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Digital Holographic Microscopy for Quantitative Visualization

Li, Yan GERI weekly seminar 13-11-2009. Digital Holographic Microscopy for Quantitative Visualization. Outline. Introduction Quantitative phase-contrast imaging Experiments Conclusions Future work. Holography. A method for obtaining the amplitude and phase of a wavefield

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Digital Holographic Microscopy for Quantitative Visualization

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  1. Li, Yan GERI weekly seminar 13-11-2009 Digital Holographic Microscopy for Quantitative Visualization

  2. Outline Introduction Quantitative phase-contrast imaging Experiments Conclusions Future work

  3. Holography • A method for obtaining the amplitude and phase of a wavefield • Two-step imaging • Recording: interference pattern • Reconstruction • Digital holography • Recording medium: CCD or CMOS camera • Reconstruction: numerical algorithms

  4. General methods • Phase shifting • Fourier transform analysis • Digital holography • Numerical reconstruction

  5. Intensity-contrast image (Photograph) Amplitude-contrast result Phase-contrast result Hologram

  6. Macroscopic object • Surface roughness • Multiple holograms: wavelength, illumination, refractive index

  7. Amplitude-contrast result Phase-contrast result • Microscopic objects: • Quantitative phase-contrast image from single hologram

  8. Three contributions to the reconstructed phase • The absolute phase of the object • The tilt aberration due to the off-axis geometry and the curvature produced by the microscopic objective • Other aberrations or wavefront deformations induced by the setup

  9. Quantitative phase-contrast imaging • Capturing a reference hologram without the object to compensate the unwanted phase terms • Digital phase mask • Manually generated • 1D fitting procedure • 2D fitting procedure

  10. 1D fitting procedure Y = a0 + a1x + a2x2 + b1y + b2y2 Yh = a0 + a1x + a2x2

  11. 2D fitting procedure

  12. Holographic microscope for reflection imaging

  13. Holographic microscope for transmission imaging

  14. Example 1: mirror

  15. Example 2: resolution test target

  16. Example 3: bull sperms slide

  17. Conclusions • Feasibility • Advantages • Noncontact, full-field, vibration insensitive, marker free, investigation of dynamic processes • Disadvantages • Speckles, compromised resolution • Approaches to deal with disadvantages • Light source • Microscope objective with higher magnification

  18. Future work 2D fitting procedure Applying digital phase mask in hologram plane Setup for living cell applications Quality microscope objectives with higher magnification Other algorithms for numerical reconstruction

  19. Thank you! Questions?

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