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Virtual Journal for Biomedical Optics

Virtual Journal for Biomedical Optics


  • Editors: Andrew Dunn and Anthony Durkin
  • Vol. 6, Iss. 8 — Aug. 26, 2011

Automated imaging, identification, and counting of similar cells from digital hologram reconstructions

Mona Mihailescu, Mihaela Scarlat, Alexandru Gheorghiu, Julia Costescu, Mihai Kusko, Irina Alexandra Paun, and Eugen Scarlat  »View Author Affiliations

Applied Optics, Vol. 50, Issue 20, pp. 3589-3597 (2011)

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This paper presents our method, which simultaneously combines automatic imaging, identification, and counting with the acquisition of morphological information for at least 1000 blood cells from several three-dimensional images of the same sample. We started with seeking parameters to differentiate between red blood cells that are similar but different with respect to their development stage, i.e., mature or immature. We highlight that these cells have different diffractive patterns with complementary central intensity distribution in a given plane along the propagation axis. We use the Fresnel approximation to simulate propagation through cells modeled as spheroid-shaped phase objects and to find the cell property that has the dominant influence on this behavior. Starting with images obtained in the reconstruction step of the digital holographic microscopy technique, we developed a code for automated simultaneous individual cell image separation, identification, and counting, even when the cells are partially overlapped on a slide, and accurate measuring of their morphological features. To find the centroids of each cell, we propose a method based on analytical functions applied at threshold intervals. Our procedure separates the mature from the immature red blood cells and from the white blood cells through a decision based on gradient and radius values.

© 2011 Optical Society of America

OCIS Codes
(090.0090) Holography : Holography
(090.1970) Holography : Diffractive optics
(170.0180) Medical optics and biotechnology : Microscopy
(090.1995) Holography : Digital holography

ToC Category:

Original Manuscript: February 28, 2011
Manuscript Accepted: May 4, 2011
Published: July 7, 2011

Virtual Issues
Vol. 6, Iss. 8 Virtual Journal for Biomedical Optics
July 6, 2011 Spotlight on Optics

Mona Mihailescu, Mihaela Scarlat, Alexandru Gheorghiu, Julia Costescu, Mihai Kusko, Irina Alexandra Paun, and Eugen Scarlat, "Automated imaging, identification, and counting of similar cells from digital hologram reconstructions," Appl. Opt. 50, 3589-3597 (2011)

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  1. E. B. Krumbhaar, “Reticulosis-increased percentage in the peripheral blood,” in Hematology: Landmark Papers of the Twentieth Century, M.A.Lichtman, L.A.Boxer, E.Henderson, and J.L.Spivak, eds. (Academic, 2000), p. 805.
  2. L. M. Lowenstein, “The Mammalian reticulocyte,” in International Review of Cytology, G.H.Bourne and J.F.Danielli, eds. (Academic, 1959), Vol.  8. [CrossRef] [PubMed]
  3. L. Blanc, A. De Gassart, C. Géminard, P. Bette-Bobillo, and M. Vidal, “Exosome release by reticulocytes—an integral part of the red blood cell differentiation system,” Blood Cells Mol. Dis. 35, 21–26 (2005). [CrossRef] [PubMed]
  4. J. G. Stephens, “Surface and fragility differences between mature and immature red cells,” J. Physiol. 99, 30–48 (1940). [PubMed]
  5. A. K. Percy, E. Schmell, B. J. Earles, and W. J. Lennarz, “Phospholipid biosynthesis in the membranes of immature and mature red blood cells,” Biochemistry 12, 2456–2461 (1973). [CrossRef] [PubMed]
  6. T. Lecklin, A. Tuominen, and M. Nikinmaa, “The adrenergic volume changes of immature and mature rainbow trout (Oncorhynchus mykiss) erythrocytes,” J. Exp. Biol. 203, 3025–3031(2000). [PubMed]
  7. Y. M. Serebrennikova, J. Patel, W. K. Milhous, and L. H. Garc?a-Rubio, “Quantitative analysis of morphological alterations in Plasmodium falciparum infected red blood cells through theoretical interpretation of spectral measurements,” J. Therm. Biol. 265, 493–500 (2010). [CrossRef]
  8. J. C. Thompson and B. W. Manktelow, “Pathogenesis and red blood cell destruction in hemoglobinemic leptospirosis,” J. Comp. Pathol. 96, 529–540 (1986). [CrossRef] [PubMed]
  9. S. Christel and C. Little, “Morphological changes during heating of erythrocytes from stored human blood,” J. Therm. Biol. 9, 221–228 (1984). [CrossRef]
  10. I. A. Kabat, W. Leyko, B. Kwiatkowski, and I. Zakrzewska, “Osmotic properties and morphological changes of submicroscopic surface structures of mammalian red blood cells subjected to UV-irradiation in vitro,” Zentralbl. Bakteriol. Orig. B 159, 88–94 (1974). [PubMed]
  11. A. J. McGoron, C. H. Joiner, M. B. Palascak, W. J. Claussen, and R. S. Franco, “Dehydration of mature and immature sickle red blood cells during fast oxygenation/deoxygenation cycles: role of KCl cotransport and extracellular calcium,” Blood 95, 2164–2168 (2000). [PubMed]
  12. N. Mochandas, M. R. Clark, M. S. Jacobs, and S. B. Shohet, “Analysis of factors regulating erythrocyte deformability,” J. Clin. Invest. 66, 563–573 (1980). [CrossRef]
  13. M. Mir, H. Ding, Z. Wang, J. Reedy, K. Tangella, and G. Popescu, “Blood screening using diffraction phase cytometry,” J. Biomed. Opt. 15, 027016 (2010). [CrossRef] [PubMed]
  14. S. Rancourt-Grenier, M.-T. Wei, J.-J. Bai, A. Chiou, P. P. Bareil, P.-L. Duval, and Y. Sheng, “Dynamic deformation of red blood cell in dual-trap optical tweezers,” Opt. Express 18, 10462–10472 (2010). [CrossRef] [PubMed]
  15. C. Fang-Yen, S. Oh, Y. Park, W. Choi, S. Song, H. S. Seung, R. R. Dasari, and M. S. Feld, “Imaging voltage-dependent cell motions with heterodyne Mach–Zehnder phase microscopy,” Opt. Lett. 32, 1572–1574 (2007). [CrossRef] [PubMed]
  16. B.-W. Yang and Z. Li, “Measuring microinteractions between coagulating red blood cells using optical tweezers,” Biomed. Opt. Express 1, 1217–1224 (2010). [CrossRef]
  17. Y. K. Park, C. A. Best, K. Badizadegan, R. R. Dasari, M. S. Feld, T. Kuriabova, M. L. Henle, A. J. Levine, and G. Popescu, “Measurement of red blood cell mechanics during morphological changes,” Proc. Natl. Acad. Sci. USA 107, 6731–6736 (2010). [CrossRef] [PubMed]
  18. Y.-S. Choi and S. Lee, “Three-dimensional volumetric measurement of red blood cell motion using digital holographic microscopy,” Appl. Opt. 48, 2983–2990 (2009). [CrossRef] [PubMed]
  19. B. Rappaz, A. Barbul, A. Hoffmann, D. Boss, R. Korenstein, C. Depeursinge, P. J. Magistretti, and P. Marquet, “Spatial analysis of erythrocyte membrane fluctuations by digital holographic microscopy,” Blood Cells Mol. Dis. 42, 228–232 (2009). [CrossRef] [PubMed]
  20. M. Paturzo, F. Merola, S. Grilli, S. De Nicola, A. Finizio, and P. Ferraro, “Super-resolution in digital holography by a two-dimensional dynamic phase grating,” Opt. Express 16, 17107–17118 (2008). [CrossRef] [PubMed]
  21. V. Mico, Z. Zalevsky, C. Ferreira, and J. García, “Super-resolution digital holographic microscopy for three-dimensional samples,” Opt. Express 16, 19260–19270 (2008). [CrossRef]
  22. G. J. Streekstra1, J. G. G. Dobbe, and A. G. Hoekstra, “Quantification of the fraction poorly deformable red blood cells using ektacytometry,” Opt. Express 18, 14173–14182 (2010). [CrossRef]
  23. J. W. Goodman, Introduction to Fourier Optics (McGraw-Hill, 1968).
  24. M. Mihailescu, “Natural quasi-periodic binary structure with focusing property in near-field diffraction pattern,” Opt. Express 18, 12526–12536 (2010). [CrossRef] [PubMed]
  25. B. Rappaz, A. Barbul, Y. Emery, R. Korenstein, C. Depeursinge, P. J. Magistretti, and P. Marquet, “Comparative study of human erythrocytes by digital holographic microscopy, confocal microscopy, and impedance volume analyzer,” Cytometry Part A 73A, 895–903 (2008). [CrossRef]
  26. W. Groner, N. Mohandas, and M. Bessis, “New optical technique for measuring erythrocyte deformability with the ektacytometer,” Clin. Chem. 26, 1435–1442 (1980). [PubMed]
  27. F. M. Gaffney, “Experimental hemolytic anemia with particular reference to the corpuscular hemoglobin concentrations of the erythrocytes,” Br. J. Haematol. 3, 311–319 (1957). [CrossRef] [PubMed]
  28. G. d’Onofrio, R. Chirillo, G. Zini, G. Caenaro, M. Tommasi, and G. Micciulli, “Simultaneous measurement of reticulocyte and red blood cell indices in healthy subjects and patients with microcytic and macrocytic anemia,” Blood 85, 818–823 (1995). [PubMed]
  29. F. Montfort, F. Charrière, T. Colomb, E. Cuche, P. Marquet, and C. Depeursinge, “Purely numerical compensation for microscope objective phase curvature in digital holographic microscopy: influence of digital phase masks position,” J. Opt. Soc. Am. A 23, 2944–2953 (2006). [CrossRef]
  30. M. Mir, Z. Wang, K. Tangella, and G. Popescu, “Phase cytometry: blood on a CD-ROM,” Opt. Express 17, 2579–2585 (2009). [CrossRef] [PubMed]

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