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