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Optics Express

Optics Express

  • Editor: Andrew M. Weiner
  • Vol. 21, Iss. 5 — Mar. 11, 2013
  • pp: 6466–6479

Separation of overlapped particles in digital holographic microscopy

Ahmed El Mallahi and Frank Dubois  »View Author Affiliations

Optics Express, Vol. 21, Issue 5, pp. 6466-6479 (2013)

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In this paper, we present a procedure to separate aggregates of overlapped particles in digital holograms, based on a focus plane analysis applied to each particle. The method can be applied either on phase or on amplitude objects, according that each object has a border in one focus plane. Numerical simulations are performed to quantify the robustness of the process by increasing the overlapping areas between the particles. The separation algorithm is successfully demonstrated experimentally on different types of aggregates.

© 2013 OSA

OCIS Codes
(090.0090) Holography : Holography
(100.0100) Image processing : Image processing
(100.2000) Image processing : Digital image processing
(100.6890) Image processing : Three-dimensional image processing
(090.1995) Holography : Digital holography

ToC Category:

Original Manuscript: February 22, 2013
Revised Manuscript: February 27, 2013
Manuscript Accepted: February 27, 2013
Published: March 7, 2013

Virtual Issues
Vol. 8, Iss. 4 Virtual Journal for Biomedical Optics

Ahmed El Mallahi and Frank Dubois, "Separation of overlapped particles in digital holographic microscopy," Opt. Express 21, 6466-6479 (2013)

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  1. U. Schnars and W. Jüptner, “Digital recording and numerical reconstruction of holograms,” Meas. Sci. Technol.13(9), R85–R101 (2002). [CrossRef]
  2. P. Ferraro, S. De Nicola, A. Finizio, G. Coppola, S. Grilli, C. Magro, and G. Pierattini, “Compensation of the inherent wave front curvature in digital holographic coherent microscopy for quantitative phase-contrast imaging,” Appl. Opt.42(11), 1938–1946 (2003). [CrossRef] [PubMed]
  3. P. Marquet, B. Rappaz, P. J. Magistretti, E. Cuche, Y. Emery, T. Colomb, and C. Depeursinge, “Digital holographic microscopy: a noninvasive contrast imaging technique allowing quantitative visualization of living cells with subwavelength axial accuracy,” Opt. Lett.30(5), 468–470 (2005). [CrossRef] [PubMed]
  4. F. Dubois, C. Yourassowsky, O. Monnom, J.-C. Legros, O. Debeir, P. Van Ham, R. Kiss, and C. Decaestecker, “Digital holographic microscopy for the three-dimensional dynamic analysis of in vitro cancer cell migration,” J. Biomed. Opt.11(5), 054032 (2006). [CrossRef] [PubMed]
  5. B. Kemper and G. von Bally, “Digital holographic microscopy for live cell applications and technical inspection,” Appl. Opt.47(4), A52–A61 (2008). [CrossRef] [PubMed]
  6. D. Allano, M. Malek, F. Walle, F. Corbin, G. Godard, S. Coëtmellec, B. Lecordier, J.-M. Foucaut, and D. Lebrun, “Three-dimensional velocity near-wall measurements by digital in-line holography: calibration and results,” Appl. Opt.52(1), A9–A17 (2013). [CrossRef] [PubMed]
  7. A. El Mallahi, C. Minetti, and F. Dubois, “Automated three-dimensional detection and classification of living organisms using digital holographic microscopy with partial spatial coherent source: Application to the monitoring of drinking water resources,” Appl. Opt.52(1), A68–A80 (2013). [CrossRef] [PubMed]
  8. S. Beucher, “The watershed transformation applied to image segmentation,” Scanning Microsc. Suppl.6, 299–314 (1992).
  9. N. Malpica, C. O. de Solórzano, J. J. Vaquero, A. Santos, I. Vallcorba, J. M. García-Sagredo, and F. del Pozo, “Applying watershed algorithms to the segmentation of clustered nuclei,” Cytometry28(4), 289–297 (1997). [CrossRef] [PubMed]
  10. F. Long, H. Peng, and E. Myers, “Automatic segmentation of nuclei in 3D microscopy images of C. Elegans,” in Proceedings of IEEE Conference on Biomedical Imaging: From Nano to Macro (IEEE, 2007), pp 536–539. [CrossRef]
  11. A. Pinidiyaarachchi and C. Wählby, “Seeded watersheds for combined segmentation and tracking of cells,” in Proceedings of the 13th international conference on Image Analysis and Processing, (Springer Berlin, 2005), pp. 336–343. [CrossRef]
  12. P. Soille, Morphological Image Analysis: Principle and Applications (Springer, 1999).
  13. J. Cheng and J. C. Rajapakse, “Segmentation of clustered nuclei with shape markers and marking function,” IEEE Trans. Biomed. Eng.56(3), 741–748 (2009). [CrossRef] [PubMed]
  14. C. Jung and C. Kim, “Segmenting clustered nuclei using H-minima transform-based marker extraction and contour parameterization,” IEEE Trans. Biomed. Eng.57(10), 2600–2604 (2010). [CrossRef] [PubMed]
  15. P. V. C. Hough, “Method and means for recognizing complex patterns,” U.S. Patent 3 069 654 (1969).
  16. D. H. Ballard, “Generalizing the Hough transform to detect arbitrary shapes,” Pattern Recognit.13(2), 111–122 (1981). [CrossRef]
  17. M. Smereka and I. Duleba, “Circular object detection using a modified Hough transform,” Int. J. Appl. Math. Comput. Sci.18(1), 85–91 (2008). [CrossRef]
  18. B. Parvin, Q. Yang, J. Han, H. Chang, B. Rydberg, and M. H. Barcellos-Hoff, “Iterative voting for inference of structural saliency and characterization of subcellular events,” IEEE Trans. Image Process.16(3), 615–623 (2007). [CrossRef] [PubMed]
  19. Q. Wen, H. Chang, and B. Parvin, “A Delaunay triangulation approach for segmenting clumps of nuclei,” in Proceedings of IEEE Conference on Biomedical Imaging: From Nano to Macro (IEEE, 2009), pp 9–12.
  20. S.-H. Lee and D. G. Grier, “Holographic microscopy of holographically trapped three-dimensional structures,” Opt. Express15(4), 1505–1512 (2007). [CrossRef] [PubMed]
  21. L. Dixon, F. C. Cheong, and D. G. Grier, “Holographic deconvolution microscopy for high-resolution particle tracking,” Opt. Express19(17), 16410–16417 (2011). [CrossRef] [PubMed]
  22. J. Fung, K. E. Martin, R. W. Perry, D. M. Kaz, R. McGorty, and V. N. Manoharan, “Measuring translational, rotational, and vibrational dynamics in colloids with digital holographic microscopy,” Opt. Express19(9), 8051–8065 (2011). [CrossRef] [PubMed]
  23. J. Fung, R. W. Perry, T. G. Dimiduk, and V. N. Manoharan, “Imaging multiple colloidal particles by fitting electromagnetic scattering solutions to digital holograms,” J. Quant. Spectrosc. Radiat. Transf.113(18), 2482–2489 (2012). [CrossRef]
  24. F. Dubois, C. Schockaert, N. Callens, and C. Yourassowsky, “Focus plane detection criteria in digital holography microscopy by amplitude analysis,” Opt. Express14(13), 5895–5908 (2006). [CrossRef] [PubMed]
  25. A. El Mallahi and F. Dubois, “Dependency and precision of the refocusing criterion based on amplitude analysis in digital holographic microscopy,” Opt. Express19(7), 6684–6698 (2011). [CrossRef] [PubMed]
  26. M. Sezgin and B. Sankur, “Survey over image thresholding techniques and quantitative performance evaluation,” J. Electron. Imaging13, 145–165 (2004).
  27. M. Nazarathy and J. Shamir, “Fourier optics described by operator,” J. Opt. Soc. Am. A70(2), 150–159 (1980). [CrossRef]
  28. M. Born and E. Wolf, Principles of Optics-Electromagnetic Theory of Propagation, Interference and Diffraction of Light (Cambridge, 1980).
  29. J.-C. Diels and W. Rudolph, Ultrashort Laser Pulse Phenomena (Elsevier, 2006).
  30. J. W. Goodman, Introduction to Fourier Optics (McGraw-Hill, 1996).
  31. N. Callens, C. Minetti, G. Coupier, M.-A. Mader, F. Dubois, C. Misbah, and T. Podgorski, “Hydrodynamic lift of vesicles under shear flow in microgravity,” Europhys. Lett.83(2), 24002 (2008). [CrossRef]
  32. X. Grandchamp, G. Coupier, A. Srivastav, C. Minetti, and T. Podgorski, “Lift and down-gradient shear-induced diffusion in red blood cell suspensions,” Phys. Rev. Lett. (to be published).
  33. I. Moon, B. Javidi, F. Yi, D. Boss, and P. Marquet, “Automated statistical quantification of three-dimensional morphology and mean corpuscular hemoglobin of multiple red blood cells,” Opt. Express20(9), 10295–10309 (2012). [CrossRef] [PubMed]

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