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Adaptive optics: optimization and wavefront sensing Novel microscope enhancements. confocal. widefield. Spherical Aberration (on axis). Constant optical Path difference Every ray arrives At same focal point. Perfect lens. Real lens. 2 related types, lateral and transverse - PowerPoint PPT Presentation
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1) Adaptive optics: optimization and wavefront sensing
2) Novel microscope enhancements
widefield confocal
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
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
37 element micromachined deformable mirrorCan travel 6 microns
Norris. J. Microcopy 2002
Performance for TPEF of coumarin dye solution
Good agreement with calculated, measured in simple specimen
Adaptive optics on non-scanning 2-photon microscope
600 microns into solution:PSF greatly improved
Lateral PSFs (measured by THG)
Adaptive optics improves resolution and signal strengthFor nonlinear optical processes (TPEF, SHG, THG, CARS)
Girkin, OPEX
Optimize feedback based on two-photon fluorescence intensity
Setup for adaptive optics on laser scanning microscope
Correction for TPEF of sub-resolution bead
x-y optical section
Significant improvement even for beads in water
Correction for TPEF of sub-resolution bead
x-z cross section
Significant improvement even for beads into 30 microns of water
Improvement in PSF important for multiphoton processes
TPEF of guinea pig bladder1.3 NA 40x
30 microns into the tissue
Surfaceoptimized
Optimized for30 microns
Need to optimize at every depth
CARS and adaptive optics
Xie and GirkinOpex
Non-resonant CARS from glass-air interface
Depth dependence of CARS for beads in agarose
Optimizing at greatest depth works bestSystems aberrations also very important
Comparison of CARS image with system, sample induced aberrations
600 microns into solution
Comparison of CARS image with system, sample induced aberrations from tissue
Radial Dependence of correction
Best response when optimize at every point But very slow
Adaptive Optics by Wavefront correction
Denk, PNAS, 2006
Astigmatism
Different planesHave differentFocal lengths
Correction of Astigmatism
AO on zebrafish larvaeOlfactory bulb:GFP
50 microns
200 microns
Imaging bloodflow
Wavefront sensing and correction using Spatial Light Modulator
SLM larger range than Deformable mirror: better depth
Eliceiritbp
MPE in vivo live animal imaging
Flexible periscope converts inverted to upright microscope
Difficulties with live animal imaging: respiration
8 second intervals, each scan 2 secondsFew micron motion, even anesthetized
Performance for in vivo imaging of muscle
Imaging through 200 microns of tissue
TPEF of kidney of anesthetized rabbit kidney
Breath-holding for one minute:Necessary for internal organ imaging
Fraction of light collected in epi-illumination geometry
High NA only collects 30% of available light (ideal limit without absorption and scattering)
Parabolic reflector to enhance light collection
Balaban, J. Microscopy (2007)
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
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
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
Calculation of enhancements basedOn Monte Carlo simulation
Muscle more absorbing than brain: limits enhancement Over purely scattering tissues
Comparison of gain in simulation and experimentfor beads in phantom using optical parameters in literature
Gain over epi-detection is substantial
Gasi
Gain is ~8 foldPredicted ~12 fold
Discrepancy probably due to imperfect optics