OSA's Digital Library

Virtual Journal for Biomedical Optics

Virtual Journal for Biomedical Optics


  • Editors: Andrew Dunn and Anthony Durkin
  • Vol. 7, Iss. 10 — Oct. 5, 2012

Dynamic three-dimensional tracking of single fluorescent nanoparticles deep inside living tissue

Jan-Hendrik Spille, Tim Kaminski, Heinz-Peter Königshoven, and Ulrich Kubitscheck  »View Author Affiliations

Optics Express, Vol. 20, Issue 18, pp. 19697-19707 (2012)

View Full Text Article

Enhanced HTML    Acrobat PDF (1928 KB) Open Access

Browse Journals / Lookup Meetings

Browse by Journal and Year


Lookup Conference Papers

Close Browse Journals / Lookup Meetings

Article Tools



Three-dimensional (3D) spatial information can be encoded in two-dimensional images of fluorescent nanoparticles by astigmatic imaging. We combined this method with light sheet microscopy for high contrast single particle imaging up to 200 µm deep within living tissue and real-time image analysis to determine 3D particle localizations with nanometer precision and millisecond temporal resolution. Axial information was instantly directed to the sample stage to keep a moving particle within the focal plane in an active feedback loop. We demonstrated 3D tracking of nanoparticles at an unprecedented depth throughout large cell nuclei over several thousand frames and a range of more than 10 µm in each spatial dimension, while simultaneously acquiring optically sectioned wide field images. We conclude that this 3D particle tracking technique employing light sheet microscopy presents a valuable extension to the nanoscopy toolbox.

© 2012 OSA

OCIS Codes
(110.0110) Imaging systems : Imaging systems
(110.6880) Imaging systems : Three-dimensional image acquisition
(180.2520) Microscopy : Fluorescence microscopy
(180.6900) Microscopy : Three-dimensional microscopy

ToC Category:
Imaging Systems

Original Manuscript: June 28, 2012
Revised Manuscript: August 3, 2012
Manuscript Accepted: August 4, 2012
Published: August 13, 2012

Virtual Issues
Vol. 7, Iss. 10 Virtual Journal for Biomedical Optics

Jan-Hendrik Spille, Tim Kaminski, Heinz-Peter Königshoven, and Ulrich Kubitscheck, "Dynamic three-dimensional tracking of single fluorescent nanoparticles deep inside living tissue," Opt. Express 20, 19697-19707 (2012)

Sort:  Author  |  Year  |  Journal  |  Reset  


  1. M. A. Thompson, J. M. Casolari, M. Badieirostami, P. O. Brown, and W. E. Moerner, “Three-dimensional tracking of single mRNA particles in Saccharomyces cerevisiae using a double-helix point spread function,” Proc. Natl. Acad. Sci. U.S.A.107(42), 17864–17871 (2010). [CrossRef] [PubMed]
  2. J. P. Siebrasse, R. Veith, A. Dobay, H. Leonhardt, B. Daneholt, and U. Kubitscheck, “Discontinuous movement of mRNP particles in nucleoplasmic regions devoid of chromatin,” Proc. Natl. Acad. Sci. U.S.A.105(51), 20291–20296 (2008). [CrossRef] [PubMed]
  3. N. Arhel, A. Genovesio, K.-A. Kim, S. Miko, E. Perret, J.-C. Olivo-Marin, S. Shorte, and P. Charneau, “Quantitative four-dimensional tracking of cytoplasmic and nuclear HIV-1 complexes,” Nat. Methods3(10), 817–824 (2006). [CrossRef] [PubMed]
  4. M. J. Saxton and K. Jacobson, “Single-particle tracking: applications to membrane dynamics,” Annu. Rev. Biophys. Biomol. Struct.26(1), 373–399 (1997). [CrossRef] [PubMed]
  5. M. A. Thompson, M. D. Lew, M. Badieirostami, and W. E. Moerner, “Localizing and tracking single nanoscale emitters in three dimensions with high spatiotemporal resolution using a double-helix point spread function,” Nano Lett.10(1), 211–218 (2010). [CrossRef] [PubMed]
  6. H. P. Kao and A. S. Verkman, “Tracking of single fluorescent particles in three dimensions: use of cylindrical optics to encode particle position,” Biophys. J.67(3), 1291–1300 (1994). [CrossRef] [PubMed]
  7. Y. Katayama, O. Burkacky, M. Meyer, C. Bräuchle, E. Gratton, and D. C. Lamb, “Real-time nanomicroscopy via three-dimensional single-particle tracking,” ChemPhysChem10(14), 2458–2464 (2009). [CrossRef] [PubMed]
  8. G. A. Lessard, P. M. Goodwin, and J. H. Werner, “Three-dimensional tracking of individual quantum dots,ˮ,” Appl. Phys. Lett.91(22), 224106 (2007). [CrossRef]
  9. K. McHale, A. J. Berglund, and H. Mabuchi, “Quantum dot photon statistics measured by three-dimensional particle tracking,” Nano Lett.7(11), 3535–3539 (2007). [CrossRef] [PubMed]
  10. N. P. Wells, G. A. Lessard, P. M. Goodwin, M. E. Phipps, P. J. Cutler, D. S. Lidke, B. S. Wilson, and J. H. Werner, “Time-resolved three-dimensional molecular tracking in live cells,” Nano Lett.10(11), 4732–4737 (2010). [CrossRef] [PubMed]
  11. M. D. McMahon, A. J. Berglund, P. Carmichael, J. J. McClelland, and J. A. Liddle, “3D particle trajectories observed by orthogonal tracking microscopy,” ACS Nano3(3), 609–614 (2009). [CrossRef] [PubMed]
  12. E. Toprak, H. Balci, B. H. Blehm, and P. R. Selvin, “Three-dimensional particle tracking via bifocal imaging,” Nano Lett.7(7), 2043–2045 (2007). [CrossRef] [PubMed]
  13. S. Ram, P. Prabhat, J. Chao, E. S. Ward, and R. J. Ober, “High accuracy 3D quantum dot tracking with multifocal plane microscopy for the study of fast intracellular dynamics in live cells,” Biophys. J.95(12), 6025–6043 (2008). [CrossRef] [PubMed]
  14. Y. Sun, J. D. McKenna, J. M. Murray, E. M. Ostap, and Y. E. Goldman, “Parallax: high accuracy three-dimensional single molecule tracking using split images,” Nano Lett.9(7), 2676–2682 (2009). [CrossRef] [PubMed]
  15. G. Bolognesi, S. Bianchi, and R. Di Leonardo, “Digital holographic tracking of microprobes for multipoint viscosity measurements,” Opt. Express19(20), 19245–19254 (2011). [CrossRef] [PubMed]
  16. L. Holtzer, T. Meckel, and T. Schmidt, “Nanometric three-dimensional tracking of individual quantum dots in cells,ˮ,” Appl. Phys. Lett.90(5), 053902 (2007). [CrossRef]
  17. D. Ernst, S. Hain, and J. Köhler, “Setup for single-particle orbit tracking: artifacts and corrections,” J. Opt. Soc. Am. A29(7), 1277–1287 (2012). [CrossRef] [PubMed]
  18. M. F. Juette and J. Bewersdorf, “Three-dimensional tracking of single fluorescent particles with submillisecond temporal resolution,” Nano Lett.10(11), 4657–4663 (2010). [CrossRef] [PubMed]
  19. J. G. Ritter, R. Veith, A. Veenendaal, J. P. Siebrasse, and U. Kubitscheck, “Light sheet microscopy for single molecule tracking in living tissue,” PLoS ONE5(7), e11639 (2010). [CrossRef] [PubMed]
  20. J. P. Siebrasse, T. Kaminski, and U. Kubitscheck, “Nuclear export of single native mRNA molecules observed by light sheet fluorescence microscopy,” Proc. Natl. Acad. Sci. U.S.A.109(24), 9426–9431 (2012). [CrossRef] [PubMed]
  21. J. G. Ritter, R. Veith, J.-P. Siebrasse, and U. Kubitscheck, “High-contrast single-particle tracking by selective focal plane illumination microscopy,” Opt. Express16(10), 7142–7152 (2008). [CrossRef] [PubMed]
  22. Y. Deng and J. W. Shaevitz, “Effect of aberration on height calibration in three-dimensional localization-based microscopy and particle tracking,” Appl. Opt.48(10), 1886–1890 (2009). [CrossRef] [PubMed]
  23. J. G. Ritter, J.-H. Spille, T. Kaminski, and U. Kubitscheck, “A cylindrical zoom lens unit for adjustable optical sectioning in light sheet microscopy,” Biomed. Opt. Express2(1), 185–193 (2011). [CrossRef] [PubMed]
  24. J. Huisken and D. Y. Stainier, “Even fluorescence excitation by multidirectional selective plane illumination microscopy (mSPIM),” Opt. Lett.32(17), 2608–2610 (2007). [CrossRef] [PubMed]
  25. M. K. Cheezum, W. F. Walker, and W. H. Guilford, “Quantitative comparison of algorithms for tracking single fluorescent particles,” Biophys. J.81(4), 2378–2388 (2001). [CrossRef] [PubMed]
  26. I. Izeddin, M. El Beheiry, J. Andilla, D. Ciepielewski, X. Darzacq, and M. Dahan, “PSF shaping using adaptive optics for three-dimensional single-molecule super-resolution imaging and tracking,” Opt. Express20(5), 4957–4967 (2012). [CrossRef] [PubMed]
  27. H. Deschout, K. Neyts, and K. Braeckmans, “The influence of movement on the localization precision of sub-resolution particles in fluorescence microscopy,” J Biophotonics5(1), 97–109 (2012). [CrossRef] [PubMed]
  28. M. J. Mlodzianoski, M. F. Juette, G. L. Beane, and J. Bewersdorf, “Experimental characterization of 3D localization techniques for particle-tracking and super-resolution microscopy,” Opt. Express17(10), 8264–8277 (2009). [CrossRef] [PubMed]
  29. C. von Middendorff, A. Egner, C. Geisler, S. W. Hell, and A. Schönle, “Isotropic 3D nanoscopy based on single emitter switching,” Opt. Express16(25), 20774–20788 (2008). [CrossRef] [PubMed]
  30. R. Veith, T. Sorkalla, E. Baumgart, J. Anzt, H. Häberlein, S. Tyagi, J. P. Siebrasse, and U. Kubitscheck, “Balbiani ring mRNPs diffuse through and bind to clusters of large intranuclear molecular structures,” Biophys. J.99(8), 2676–2685 (2010). [CrossRef] [PubMed]
  31. D. Ernst, M. Hellmann, J. Kohler, and M. Weiss, “Fractional Brownian motion in crowded fluids,ˮ,” Soft Matter8(18), 4886–4889 (2012). [CrossRef]
  32. F. Cella Zanacchi, Z. Lavagnino, M. Perrone Donnorso, A. Del Bue, L. Furia, M. Faretta, and A. Diaspro, “Live-cell 3D super-resolution imaging in thick biological samples,” Nat. Methods8(12), 1047–1049 (2011). [CrossRef] [PubMed]
  33. A. P. Fields and A. E. Cohen, “Electrokinetic trapping at the one nanometer limit,” Proc. Natl. Acad. Sci. U.S.A.108(22), 8937–8942 (2011). [CrossRef] [PubMed]

Cited By

Alert me when this paper is cited

OSA is able to provide readers links to articles that cite this paper by participating in CrossRef's Cited-By Linking service. CrossRef includes content from more than 3000 publishers and societies. In addition to listing OSA journal articles that cite this paper, citing articles from other participating publishers will also be listed.


Fig. 1 Fig. 2 Fig. 3
Fig. 4

Supplementary Material

» Media 1: MPG (3538 KB)     
» Media 2: MPG (5842 KB)     
» Media 3: MPG (4618 KB)     

« Previous Article  |  Next Article »

OSA is a member of CrossRef.

CrossCheck Deposited