OSA's Digital Library

Optics Express

Optics Express

  • Editor: Andrew M. Weiner
  • Vol. 22, Iss. 3 — Feb. 10, 2014
  • pp: 2358–2375

Dual-Color Fluorescence Cross-Correlation Spectroscopy on a Single Plane Illumination Microscope (SPIM-FCCS)

Jan Wolfgang Krieger, Anand Pratap Singh, Christoph S. Garbe, Thorsten Wohland, and Jörg Langowski  »View Author Affiliations

Optics Express, Vol. 22, Issue 3, pp. 2358-2375 (2014)

View Full Text Article

Enhanced HTML    Acrobat PDF (2786 KB)

Browse Journals / Lookup Meetings

Browse by Journal and Year


Lookup Conference Papers

Close Browse Journals / Lookup Meetings

Article Tools



Single plane illumination microscopy based fluorescence correlation spectroscopy (SPIM-FCS) is a new method for imaging FCS in 3D samples, providing diffusion coefficients, flow velocities and concentrations in an imaging mode. Here we extend this technique to two-color fluorescence cross-correlation spectroscopy (SPIM-FCCS), which allows to measure molecular interactions in an imaging mode. We present a theoretical framework for SPIM-FCCS fitting models, which is subsequently used to evaluate several test measurements of in-vitro (labeled microspheres, several DNAs and small unilamellar vesicles) and in-vivo samples (dimeric and monomeric dual-color fluorescent proteins, as well as membrane bound proteins). Our method yields the same quantitative results as the well-established confocal FCCS, but in addition provides unmatched statistics and true imaging capabilities.

© 2014 Optical Society of America

OCIS Codes
(040.1490) Detectors : Cameras
(180.2520) Microscopy : Fluorescence microscopy
(180.6900) Microscopy : Three-dimensional microscopy
(300.6280) Spectroscopy : Spectroscopy, fluorescence and luminescence

ToC Category:

Original Manuscript: October 29, 2013
Revised Manuscript: December 8, 2013
Manuscript Accepted: December 8, 2013
Published: January 28, 2014

Virtual Issues
Vol. 9, Iss. 4 Virtual Journal for Biomedical Optics

Jan Wolfgang Krieger, Anand Pratap Singh, Christoph S. Garbe, Thorsten Wohland, and Jörg Langowski, "Dual-Color Fluorescence Cross-Correlation Spectroscopy on a Single Plane Illumination Microscope (SPIM-FCCS)," Opt. Express 22, 2358-2375 (2014)

Sort:  Author  |  Year  |  Journal  |  Reset  


  1. D. Magde, E. L. Elson, W. W. Webb, “Fluorescence correlation spectroscopy I: Conceptual basis and theory,” Biopolymers 13, 1–27 (1974). [CrossRef]
  2. D. Magde, E. L. Elson, W. W. Webb, “Fluorescence correlation spectroscopy. II. an experimental realization.” Biopolymers 13, 29–61 (1974). [CrossRef] [PubMed]
  3. K. M. Berland, P. T. So, E. Gratton, “Two-photon fluorescence correlation spectroscopy: method and application to the intracellular environment,” Biophys. J. 68, 694–701 (1995). [CrossRef] [PubMed]
  4. P. Schwille, F. Meyer-Almes, R. Rigler, “Dual-color fluorescence cross-correlation spectroscopy for multi-component diffusional analysis in solution,” Biophys. J. 72, 1878–1886 (1997). [CrossRef] [PubMed]
  5. U. Kettling, A. Koltermann, P. Schwille, M. Eigen, “Real-time enzyme kinetics monitored by dual-color fluorescence cross-correlation spectroscopy,” Proc. Natl. Acad. Sci. 95, 1416–1420 (1998). [CrossRef] [PubMed]
  6. K. G. Heinze, M. Jahnz, P. Schwille, “Triple-color coincidence analysis: One step further in following higher order molecular complex formation,” Biophys. J. 86, 506–516 (2004). [CrossRef]
  7. L. C. Hwang, M. Gösch, T. Lasser, T. Wohland, “Simultaneous multicolor fluorescence cross-correlation spectroscopy to detect higher order molecular interactions using single wavelength laser excitation,” Biophys. J. 91, 715–727 (2006). [CrossRef] [PubMed]
  8. F. Bestvater, Z. Seghiri, M. S. Kang, N. Gröner, J. Y. Lee, I. Kang-Bin, M. Wachsmuth, “EMCCD-based spectrally resolved fluorescence correlation spectroscopy,” Opt. Express 18, 23818–23828 (2010). [CrossRef]
  9. D. M. Shcherbakova, M. A. Hink, L. Joosen, T. W. J. Gadella, V. V. Verkhusha, “An orange fluorescent protein with a large stokes shift for single-excitation multicolor FCCS and FRET imaging,” J. Am. Chem. Soc. 134, 7913–7923 (2012). [CrossRef]
  10. O. Krichevsky, G. Bonnet, “Fluorescence correlation spectroscopy: the technique and its applications,” Rep. Prog. Phys. 65, 251–297 (2002). [CrossRef]
  11. K. Bacia, S. A. Kim, P. Schwille, “Fluorescence cross-correlation spectroscopy in living cells,” Nat. Methods 3, 83–89 (2006). [CrossRef]
  12. E. Haustein, P. Schwille, “Fluorescence correlation spectroscopy: novel variations of an established technique,” Annu. Rev. Biophys. Biomol. Struct. 36, 151–169 (2007). [CrossRef] [PubMed]
  13. N. Dross, C. Spriet, M. Zwerger, G. Müller, W. Waldeck, J. Langowski, “Mapping eGFP oligomer mobility in living cell nuclei,” PLoS ONE 4, e5041 (2009). [CrossRef]
  14. J. Ries, S. Chiantia, P. Schwille, “Accurate determination of membrane dynamics with line-scan FCS,” Biophys. J. 96, 1999–2008 (2009). [CrossRef] [PubMed]
  15. Q. Ruan, M. A. Cheng, M. Levi, E. Gratton, W. W. Mantulin, “Spatial-temporal studies of membrane dynamics: Scanning fluorescence correlation spectroscopy (SFCS),” Biophys. J. 87, 1260–1267 (2004). [CrossRef] [PubMed]
  16. G. Heuvelman, F. Erdel, M. Wachsmuth, K. Rippe, “Analysis of protein mobilities and interactions in living cells by multifocal fluorescence fluctuation microscopy,” Eur. Biophys. J. 38, 813–828 (2009). [CrossRef] [PubMed]
  17. D. J. Needleman, Y. Xu, T. J. Mitchison, “Pin-hole array correlation imaging: Highly parallel fluorescence correlation spectroscopy,” Biophys. J. 96, 5050–5059 (2009). [CrossRef] [PubMed]
  18. R. A. Colyer, G. Scalia, I. Rech, A. Gulinatti, M. Ghioni, S. Cova, S. Weiss, X. Michalet, “High-throughput FCS using an LCOS spatial light modulator and an 8×1 spad array,” Biomed. Opt. Express 1, 1408–1431 (2010). [CrossRef]
  19. M. Kloster-Landsberg, D. Tyndall, I. Wang, R. Walker, J. Richardson, R. Henderson, A. Delon, “Note: Multi-confocal fluorescence correlation spectroscopy in living cells using a complementary metal oxide semiconductor-single photon avalanche diode array,” Rev. Sci. Instrum. 84, 076105 (2013). [CrossRef] [PubMed]
  20. B. Kannan, L. Guo, T. Sudhaharan, S. Ahmed, I. Maruyama, T. Wohland, “Spatially resolved total internal reflection fluorescence correlation microscopy using an electron multiplying charge-coupled device camera,” Anal. Chem. 79, 4463–4470 (2007). [CrossRef] [PubMed]
  21. T. Wohland, X. Shi, J. Sankaran, E. H. K. Stelzer, “Single plane illumination fluorescence correlation spectroscopy (SPIM-FCS) probes inhomogeneous three-dimensional environments,” Opt. Express 10, 10627–10641 (2010). [CrossRef]
  22. J. Capoulade, M. Wachsmuth, L. Hufnagel, M. Knop, “Quantitative fluorescence imaging of protein diffusion and interaction in living cells,” Nat. Biotechnol. 29, 835—839 (2011). [CrossRef] [PubMed]
  23. SI: The supplementary notes are available at http://www.dkfz.de/Macromol/publications/files/spimfccs2013_supplement.pdf .
  24. M. Gösch, A. Magnusson, S. Hård, H. Blom, S. Anderegg, K. Korn, P. Thyberg, M. Wells, T. Lasser, R. Rigler, “Parallel dual-color fluorescence cross-correlation spectroscopy using diffractive optical elements,” J. Biomed. Opt. 10, 054008 (2005). [CrossRef] [PubMed]
  25. J. Ries, Z. Petrášek, A. J. García-Sáez, P. Schwille, “A comprehensive framework for fluorescence cross-correlation spectroscopy,” New J. Phys. 12, 113009 (2010). [CrossRef]
  26. V. Betaneli, E. P. Petrov, P. Schwille, “The role of lipids in VDAC oligomerization,” Biophys. J. 102, 523–531 (2012). [CrossRef] [PubMed]
  27. P. W. Wiseman, J. A. Squier, K. R. Wilson, “Dynamic image correlation spectroscopy (ICS) and two-color image cross-correlation spectroscopy (ICCS): concepts and application,” in “BiOS 2000 Int. Symp. Biomed. Opt.”, (Int. Soc. Opt. Photon., 2000), pp. 14–20.
  28. T. Toplak, E. Pandzic, L. Chen, M. Vicente-Manzanares, A. R. Horwitz, P. W. Wiseman, “STICCS reveals matrix-dependent adhesion slipping and gripping in migrating cells,” Biophys. J. 103, 1672–1682 (2012). [CrossRef] [PubMed]
  29. M. A. Digman, P. W. Wiseman, A. R. Horwitz, E. Gratton, “Detecting protein complexes in living cells from laser scanning confocal image sequences by the cross correlation raster image spectroscopy method,” Biophys. J. 96, 707–716 (2009). [CrossRef] [PubMed]
  30. A. P. Singh, J. W. Krieger, J. Buchholz, E. Charbon, J. Langowski, T. Wohland, “The performance of 2D array detectors for light sheet based fluorescence correlation spectroscopy,” Opt. Express 21, 8652–8668 (2013). [CrossRef] [PubMed]
  31. J. Sankaran, X. Shi, L. Ho, E. Stelzer, T. Wohland, “ImFCS: A software for imaging FCS data analysis and visualization,” Opt. Express 18, 25468–25481 (2010). Available at http://staff.science.nus.edu.sg/~chmwt/resources/imfcs_software.html . [CrossRef]
  32. L. C. Hwang, T. Wohland, “Single wavelength excitation fluorescence cross-correlation spectroscopy with spectrally similar fluorophores: Resolution for binding studies,” J. Chem. Phys. 122, 114708 (2005). [CrossRef] [PubMed]
  33. P. Liu, T. Sudhaharan, R. M. Koh, L. C. Hwang, S. Ahmed, I. N. Maruyama, T. Wohland, “Investigation of the dimerization of proteins from the epidermal growth factor receptor family by single wavelength fluorescence cross-correlation spectroscopy,” Biophys. J. 93, 684–698 (2007). [CrossRef] [PubMed]
  34. J. Buchholz, J. W. Krieger, G. Mocsár, B. Kreith, E. Charbon, G. Vámosi, U. Kebschull, J. Langowski, “Fpga implementation of a 32×32 autocorrelator array for analysis of fast image series,” Opt. Express 20, 17767–17782 (2012). [CrossRef] [PubMed]
  35. D. W. Marquardt, “An algorithm for least-squares estimation of nonlinear parameters,” J. Soc. Ind. Appl. Math. 11, 431–441 (1963). [CrossRef]
  36. K. Levenberg, “A method for the solution of certain nonlinear problems in least squares,” Quart. Appl. Math. 2, 164–168 (1944).
  37. J. Wuttke, “lmfit 3.2 – a c/c++ routine for levenberg-marquardt minimization with wrapper for least-squares curve fitting, based on work by B.S. Garbow, K.E. Hillstrom, J.J. Moré, and S. Moshier, available at http://apps.jcns.fz-juelich.de/doku/sc/lmfit ,” (2010).
  38. A. Corana, M. Marchesi, C. Martini, S. Ridella, “Minimizing multimodal functions of continuous variables with the ’simulated annealing’ algorithm corrigenda for this article is available here,” ACM T. Math. Software 13, 262–280 (1987). [CrossRef]
  39. Y. H. Foo, N. Naredi-Rainer, D. C. Lamb, S. Ahmed, T. Wohland, “Factors affecting the quantification of biomolecular interactions by fluorescence cross-correlation spectroscopy,” Biophys. J. 102, 1174–1183 (2012). [CrossRef] [PubMed]
  40. QuickFit 3.0 can be downloaded free of charge from http://www.dkfz.de/Macromol/quickfit/ .
  41. K. Greger, J. Swoger, E. H. K. Stelzer, “Basic building units and properties of a fluorescence single plane illumination microscope,” Rev. Sci. Instrum. 78, 023705 (2007). [CrossRef] [PubMed]
  42. A. Edelstein, N. Amodaj, K. Hoover, R. Vale, N. Stuurman, “Computer control of microscopes using μManager,” Curr. Protoc. Mol. Biol. 14, 1–14 (2010).
  43. E. M. M. Manders, F. J. Verbeek, J. A. Aten, “Measurement of co-localization of objects in dual-colour confocal images,” J. Microsc. 169, 375–382 (1993). [CrossRef]
  44. The matlab scripts for the bead scan evaluation is freely available at: http://www.dkfz.de/Macromol/quickfit/beadscan.html .
  45. N. Bag, J. Sankaran, A. Paul, R. S. Kraut, T. Wohland, “Calibration and limits of camera-based fluorescence correlation spectroscopy: A supported lipid bilayer study,” ChemPhysChem 13, 2784–2794 (2012). [CrossRef] [PubMed]
  46. T. Wocjan, J. Krieger, O. Krichevsky, J. Langowski, “Dynamics of a fluorophore attached to superhelical DNA: Fcs experiments simulated by brownian dynamics,” Phys. Chem. Chem. Phys. 11, 10671–10681 (2009). [CrossRef]
  47. C.-H. Huang, “Phosphatidylcholine vesicles. formation and physical characteristics,” Biochemistry 8, 344–352 (1969). PMID: . [CrossRef] [PubMed]
  48. L. A. Maguire, H. Zhang, P. A. Shamlou, “Preparation of small unilamellar vesicles (SUV) and biophysical characterization of their complexes with poly-l-lysine-condensed plasmid DNA,” Biotechnol Appl. Biochem. 37, 73–81 (2003). [CrossRef] [PubMed]
  49. J. Sankaran, N. Bag, R. S. Kraut, T. Wohland, “Accuracy and precision in camera-based fluorescence correlation spectroscopy measurements,” Anal. Chem. 85, 3948–3954 (2013). [CrossRef] [PubMed]
  50. M. Wachsmuth, “Fluoreszenzfluktuationsmikroskopie: Entwicklung eines prototyps, theorie und messung der beweglichkeit von biomolekülen im zellkern,” Ph.D. thesis, Ruprecht-Karls-Universität, Heidelberg. (2001).
  51. J. R. Unruh, E. Gratton, “Analysis of molecular concentration and brightness from fluorescence fluctuation data with an electron multiplied ccd camera,” Biophys. J. 95, 5385–5398 (2008). [CrossRef] [PubMed]
  52. N. Baudendistel, G. Müller, W. Waldeck, P. Angel, J. Langowski, “Two-hybrid fluorescence cross-correlation spectroscopy detects protein–protein interactions in vivo,” ChemPhysChem 6, 984–990 (2005). [CrossRef] [PubMed]
  53. G. Vámosi, N. Baudendistel, C.-W. von der Lieth, N. Szalóki, G. Mocsár, G. Müller, P. Brázda, W. Waldeck, S. Damjanovich, J. Langowski, K. Tóth, “Conformation of the c-Fos/c-Jun complex in vivo: A combined FRET, FCCS, and MD-modeling study,” Biophys. J. 94, 2859–2868 (2008). [CrossRef]
  54. B. K. Müller, E. Zaychikov, C. Bräuchle, D. C. Lamb, “Pulsed interleaved excitation,” Biophys. J. 89, 3508–3522 (2005). [CrossRef] [PubMed]
  55. A. N. Kapanidis, T. A. Laurence, N. K. Lee, E. Margeat, X. Kong, S. Weiss, “Alternating-laser excitation of single molecules,” Acc. Chem. Res. 38, 523–533 (2005). [CrossRef] [PubMed]
  56. J. Roszik, D. Lisboa, J. Szöllősi, G. Vereb, “Evaluation of intensity-based ratiometric FRET in image cytometry—approaches and a software solution,” Cytometry A 75A, 761–767 (2009). [CrossRef]
  57. S. Talwar, A. Kumar, M. Rao, G. I. Menon, G. V. Shivashankar, “Correlated spatio-temporal fluctuations in chromatin compaction states characterize stem cells.” Biophys. J. 104, 553–564 (2013). [CrossRef] [PubMed]
  58. S. Burri, D. Stucki, Y. Maruyama, C. Bruschini, E. Charbon, F. Regazzoni, “Jailbreak imagers: Transforming a single-photon image sensor into a true random number generator,” in “International Image Sensor Workshop,” (2013). Snowbird, June 2013, available online http://www.imagesensors.org/PastWorkshops/2013Workshop/2013Papers/07-20_099_regazzoni_paper_revised.pdf .

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.

« Previous Article  |  Next Article »

OSA is a member of CrossRef.

CrossCheck Deposited