Single Particle Tracking Across Sequences of Microscopical Images: Application to Platelet Adhesion Under Flow

  • Marianna Machin
  • Andrea Santomaso
  • Mario Mazzucato
  • Maria Rita Cozzi
  • Monica Battiston
  • Luigi De Marco
  • Paolo CanuEmail author


A versatile and automated image processing technique and data extraction procedure from videomicroscopic data is presented. The motivation is a detailed quantification of blood platelet adhesion from laminar flow onto a surface. The characteristics of the system under observation (type of cells, their speed of movement, and the quality of the optical image to analyze) provided the criteria for developing a new procedure enabling tracking for long image sequences. Specific features of the novel method include: automatic segmentation methodology which removes operator bias; platelet recognition across the series of images based on a probability density function (two-dimensional, Gaussian-like) tailored to the physics of platelet motion on the surface; options to automatically tune the procedure parameters to explore different applications; integrated analysis of the results (platelet trajectories) to obtain relevant information, such as deposition and removal rates, displacement distributions, pause times and rolling velocities. Synthetic images, providing known reference conditions, are used to test the method. The algorithm operation is illustrated by application to images obtained by fluorescence microscopy of the interaction between platelets and von Willebrand factor-coated surfaces in parallel-plate flow chambers. Potentials and limits are discussed, together with evaluation of errors resulting from an inaccurate tracking.


Image analysis Platelets Fluorescence microscopy Perfusion chamber Gaussian distribution Image simulation 



We thank the Centro di Riferimento Oncologico (C.R.O.-I.R.C.C.S., Aviano, Italy) for supporting A.S.


  1. 1.
    Acton, S. T., K. Wethmar, and K. Ley. Automatic tracking of rolling leukocytes in vivo. Microvasc. Res. 63:139–148, 2002.PubMedCrossRefGoogle Scholar
  2. 2.
    Alon, R., S. Chen, K. D. Puri, E. B. Finger, and T. A. Springer. The kinetics of L-selectin tethers and the mechanics of selectin-mediated rolling. J. Cell. Biol. 138:1169–1180, 1997.PubMedCrossRefGoogle Scholar
  3. 3.
    Anderson, C. M., G. N. Georgiou., I. E. Morrison, G. V. Stevenson, and R. J. Cherry. Tracking of cell surface receptors by fluorescence digital imaging microscopy using a charge-coupled device camera: low-density lipoprotein and influenza virus receptor mobility at 4°C. J. Cell Sci. 101:415–425, 1992.PubMedGoogle Scholar
  4. 4.
    Cheezum, M. K., W. F. Walker, and W. H. Guilford. Quantitative comparison of algorithms for tracking single fluorescent particles. Biophys. J. 81:2378–2388, 2001.PubMedGoogle Scholar
  5. 5.
    Chen, S. and T. A. Springer. An automatic braking system that stabilizes leukocyte rolling by an increase in selectin bond number with shear. J. Cell Biol. 144:185–200, 1999.Google Scholar
  6. 6.
    Doggett, T. A., G. Girdhar, A. Lawshe, D. W. Schmidtke, I. J. Laurenzi, S. L. Diamond, and T. G. Diacovo. Selectin-like kinetics and biomechanics promote rapid platelet adhesion in flow: the GP Ibα-vWF tether bond. Biophys. J. 83:194–205, 2002.PubMedGoogle Scholar
  7. 7.
    Dow, J. A., J. M. Lackie and K. V. Crocket A simple microcomputer-based system for real-time analysis of cell behaviour. J. Cell Sci. 87:171–182, 1987.PubMedGoogle Scholar
  8. 8.
    Gelles, J., B. J. Schnapp, and M. P. Sheetz. Tracking kinesin-driven movements with nanometre-scale precision. Nature 331:450–453, 1988.PubMedCrossRefGoogle Scholar
  9. 9.
    Gerlich, D., J. Mattes, and R. Eils. Quantitative motion analysis and visualization of cellular structures. Methods 29:3–13, 2003.PubMedCrossRefGoogle Scholar
  10. 10.
    Ghosh, R. N., and W. W. Webb. Automated detection and tracking of individual and clustered cell surface low density lipoprotein receptor molecules. Biophys. J. 66:1301–1318, 1994.PubMedGoogle Scholar
  11. 11.
    Goldsmith, H. L., and V. T. Turitto. Rheological aspects of thrombosis and haemostasis: basic principles and applications. Thromb. Haemost. 55:415–435, 1986.PubMedGoogle Scholar
  12. 12.
    Kaplanski, G., C. Farnarier, O. Tissot, A. Pierres, A. M. Benoliel, M. C. Alessi, S. Kaplanski, and P. Bongrand. Granulocyte-endothelium initial adhesion. Analysis of transient binding events mediated by E-selectin in a laminar shear flow. Biophys. J. 64:1653–4, 1993.Google Scholar
  13. 13.
    Kerre, E. E., and M. Nachtegael. Fuzzy Techniques in Image Processing. Heidelberg: Physica-Verlag, 2000.Google Scholar
  14. 14.
    Knuth, D. The Art of Computer Programming, Vol. 2, 2nd ed. Reading: Addison-Wesley, 1981.Google Scholar
  15. 15.
    Kumar, R. A., J.-F. Dong, J. A. Thaggard, M. A. Cruz, J. A. Lopez, and L. V. McIntire. Kinetics of GPIbα-vWF-A1 tether bond under flow: effect of GPIbα mutations on the association and dissociation rates. Biophys. J. 85:4099–4109, 2003.PubMedCrossRefGoogle Scholar
  16. 16.
    Lim, J. S. Two-Dimensional Signal and Image Processing. Englewood Cliffs, NJ: Prentice Hall, 1990.Google Scholar
  17. 17.
    Machin, M., A. Santomaso, M. R. Cozzi, M. Battiston, M. Mazzucato, L. De Marco, and P. Canu. Characterization of platelet adhesion under flow using microscopic image sequence analysis. Int. J. Artif. Organs 28:678–685, 2005.PubMedGoogle Scholar
  18. 18.
    MATLAB, The MathWorks, Inc., version 7, Release 14 with service pack 1 and image processing toolbox version 5.0.1, 2005.Google Scholar
  19. 19.
    Mazzucato, M., P. Pradella, M. R. Cozzi, L. De Marco, and Z. M. Ruggeri. Sequential cytoplasmic calcium signals in a 2-stage platelet activation process induced by the glycoprotein Ibα mechanoreceptor. Blood 100:2793–2800, 2002.PubMedCrossRefGoogle Scholar
  20. 20.
    Mitra, S. K. Digital Signal Processing. New York: McGraw-Hill, 2001.Google Scholar
  21. 21.
    Miyata, S., and Z. M. Ruggeri. Distinct structural attributes regulating von Willebrand factor A1 domain interaction with platelet glycoprotein Ibα under flow. J. Biol. Chem. 274:6586–6593, 1999.PubMedCrossRefGoogle Scholar
  22. 22.
    Nazar, A. M., E. A. Silva, and J. J. Ammann. Image Processing for Particle Characterization. Mater. Characterization 36:165–173, 1996.CrossRefGoogle Scholar
  23. 23.
    Otsu, N. A threshold selection method from gray-level histograms. TSMCA TSMC- 9:62–66, 1979.Google Scholar
  24. 24.
    Pierres, A., A. M. Benoliel, and P. Bongrand. Measuring bonds between surface-associated molecules. J. Immunol Methods 196:105–20, 1996.PubMedCrossRefGoogle Scholar
  25. 25.
    Pierres, A., A. M. Benoliel, and P. Bongrand. Use of a laminar flow chamber to study the rate of bond formation and dissociation between surface-bound adhesion molecules: effect of applied force and distance between surfaces. Faraday Discuss. 111:321–330, 1998.PubMedCrossRefGoogle Scholar
  26. 26.
    Pierres, A., A. M. Benoliel, and P. Bongrand. Cell fitting to adhesive surfaces: a prerequisite to firm attachment and subsequent events. Eur. Cells Mater. 3:31–45, 2002.Google Scholar
  27. 27.
    Ruggeri, Z. M. Platelets in atherothrombosis. Nature Med. 8:1227–1234, 2002.PubMedCrossRefGoogle Scholar
  28. 28.
    Russ, J. C. Computer-Assisted Microscopy: the Measurement and Analysis of Images. New York, Plenum Press, 1990.Google Scholar
  29. 29.
    Sabri, S., F. Richelme, A. Pierres, A. M. Benoliel, and P. Bongrand. Interest of image processing in cell biology and immunology. J. Immunol. Methods 208:1–27, 1997.PubMedCrossRefGoogle Scholar
  30. 30.
    Sakariassen, K. S., V. T. Turitto, and H. R. Baumgartner. Recollections of the development of flow devices for studying mechanisms of hemostasis and thrombosis in flowing whole blood. J. Thromb. Haemost. 2:1681–1690, 2004.PubMedCrossRefGoogle Scholar
  31. 31.
    Savage, B., E. Saldivar, and Z. M. Ruggeri. Initiation of platelet adhesion by arrest onto fibrinogen or translocation on von Willebrand factor. Cell 84:289–297, 1996.PubMedCrossRefGoogle Scholar
  32. 32.
    Saxton, M. J., and K. Jacobson. Single particle tracking: applications to membrane dynamics. Ann. Rev. Biophys. Biomol. Struct. 26:373–399, 1997.CrossRefGoogle Scholar
  33. 33.
    Seinfeld, J. H., and S. N. Pandis. Atmospheric Chemistry and Physics: From Air Pollution to Climate Change. New York: Wiley, 1998.Google Scholar
  34. 34.
    Smith, M. J., E. L. Berg, and M. B. Lawrence. A direct comparison of selectin-mediated transient, adhesive events using high temporal resolution. Biophys. J. 77:3371–3383, 1999.PubMedGoogle Scholar
  35. 35.
    Turitto, V. T., and H. J. Weiss. Red blood cells: their dual role in thrombus formation. Science 207:541–543, 1980.PubMedCrossRefGoogle Scholar
  36. 36.
    Tvarusko, W., M. Bentele, T. Misteli, R. Rudolf, C. Kaether, D. L. Spector, H. H. Gerdes, and R. Eils. Time-resolved analysis and visualization of dynamic processes in living cells. Proc. Natl. Acad. Sci. 96:7950–7955, 1999.PubMedCrossRefGoogle Scholar
  37. 37.
    Usami, S., H. H. Chen, Y. Zhao, S. Chien, and R. Skalak. Design and construction of a linear shear stress flow chamber. Ann. Biomed. Eng. 21:77–83, 1999.CrossRefGoogle Scholar
  38. 38.
    Vincent, L., and P. Soille. Watersheds in Digital Spaces: An Efficient Algorithm Based on Immersion Simulations. TPAMI 13:583–598, 1991.CrossRefGoogle Scholar
  39. 39.
    Wilson, K. M., I. E. G. Morrison, P. R. Smith, N. Fernandez, and R. J. Cherry. Single particle tracking of cell-surface HLA-DR molecules using R-phycoerythrin labeled monoclonal antibodies and fluorescence digital imaging. J. Cell Sci. 109:2101–2109, 1996.PubMedGoogle Scholar
  40. 40.
    Wit, P. J., J. Noordmans, and H. J. Busscher. Tracking of colloidal particles using microscopic image sequence analysis. Application to particulate microelectrophoresis and particle deposition. Coll. Surf. A 125:85–92, 1997.CrossRefGoogle Scholar
  41. 41.
    Work, S. S., and D. M. Warshaw. Computer-assisted tracking of actin filament motility. Anal. Biochem. 202:275–285, 1992.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science + Business Media, Inc. 2006

Authors and Affiliations

  • Marianna Machin
    • 1
  • Andrea Santomaso
    • 1
  • Mario Mazzucato
    • 2
  • Maria Rita Cozzi
    • 2
  • Monica Battiston
    • 2
  • Luigi De Marco
    • 2
  • Paolo Canu
    • 1
    Email author
  1. 1.Department of Chemical Engineering Principles and Practice (DIPIC)University of Padova, Via MarzoloPadovaItaly
  2. 2.Servizio Immunotrasfusionale e Analisi Cliniche C.R.O.-I.R.C.C.S.Aviano (PN)Italy

Personalised recommendations