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A New Principle for Contrast Microscopy

A New Principle for Contrast Microscopy. Holography with Second Harmonic Signals. Ye Pu and Demetri Psaltis Department of Electrical Engineering Periklis Pantazis and Scott Fraser Beckman Institute California Institute of Technology Pasadena, CA 91125.

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A New Principle for Contrast Microscopy

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  1. A New Principle for Contrast Microscopy Holography with Second Harmonic Signals Ye Pu and Demetri Psaltis Department of Electrical Engineering Periklis Pantazis and Scott Fraser Beckman Institute California Institute of Technology Pasadena, CA 91125

  2. The Ubiquity of 4-Dimensional Dynamics Complex Dynamics • Ubiquitous • Often involves 4-D function of space and time in wide scales • Great impacts to many sciences and industries

  3. Holography: Capturing Fast4-Dimensional Dynamics HPIV Recording Reconstruction Holographic Particle Image Velocimetry (HPIV) • Samples the dynamic system with microparticles • Records 3D particle images in one snapshot with holography • Extracts particle velocity through double-exposure • Temporal sampling window: 10 ns – 100 fs

  4. Holography: Capturing Fast4-Dimensional Dynamics Y. Pu and H. Meng, Appl. Opt. 44, 7697 (2005).

  5. Holography Going Second Harmonic Harmonic Holography (H2) • Imaging at 2nd harmonic frequency • Femtosecond NIR excitation • Non-centrosymmetric nanocrystal markers • Dark-field imaging Holography for Microscopy • Imaging at fundamental frequency • Nanoparticles as tracers • Background scattering

  6. Why Second Harmonic? Second Harmonic Generation… • is a coherent two-photon process • requires non-centrosymmetric crystal structures • is not allowed in centrosymetric and isotropic materials (gases, liquids, and most biological tissues)

  7. Compared to Fluorescence …

  8. Principle of H2 Microscopy Fluorescence Microscopy (2D) H2 Microscopy (Whole-field 3D)

  9. H2 Imaging @ 2nd Harmonic Sample: • 100 nm BaTiO3 in cubic phase (centrosymmetric) • Sintered at 1000ºC for one hour • Converted to tetragonal phase (non-centrosymmetric) • Forms large size (microns) clusters Holography: • Digitally recorded hologram • Computational reconstruction of images • Numerical correction of aberration by ray-tracing

  10. Experimental Results (1 pulse) Direct Image Holographic Reconstruction

  11. Experimental Results (5 pulses) Direct Image Holographic Reconstruction

  12. Experimental Results (10 pulses) Direct Image Holographic Reconstruction

  13. Signal to Noise Ratio SNR Advantage: 4.36 (1 pulse) 2.32 (5 pulses) 1.83 (10 pulses) Aberration Correction + Read Noise Removal Aberration Correction

  14. Field Resonance Enhanced Second Harmonic (FRESH) FRESH Principle • Analogous to Fluorescent Resonance Energy Transfer (FRET) • Exciter (metal nanostructure) enhances local electric field • Probe (non-centrosymmetric nanocrystal) probes the enhanced field and emits drastically increased second harmonic signal • I(2w) E(w)4

  15. Survey of 3D Imaging Techniques ACRONYMS: AFM: Atomic force microscopy CLSM: Confocal scanning microscopy Cryo-EM: Cryogenic electron microscopy DC-WFM: Deconvolution wide-field microscopy MMM: Multiphoton multifocal microscopy SD-CLSM: Scanning disk CLSM SHIM: Second harmonic imaging microscopy STM: Scanning tunneling microscopy TIR-FM: Total internal reflection fluorescence microscopy 2PLSM: Two-photon laser scanning microscopy

  16. Holographic Particle Imaging for 4-D Dynamic System Measurement Thank You!

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