Adaptive optics: optimization and wavefront sensing Novel microscope enhancements
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Transcript of Adaptive optics: optimization and wavefront sensing Novel microscope enhancements
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1) Adaptive optics: optimization and wavefront sensing
2) Novel microscope enhancements
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widefield confocal
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Spherical Aberration (on axis)
Perfect lens
Real lens
2 related types, lateral and transverseDifferent effective focal lengths, positions
Constant opticalPath differenceEvery ray arrivesAt same focal point
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Adaptive optics idea
Active element undoes what microscope, specimen does to PSF
Correction is determined by iteration: genetic algorithms, random searchesMore correction takes more time
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37 element micromachined deformable mirrorCan travel 6 microns
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Norris. J. Microcopy 2002
Performance for TPEF of coumarin dye solution
Good agreement with calculated, measured in simple specimen
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Adaptive optics on non-scanning 2-photon microscope
600 microns into solution:PSF greatly improved
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Lateral PSFs (measured by THG)
Adaptive optics improves resolution and signal strengthFor nonlinear optical processes (TPEF, SHG, THG, CARS)
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Girkin, OPEX
Optimize feedback based on two-photon fluorescence intensity
Setup for adaptive optics on laser scanning microscope
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Correction for TPEF of sub-resolution bead
x-y optical section
Significant improvement even for beads in water
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Correction for TPEF of sub-resolution bead
x-z cross section
Significant improvement even for beads into 30 microns of water
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Improvement in PSF important for multiphoton processes
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TPEF of guinea pig bladder1.3 NA 40x
30 microns into the tissue
Surfaceoptimized
Optimized for30 microns
Need to optimize at every depth
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CARS and adaptive optics
Xie and GirkinOpex
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Non-resonant CARS from glass-air interface
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Depth dependence of CARS for beads in agarose
Optimizing at greatest depth works bestSystems aberrations also very important
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Comparison of CARS image with system, sample induced aberrations
600 microns into solution
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Comparison of CARS image with system, sample induced aberrations from tissue
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Radial Dependence of correction
Best response when optimize at every point But very slow
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Adaptive Optics by Wavefront correction
Denk, PNAS, 2006
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Astigmatism
Different planesHave differentFocal lengths
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Correction of Astigmatism
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AO on zebrafish larvaeOlfactory bulb:GFP
50 microns
200 microns
Imaging bloodflow
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Wavefront sensing and correction using Spatial Light Modulator
SLM larger range than Deformable mirror: better depth
Eliceiritbp
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MPE in vivo live animal imaging
Flexible periscope converts inverted to upright microscope
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Difficulties with live animal imaging: respiration
8 second intervals, each scan 2 secondsFew micron motion, even anesthetized
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Performance for in vivo imaging of muscle
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Imaging through 200 microns of tissue
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TPEF of kidney of anesthetized rabbit kidney
Breath-holding for one minute:Necessary for internal organ imaging
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Fraction of light collected in epi-illumination geometry
High NA only collects 30% of available light (ideal limit without absorption and scattering)
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Parabolic reflector to enhance light collection
Balaban, J. Microscopy (2007)
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zeffeIzI
)0()(
Light Attenuation in tissue
Z= depth from surface
Simplest case fit to µs [cm-1]1/ µs =scattering length, or mean free path
Multiple scattering in thick, turbid media
)1(' gss g=anisotropy, avg cos0=isotropic1=all forward
Tendon~0.9Brain=0.1
sat
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Photon Transport Theory
4
)',()',(4
),(),(
dsrJsspsrJds
srdJ st
J(r,s) in a specific direction s within a unit solid angle dω
2/32
2
)cos21(
1)(
gg
gp
Anisotropy around propagation axis
radiance J(r,s) relates to the observable quantity, intensity I through the relation
4
),( dsrJI
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S ta rt P h o to n
E nd
F lo w C h artSta rt P ho ton
E nd
S et s tep s izew h en requ ired
M o ve P h o ton
M ove P ho ton to bo und aryP a rtia l Transm it
A b so rb
S catte r
Te rm in ate P ho to n
A no the r P ho to n
H it B o und ary
N
N
Y
Y
R
T
S e t rem ain ing s tepto new step size ,reve rse d irec tion
Absorption weakens intensityScattering changes direction
Calculate photon weight by albedo
New direction based on g
Continue until photon escapesForward or backwards
Monte Carlo Simulation of Irradiance:Based on probabilities from optical parameters
as
sa
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Calculation of enhancements basedOn Monte Carlo simulation
Muscle more absorbing than brain: limits enhancement Over purely scattering tissues
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Comparison of gain in simulation and experimentfor beads in phantom using optical parameters in literature
Gain over epi-detection is substantial
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Gasi
Gain is ~8 foldPredicted ~12 fold
Discrepancy probably due to imperfect optics