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Adaptive optics: optimization and wavefront sensing Novel microscope enhancements

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

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Adaptive optics: optimization and wavefront sensing Novel microscope enhancements

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  1. Adaptive optics: optimization and wavefront sensing • Novel microscope enhancements

  2. confocal widefield

  3. 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 Different effective focal lengths, positions

  4. 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

  5. 37 element micromachined deformable mirror Can travel 6 microns

  6. Performance for TPEF of coumarin dye solution Good agreement with calculated, measured in simple specimen Norris. J. Microcopy 2002

  7. Adaptive optics on non-scanning 2-photon microscope 600 microns into solution: PSF greatly improved

  8. Lateral PSFs (measured by THG) Adaptive optics improves resolution and signal strength For nonlinear optical processes (TPEF, SHG, THG, CARS)

  9. Setup for adaptive optics on laser scanning microscope Optimize feedback based on two-photon fluorescence intensity Girkin, OPEX

  10. Correction for TPEF of sub-resolution bead x-y optical section Significant improvement even for beads in water

  11. Correction for TPEF of sub-resolution bead x-z cross section Significant improvement even for beads into 30 microns of water

  12. Improvement in PSF important for multiphoton processes

  13. 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

  14. CARS and adaptive optics Xie and Girkin Opex

  15. Non-resonant CARS from glass-air interface

  16. Depth dependence of CARS for beads in agarose Optimizing at greatest depth works best Systems aberrations also very important

  17. Comparison of CARS image with system, sample induced aberrations 600 microns into solution

  18. Comparison of CARS image with system, sample induced aberrations from tissue

  19. Radial Dependence of correction Best response when optimize at every point But very slow

  20. Adaptive Optics by Wavefront correction Denk, PNAS, 2006

  21. Astigmatism Different planes Have different Focal lengths

  22. Correction of Astigmatism

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

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

  25. MPE in vivo live animal imaging Flexible periscope converts inverted to upright microscope

  26. Difficulties with live animal imaging: respiration 8 second intervals, each scan 2 seconds Few micron motion, even anesthetized

  27. Performance for in vivo imaging of muscle

  28. Imaging through 200 microns of tissue

  29. TPEF of kidney of anesthetized rabbit kidney Breath-holding for one minute: Necessary for internal organ imaging

  30. Fraction of light collected in epi-illumination geometry High NA only collects 30% of available light (ideal limit without absorption and scattering)

  31. Parabolic reflector to enhance light collection Balaban, J. Microscopy (2007)

  32. 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 g=anisotropy, avg cos 0=isotropic 1=all forward Tendon~0.9 Brain=0.1

  33. 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

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

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

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

  37. Gain is ~8 fold Predicted ~12 fold Gasi Discrepancy probably due to imperfect optics

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