1. Digital Holographic Microscopy for Quantitative Visualization
Li, Yan
GERI weekly seminar
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 Digital holography 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
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.

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

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
Object Standard deviation Max value Min value
Mirror
Test target
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

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

17. 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

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?