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 Two-step imaging Recording: interference pattern Reconstruction Digital holography Recording medium: CCD or CMOS camera Reconstruction: numerical algorithms

General methods Digital holography Phase shifting Fourier transform analysis Digital holography Numerical reconstruction

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

Macroscopic object Surface roughness Multiple holograms: wavelength, illumination, refractive index Macroscopic objects may be defined as objects with dimensions at least 4 orders of magnitude greater than the wavelength of the optical field. Because their roughness is comparable to the wavelength of the optical wave, their phase-contrast images consist of values randomly distributed between the range and . Therefore, a point-to-point subtraction of two phase-contrast images is necessary to determine the 3D contours of large objects. The result of this subtraction produces fringes which are absolute contours of surface height above some reference surface. Usually these two phase-contrast images of the same macroscopic object are obtained by using either two different illumination sources, two different wavelengths or two different refractive indices.

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

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

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

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

2D fitting procedure

Holographic microscope for reflection imaging

Holographic microscope for transmission imaging

Example 1: mirror

Example 2: resolution test target Object Standard deviation Max value Min value Mirror 0.236485 3.35238 -0.141377 Test target 0.713149 8.55379 -5.66697 The physical resolutions of the bar patterns in ROI are as below: Resolution(line pair per millimetre) Bars with “3” aside          40.32 Bars with “4” aside          45.25 Bars with “5” aside          50.80

Example 3: bull sperms slide 20x magnification Size of the sperm: about 20~30 microns

Conclusions Feasibility Advantages Disadvantages 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

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

Thank you! Questions?