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 transverse Different effective focal lengths, positions Constant optical Path difference Every ray arrives At same focal point

Adaptive optics idea Active element undoes what microscope, specimen does to PSF Correction is determined by iteration: genetic algorithms, random searches More correction takes more time

37 element micromachined deformable mirror Can 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 strength For 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 bladder 1.3 NA 40x 30 microns into the tissue Surface optimized Optimized for 30 microns Need to optimize at every depth

CARS and adaptive optics Xie and Girkin Opex

Non-resonant CARS from glass-air interface

Depth dependence of CARS for beads in agarose Optimizing at greatest depth works best Systems 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 planes Have different Focal lengths

Correction of Astigmatism

AO on zebrafish larvae Olfactory bulb:GFP 50 microns 200 microns Imaging bloodflow

Wavefront sensing and correction using Spatial Light Modulator SLM larger range than Deformable mirror: better depth Eliceiri tbp

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 seconds Few 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)

Light Attenuation in tissue Z= depth from surface Simplest case fit to µ s [cm -1 ] 1/ µ s =scattering length, or mean free path Multiple scattering in thick, turbid media g=anisotropy, avg cos 0=isotropic 1=all forward Tendon~0.9 Brain=0.1

Photon Transport Theory J(r,s) in a specific direction s within a unit solid angle dω Anisotropy around propagation axis radiance J(r,s) relates to the observable quantity, intensity I through the relation

Absorption weakens intensity Scattering changes direction Calculate photon weight by albedo New direction based on g Continue until photon escapes Forward or backwards Monte Carlo Simulation of Irradiance: Based on probabilities from optical parameters

Calculation of enhancements based On Monte Carlo simulation Muscle more absorbing than brain: limits enhancement Over purely scattering tissues

Comparison of gain in simulation and experiment for beads in phantom using optical parameters in literature Gain over epi-detection is substantial

GasiGasi Gain is ~8 fold Predicted ~12 fold Discrepancy probably due to imperfect optics