
0
0.2
0.4
0.6
0.8
1
1.2
wtele
[°]
y'/y'max
centre ray with
vignetting
chief
ray
centre ray
with coma
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
2 Optical System of the Microscope II
Confocal Microscope
'
objective
lens
pinhole
lens pinhole CCD
in focus
out of focus
laser
illumination
Depth resolved
images
2 Optical System of the Microscope II
Confocal Images
Ref.: M. Kempe
2 Optical System of the Microscope II
Confocal Laser Scan Microscope
Complete setup: objective / tube lens / scan lens / pinhole lens
Scanning of illumination / descanning of signal
Scan mirror conjugate to system pupil plane
Digital image processing necessary
object
plane
objective
lens
pupil
plane
tube
lens
intermediate
image
scan
lens
scan
mirror
laser
source
beam
forming
pinhole
lens
pupil imaging
axis point
field point
2 Optical System of the Microscope II
Confocal Laser Scan Microscope
Scan lens
Diffraction limited
Change in pupil location of
objective lenses is critical
perturbation of telecentricity
scan
mirror
intermediate
image
0.2
480 nm
546 nm
644 nm
0.1
0
Wrms
diffraction limit
0 5 10 in °
w [°]
0 0.2 0.4 0.6 0.8 1-3
-2
-1
0
1
2
3
4
5
y/ymax
paraxial
exact
+ 3 mm
- 3 mm
pupil
location:
2 Optical System of the Microscope II
Confocal Laser Scan Microscope
Pinhole lens
Only axial colour is essential
Usage on axis only due to
descanning
Variable pinhole size not too small:
small aperture, retrofocus lens
pinhole
-0.5 0 0.5
z in
[mm]
yp
1
0.5
480 nm
546 nm
644 nm
Normalized transverse coordinate v
Usual PSF: Airy
Confocal imaging:
Identical PSF for illumination and observation
assumed
Resolution improvement be factor 1.4 for
FWhM
sin'
2 xv
4
1 )(2)(
v
vJvI
2
1 )(2)(
v
vJvI
2 Optical System of the Microscope II
Confocal Microscopy: PSF and Lateral Resolution
-8 -6 -4 -2 0 2 4 6 80
0,1
0,2
0,3
0,4
0,5
0,6
0,7
0,8
0,9
1
I(v)
incoherent
coherent
Normalized axial coordinate
Conventional wide field imaging:
Intensity on axis
Axial resolution
Confocal imaging:
Intensity on axis
Axial resolution improved by factor 1.41
for FWhM
4
2/
)2/sin()(
u
uuI
2
2/
)2/sin()(
u
uuI
2 Optical System of the Microscope II
Confocal Microscopy: Axial Sectioning
cos1'
45.0)(
nz approx
wide
cos1'
319.0
nzconfo
)2/(sin8 2
zu
u
I(u)
-5 -4 -3 -2 -1 0 1 2 3 4 50,
0,1
0,2
0,3
0,4
0,5
0,6
0,7
0,8
0,9
1,
incoherent
coherent
Large pinhole: geometrical optic
Small pinhole:
- Diffraction dominates
- Scaling by Airy diameter a = D/DAiry
- diffraction relevant for pinholes
D < Dairy
Confocal signal:
Integral over pinhole size
2 Optical System of the Microscope II
Size of Pinhole and Cnfocality
a
dvvvuUuS0
22),()(
x / DAiry
0 0.5 1 1.5 2 2.50
0.5
1
1.5
2
2.5
3
3.5
4
4.5
5
DPH / DAiry
NA = 0.30
NA = 0.60
NA = 0.75
NA = 0.90
geometrical
-25 -20 -15 -10 -5 0 5 10 15 20 250
2
4
6
8
10
12
S(u)
u
a = 3
a = 2
a = 1
a = 0.5
2 Optical System of the Microscope II
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
2 Optical System of the Microscope II
Confocal Laser Scan Microscope
Depth signal as a function of wavelength
Disturbance by axial colour aberration
z/Ru
Dph
= Dairy
3
2
1
0
-1
-2
0.4 0.45 0.5 0.55 0.6 0.65 0.7
zestim
image
plane
2 Optical System of the Microscope II
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
2 Optical System of the Microscope II
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
2 Optical System of the Microscope II
Illumination Optics: Overview
Typical images for different illuminations
bright
dark
epi trans
source
collector condenser objective
lens
object
plane
image
plane
field stop aperture stopback focal plane -
pupil
2 Optical System of the Microscope II
Köhler Illumination Principle
Principle of Köhler
illumination:
Alternating beam paths
of field and pupil
No source structure in
image
Light source conjugated
to system pupil
Differences between
ideal and real ray paths
condenser
object
plane
aperture
stopfield
stop filter
collector
source
2 Optical System of the Microscope II
Illumination Optics: Overview
Types of settings :
1. Köhler :
source into pupil,
mostly used
2. Critical :
source into field of view,
source structure disturbs image
3. Projection :
source into condenser
4. Arbitrary
condenser object
plane
collectorsource
a) Köhler = 1 fcon f
con
b) = 0.5
c) Projection = 0
d) = -0.5
e) Critical = -1
auxiliary
lens
2 Optical System of the Microscope II
Illumination Optics: Collector
Requirements and aspects:
1. Large collecting solid angle
2. Correction not critical
3. Thermal loading
large
4. Mostly shell-structure
for high NA
W(yp)
200
a) axis b) field
200
W(yp)
yp
480 nm
546 nm
644 nm
yp
W(yp)
200
a) axis b) field
200
W(yp)
yp
yp
480 nm
546 nm
644 nm
2 Optical System of the Microscope II
Illumination Optics: Condenser
2. Abbe type, achromatic, NA = 0.9 , aplanatic, residual spherical
3. Aplanatic achromatic, NA = 0.85
y'100m
a) axis b) field
yp
480 nm
546 nm
644 nm
y'100m
yp
x'100m
xp
tangential sagittal
y'100m
a) axis b) field
yp
480 nm
546 nm
644 nm
y'100m
yp
x'100m
xp
tangential sagittal
2 Optical System of the Microscope II
Illumination Optics: Condenser
Dark-field illumination systems
1. Trans-illumination
Cardioid-mirror
Realizations: approximation
of Cardioid curve
C
R
R/2 P
R/2
circle
cardioide2R
object
plane
cardiode
cover slideillumination
cone
object carrier
condenser
immersion
2 Optical System of the Microscope II
Illumination Optics: Condenser
2. Epi-illumination
Complicated ring-shaped components
around objective lens
object
ring lens
illuminationobservation
object
ring lens
illuminationobservationcircle 1
circle 2
2 Optical System of the Microscope II
Illumination Optics: TIRF-Illumination
cover glass
substrate
illumination
signal
Epi- and trans illumination for TIRF
cover glass
total
internal
reflexion
probe
carrier
illumination
observation
prism
objective lens
2 Optical System of the Microscope II
Fluorescence Microscopy
Fluorescence microscopy is the most frequently employed mode of light microscopy
used in biomedical reserach 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
400350 450 500 550 600 650 700
[nm]0
0.5
1
emission
absorption
Fluorescein
Iso Thio
Cyanate
2 Optical System of the Microscope II
TIRF Microscopy
Total internal reflection microscopy:
Excitation with evanescent field
Advantages:
1. better axial resolution
2. better SNR, no fluorescence background
Problem in optical design:
Extremly small illumination ring-shaped
channel around the observation light cone
cover
glass
illumination
ring channel
observation
total
internal
reflexion
probe
Objective lens
TIRF 100x1.45
Epi-Fluorescence TIRF
2 Optical System of the Microscope II
Adjustment of Objective Lenses
Adjustment of air gaps to
optimize spherical aberration
Reduced optimization setup
Compensates residual aberrations due to tolerances (radii, thicknesses,
refractive indices)
8,6,4,2,4,1
jt
cccc
k k
j
jjoj
t2 t
4t6
t8
d2 d4 d6 d8 c20 c40 c60 c80 Wrms
nominal 0.77300 0.17000 3.2200 2.0500 0.00527 -0.0718 0.00232 0.01290 0.0324
d2 varied 0.77320 0.17000 3.2200 2.0500 0.04144 -0.07586 0.00277 0.12854
d4 varied 0.77300 0.17050 3.2200 2.0500 0.03003 -0.07461 0.00264 0.01286
d6 varied 0.77300 0.17000 3.2250 2.0500 0.00728 -0.07367 0.00275 0.01284
d8 varied 0.77300 0.17000 3.2200 2.0550 0.005551 -0.0717 0.00235 0.01290
optimized 0.77297 0.16942 3.12670 3.2110 0.000414 0.00046 0.00030 0.01390 0.00468
2 Optical System of the Microscope II
Adjustment of Objective Lenses
Significant improvement for one wavelength on axis
Possible decreased performance in the field
Wrms
in
0.3
0
0.15
y/ymax
480 nm
546 nm
644 nm
improve-
ment
0.5 10
solid lines : nominal
dashed lines : adjusted
Example microscopic lens
Adjusting:
1. Axial shifting lens : focus
2. Clocking: astigmatism
3. Lateral shifting lens: coma
Ideal : Strehl DS = 99.62 %
With tolerances : DS = 0.1 %
After adjusting : DS = 99.3 %
2 Optical System of the Microscope II
Adjustment and Compensation
System with tolerancesStrehl: 0,2%
PSF (intensity normalized)
PSF(energy normalized)
Spherical aberrationcompensator
On axis comacompensator
StopOb
ject pl
ane
Defoc
uscom
pensat
orOn
axis a
stigma
tismcom
pensat
or Nr.
1
On axis astigmatismcompensator Nr. 2
Ref.: M. Peschka
Sucessive steps of improvements
2 Optical System of the Microscope II
Adjustment and Compensation
PSF(intensitynormalized)
With Tolerances
Step 1 (Z, Z)4 9
Step 2 (Z, Z)7 8
Step 3 (Z, Z)5 6
Step 4 ~ Step 2 (Z, Z)7 8
PSF(energynormalized) Not
adjusted
0.2%
21.77%25.48%
97.2%99.32%
Stre
hl ra
tio [%
]
0
20
40
60
80
100
Step 1Z, Z4 9
Step 2Z, Z7 8
Step 3Z, Z5 6
Step 4~ Step 2Z, Z
7 8
Ref.: M. Peschka
top related