Download - 4D Imaging and Tracking
24 June 2009 http://waf.eps.hw.ac.uk/ 1
4D Imaging and Tracking
Alan Greenaway
24 June 2009 http://waf.eps.hw.ac.uk/ 2
Imaging
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Basic principles• Derived from wavefront sensing in
astronomical adaptive optics• Conventional imaging system gives in-focus
image of a single object plane
IM AGE
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Basic principles• Conventional imaging system gives in-focus
image of a single object plane• Combined with conventional grating gives
multiple images of single object plane
Diffrac tion Order-1 0 + 1
IM AG E
24 June 2009 http://waf.eps.hw.ac.uk/ 5
Diffractive optics• Distorted grating gives
different phase shift in each diffraction order
• Principle of detour phase holography
• Quadratic distortion wavefront curvature
• Acts like lens with different focal length in each diffraction order
Off-axis Fresnel lens
24 June 2009 http://waf.eps.hw.ac.uk/ 6
3-D Snapshot Imaging
-1
0
+1
-1 0 +1
Simple & cheap to manufactureGood control of divergencePhase grating etch gives energy- balance controlHigh optical efficiency from binary grating
Blanchard & Greenaway
App.Opt. 38(1999)6692
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3-D Snapshot Imaging
-1
0
+1
-1 0 +1
Simple & cheap to manufactureGood control of divergencePhase grating etch gives energy- balance controlHigh optical efficiency from binary grating
In-focus images of various z-planes are at different magnification
24 June 2009 http://waf.eps.hw.ac.uk/ 8
Telecentricity
-1
0
+1
Combination optical system
2
1 2 and and 0cm g
fs f f f p p
f
1 2
m
and and
m g
cm g g m g
f f s f ssff p p
f f s f f s f f s
Djidel, Gansel, Campbell & Greenaway, Opt Exp 14(2006)8269-8277
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Telecentricity
-1
0
+1
Telecentric performances f
0.01 0.02 0.03 0.04 0.05 0.06 0.07 0.08 0.09 0.1
-4.2
-4.1
-4
-3.9
-3.8
-3.7
-3.6
-3.5
Lens to Off-Axis Fresnel Lens Separation (m)
Mag
nific
atio
n
m+ theoretical
m- theoretical
measured data
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200 400 60 0 800 100 0
0 .2
0 .4
0 .6
0 .8
1 .0
Practicality & Efficiency?Practicality
• Used on inverted and upright systems
Efficiency• Optical efficiency
~84% overall
• (~28% each order)nm etch depth
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First Biology Images
GFP in plant-cell mitochondria bright filed (upper row) and epi-fluorescence (bottom row)
1µm separation between in-focus planes (with Logan, St Andrews)
GFP in drosophila ovary
Epi-fluorescence images with 7.3µm between in-focus planes
Principal application microtubule dynamics through EB1 tracks(with Davis, Oxford)
24 June 2009 http://waf.eps.hw.ac.uk/ 12
3D movies• 9 z-plane phase-
contrast images of Hela cells
• Z-plane separation 810nm
• 3 z-plane DIC images from movie of Hela mitosis
With Allan, Manchester
24 June 2009 http://waf.eps.hw.ac.uk/ 13
Motility Measurement• Sperm motility from
phase-contract and/or dark-field imaging
• Current approach uses apparent length as a proxy for out-of-plane deformation of tail
• Multi z-plane images provide extra information (with Kirkman-Brown, Birmingham)
24 June 2009 http://waf.eps.hw.ac.uk/ 14
Imaging- summary• 3D ‘snapshot’ images
on commercial microscopes
• All image modes but scanning demonstrated
• 3D tracking (4D data)
Z-plane separations 0 to many microns
Simultaneous z-stacks Up to 9 simultaneous
planes demonstrated Low-cost add on
Work on sperm motility starts in September
24 June 2009 http://waf.eps.hw.ac.uk/ 15
Broadband Application
• Careful design achieve ‘white light’ imaging
• Unfiltered halogen light
Blanchard & Greenaway, Opt. Commun. 183(2000)29-36
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Tracking
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Ranging in Depth with Sharpness
• Beam divergence from source depends on optical aperture
• Defocused image on non-source planes reduces intensity…
• …thus to a reduction in sharpness (integral of intensity squared)
• Suitable for real-time analysis and CMOS detector technologies 1 0 1 2 1 4 1 6 18
20
40
60
80
1 00
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Experimental Setup
Camera
Iris
Mirror
He-NeLaser
Illumination
Nanoholes
x, y, zNano-
positioners
Objective
f = 200 mm f = 215 mm
Telecentric: Magnification equal in all diffraction orders
215 mm
Experimental arrangement
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• 210 nm diameter holes in Al foil
• Single point source
• Simulates fluorescent particle
• Mask / hole contrast >104
• Brightness limited only by illumination source
Nanohole test objects25 m
1.5 m
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Resolution on 3 planes-1 order in focus
+1 order in focus
0th order in focus
+1 order in focus
0th order in focus
-1 order in focus
+1 order in focus
0th order in focus
Image Resolution: No grating = 233nmWith grating = 226nm and 231nm (for 0th and ±1orders)
-1 order in focus
+1 order in focus
0th order in focus
24 June 2009 http://waf.eps.hw.ac.uk/ 21
Sharpness
Problem:A single mage sharpness measurement gives ambiguous depth position
-6 -5 -4 -3 -2 -1 0 1 2 3 4
101
102
Nanohole Displacement (m)
Imag
e S
harp
ness
Image Sharpness vs. Nanohole Displacement
24 June 2009 http://waf.eps.hw.ac.uk/ 22
Unique depth indication…
-6 -5 -4 -3 -2 -1 0 1 2 3 4
101
102
Nanohole Displacement (m)
Imag
e S
harp
ness
Image Sharpness vs. Nanohole Displacement
-1 Order
0th Order+1 Order Solution:
QD grating method gives 3 simultaneous image sharpness (one from each order) for each particle.
2 or more images resolve ambiguity
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ML depth estimator
24 June 2009 http://waf.eps.hw.ac.uk/ 24
Principle• For any given z-
position of source– PDF for sharpness
dependence on photon flux and optics is ‘known’
– Particle depth is ‘most likely’ value that is consistent with measured data – choose z to maximise
2P s z
1P s z
2s 1s
2P s z
1 2P s z P s z
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o o o o
o o o o
o o o o2.0 2 .5 3 .0 3 .5 4 .0 4 .5
0 .0 2
0 .0 4
0 .0 6
0 .0 8
0 .1 0
Sharpness SNR
Solid – photon bias
Dotted – actual sharpness
Circles – photon subtractedDainty & GreenawayJOSA 69(1979)786-790
Log n
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What is possible?• MVB model
• SNR per image
• Many approximations – check…
2 0 2 4
0 .0 04
0 .0 05
0 .0 06
0 .0 07
0 .0 08
0 .0 09
0 .0 1
0 .0 11
2
221
1
8 jm
jj
MVB zn z zm
a z z
nSource z-position
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Tracking - summary• Simple algorithm
tracking points with high resolution
• More analytic work needed
• Cluttered backgrounds yet to be tried
+8.1nm demonstrated in first experiment
nm depth resolution with ~thousand detected photons
Ad-hoc calibration Work on models
continues
24 June 2009 http://waf.eps.hw.ac.uk/ 28
Thanks…STFC for PIPPS/Bio-Mini-PIPSS funding… EPSRC bio-photonics platform funding…
P.A. Dalgarno1, H.I.C. Dalgarno1 , R.J. Warburton1, A. Putod1, S. Aitken1, A Weis1, C Diez1, Alan Baird1
D.P. Towers2, C.E. Towers2, N.C Angarita-Jaimes2, S Chen2
D.J. Smith3, J.C. Kirkman-Brown3, J. Gaham4,V J Allan5, R Southern5, P Marsh5,I Davis6, R Parton6, K Lillie6
1 Dept Physics, SUPA/IIS, Heriot-Watt University, UK2 School of Mechanical Engineering, University of Leeds, Leeds, UK3 Centre for Human Reproductive Science, Birmingham Women’s NHS Foundation Trust,
Edgbaston Birmingham, UK4 Cairn Research Ltd, UK5 Faculty of Life Sciences, University of Manchester6 Dept of Biochemistry, University of Oxford
24 June 2009 http://waf.eps.hw.ac.uk/ 29
Questions??