European Biophysics Journal

, Volume 38, Issue 5, pp 679–686 | Cite as

Live biospeckle laser imaging of root tissues

  • Roberto A. Braga
  • L. Dupuy
  • M. Pasqual
  • R. R. Cardoso
Original Paper


Live imaging is now a central component for the study of plant developmental processes. Currently, most techniques are extremely constraining: they rely on the marking of specific cellular structures which generally apply to model species because they require genetic transformations. The biospeckle laser (BSL) system was evaluated as an instrument to measure biological activity in plant tissues. The system allows collecting biospeckle patterns from roots which are grown in gels. Laser illumination has been optimized to obtain the images without undesirable specular reflections from the glass tube. Data on two different plant species were obtained and the ability of three different methods to analyze the biospeckle patterns are presented. The results showed that the biospeckle could provide quantitative indicators of the molecular activity from roots which are grown in gel substrate in tissue culture. We also presented a particular experimental configuration and the optimal approach to analyze the images. This may serve as a basis to further works on live BSL in order to study root development.


Tissue culture Biospeckle Roots Image analysis Live imaging 



Biospeckle laser system


Generalized differences


Laser speckle contrast analysis


Laser speckle velocimetry


Particle image velocimetry



This study was by the Federal University of Lavras, FAPEMIG, CNPq DT, Capes and by the Scottish Executive Environment and Rural Affairs Department.


  1. Arizaga R, Trivi M, Rabal H (1999) Speckle time evolution characterization by the co-occurrence matrix analysis. Opt Laser Technol 31:163–169. doi: 10.1016/S0030-3992(99)00033-X CrossRefGoogle Scholar
  2. Arizaga R et al (2002) Display of the local activity using dynamical speckle patterns. Opt Eng 41:287–294. doi: 10.1117/1.1428739 CrossRefGoogle Scholar
  3. Bazylev N, Formin N, Hirano T, Lavinskaya E, Mizukaki T, Nakagawa A, Rubnikovich S, Takayama K (2003) Quasi-real time bio-tissues monitoring using dynamic laser speckle photography. J Flow Visual 6:371–380CrossRefGoogle Scholar
  4. Beemster TSG, Baskin TI (1998) Analysis of cell division and elongation underlying the developmental acceleration of root growth in Arabidopsis thaliana1. Plant Physiol 116:1515–1526. doi: 10.1104/pp.116.4.1515 PubMedCrossRefGoogle Scholar
  5. Bengough AG, Bransby MF, Hans J, McKenna SJ, Roberts TJ, Valentine TA (2006) Root responses to soil physical conditions; growth dynamics from field to cell. J Exp Bot 57:437–447. doi: 10.1093/jxb/erj003 PubMedCrossRefGoogle Scholar
  6. Braga RA, DalFabbro IM, Borem FM, Rabelo G, Arizaga R, Rabal HJ, Trivi M (2003) Assessment of seed viability by laser speckle techniques. Biosyst Eng 86(3):287–294. doi: 10.1016/j.biosystemseng.2003.08.005 CrossRefGoogle Scholar
  7. Braga RA, Rabelo GF, Granato LR, Santos EF, Machado JC, Arizaga R, Rabal HJ, Trivi M (2005) Detection of fungi in beans by the laser biospeckle technique. Biosyst Eng 91:465–469. doi: 10.1016/j.biosystemseng.2005.05.006 CrossRefGoogle Scholar
  8. Braga RA, Horgan GW, Enes AM, Miron D, Rabelo GF, Barreto JB (2007) Biological feature isolation by wavelets in biospeckle laser images. Comp Electr Agric 58:123–132. doi: 10.1016/j.compag.2007.03.009 CrossRefGoogle Scholar
  9. Briers JD (1975) Wavelength dependence of intensity fluctuations in laser speckle patterns form biological specimens. Opt Commun 13:324–326. doi: 10.1016/0030-4018(75)90111-X CrossRefGoogle Scholar
  10. Briers JD, Webster S (1996) Laser speckle contrast analyis (LASCA): a non scanning full field technique for monitoring capillary blood flow. J Biomed Opt 1:174–179. doi: 10.1117/12.231359 CrossRefGoogle Scholar
  11. Carvalho PHA, Barreto JB, Braga RA, Rabelo GF (2009) Motility parameters assessment of bovine frezen semen by biospeckle laser (BSL) system. Biosyst Eng 102:31–35. doi: 10.1007/978-3-540-77578-2 CrossRefGoogle Scholar
  12. Dumais J, Kwiatkowska D (2001) Analysis of surface growth in shoot apices. Plant J 31:229–241. doi: 10.1046/j.1365-313X.2001.01350.x CrossRefGoogle Scholar
  13. Dupuy L, Mackenzie J, Rudge T, Haseloff J (2008) A system for modelling cell-cell interactions during plant morphogenesis. Ann Bot (Lond) 101:1255–1265. doi: 10.1093/aob/mcm235 CrossRefGoogle Scholar
  14. Formin NA (1998) Speckle photography for fluid mechanics measurements. Springer, Berlin, p 244Google Scholar
  15. Fujii H, Asakura T (1985) Blood flow observed by time-varing laser speckle. Opt Lett 10(3):104–106. doi: 10.1364/OL.10.000104 CrossRefPubMedGoogle Scholar
  16. Fujii H, Nohira K, Yamamoto Y, Ikawa H, Ohura T (1987) Evaluation of blood flow by laser speckle image sensing Part 1. Appl Opt 25:5321–5325CrossRefGoogle Scholar
  17. Haseloff J (2003) Old botanical techniques for new microscopes. BioTech 34:1174–1182Google Scholar
  18. Heisler MG, Ohno C, Das P, Sieber P, Reddy GV, Long JA, Meyerowitz EM (2005) Patterns of auxin transport and gene expression during primordium development revealed by live imaging of the arabidopsis inflorescence meristem. Curr Biol 15:1899–1911. doi: 10.1016/j.cub.2005.09.052 PubMedCrossRefGoogle Scholar
  19. Kurup S, Runions J, Köhler U, Laplaze L, Hodge S, Haseloff J (2005) Marking cell lineages in living tissues. Plant J 42:444–453. doi: 10.1111/j.1365-313X.2005.02386.x PubMedCrossRefGoogle Scholar
  20. Marcon M, Braga RA (2008) In: Rabal HJ, Braga RA (eds) Dynamic laser speckle and applications. Taylor & Francis, Boca Raton, p 304Google Scholar
  21. Moreno N, Bougourd S, Haseloff J, Feijo JA (2006) Imaging plant cells. In: Pawley JB (ed) Handbook of confocal microscopy. Springer ScienceGoogle Scholar
  22. Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15:473–497CrossRefGoogle Scholar
  23. Pajuelo M, Baldwin G, Rabal H, Cap N, Arizaga R, Trivi M (2003) Bio-speckle assessment of bruising in fruits. Opt Lasers Eng 40:13–24. doi: 10.1016/S0143-8166(02)00063-5 CrossRefGoogle Scholar
  24. Passoni I, Dai Pra A, Rabal H, Trivi M, Arizaga R (2005) Dynamic speckle processing using wavelets based entropy. Opt Comm 246:219–228. doi: 10.1016/j.optcom.2004.10.054 CrossRefGoogle Scholar
  25. Pickering CJD, Halliwell NA (1984) Laser speckle photography and particle image velocimetry: photographic film noise. Appl Opt 23:2961–2969PubMedCrossRefGoogle Scholar
  26. Pomarico JA, DiRocco HO, Alvarez L, Lanusse C, Mottier L, Saumell C, Arizaga R, Rabal H, Trivi M (2004) Speckle interferometry applied to phamacodynamic studies: evaluation of parasite motility. Eur Biophys J 33:694–699. doi: 10.1007/s00249-004-0413-4 PubMedCrossRefGoogle Scholar
  27. Rabal HJ, Braga RA (2008) Dynamic laser speckle and applications, 1st edn. Taylor & Francis/CRC, Boca Raton, p 304Google Scholar
  28. Rabelo GF, Braga RA Jr, Fabbro IMD, Arizaga R, Rabal HJ, Trivi MR (2005) Laser speckle techniques applied to study quality of fruits. Rev Bras Eng Agric Amb 9:570–575Google Scholar
  29. Rajan V, Varghese B, van Leeuwen TG, Steenbergen W (2006) Speckles in laser doppler perfusion imaging. Opt Lett 31(4):468–470. doi: 10.1364/OL.31.000468 PubMedCrossRefGoogle Scholar
  30. Reinhardt D, Pesce E-R, Stieger P, Mandel T, Baltensperger K, Bennett M, Traas J, Friml J, Kuhlemeier C (2004) Regulation of phyllotaxis by polar auxin transport. Nature 426:255–260. doi: 10.1038/nature02081 CrossRefGoogle Scholar
  31. Sendra GH, Arizaga R, Rabal HJ, Trivi M (2005) Decomposition of biospeckle images in temporary spectral bands. Opt Lett 30(13):1641–1643. doi: 10.1364/OL.30.001641 PubMedCrossRefGoogle Scholar
  32. Serov A, Lasser T (2005) High-speed laser doppler perfusion imaging using an integrating CMOS image sensor. Opt Exp 13(17):6416–6428. doi: 10.1364/OPEX.13.006416 CrossRefGoogle Scholar
  33. Sharpe J, Ahlgren U, Perry P, Hill B, Ross A, Hecksher-Sørensen J, Baldock R, Davidson D (2002) Optical projection tomography as a tool for 3D microscopy and gene expression studies. Science 5567:541–545. doi: 10.1126/science.1068206 CrossRefGoogle Scholar
  34. Truernit E, Bauby H, Dubreucq B, Grandjean O, Runions J, Barthélémy J, Palauquia J-C (2008) High-resolution whole-mount imaging of three-dimensional tissue organization and gene expression enables the study of phloem development and structure in Arabidopsis. On line publication doi: 10.1105/tpc.107.056069
  35. Wardell K, Jakobsson A, Nilsson GE (1993) Laser doppler perfusion imaging by dynamic light scattering. IEEE Trans Biomed Eng 40(4):309–316. doi: 10.1109/10.222322 PubMedCrossRefGoogle Scholar
  36. Xu Z, Joenathan C, Khorana BM (1995) Temporal and spatial properties of the time-varying speckles of botanical specimens. Opt Eng 34:1487–1502. doi: 10.1117/12.199878 CrossRefGoogle Scholar
  37. Zhao Y, Wang J, Wu X, Williams FW, Schmidt RJ (1997) Point-wise and whole-field laser speckle intensity fluctuation measurements applied to botanical specimens. Opt Lasers Eng 28:443–456. doi: 10.1016/S0143-8166(97)00056-0 CrossRefGoogle Scholar

Copyright information

© European Biophysical Societies' Association 2009

Authors and Affiliations

  • Roberto A. Braga
    • 1
  • L. Dupuy
    • 2
  • M. Pasqual
    • 1
  • R. R. Cardoso
    • 1
  1. 1.Universidade Federal de LavrasLavrasBrazil
  2. 2.Scottish Crop Research InstituteDundeeScotland, UK

Personalised recommendations