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Quantitative fluorescence imaging of protein diffusion and interaction in living cells


Diffusion processes and local dynamic equilibria inside cells lead to nonuniform spatial distributions of molecules, which are essential for processes such as nuclear organization and signaling in cell division, differentiation and migration1. To understand these mechanisms, spatially resolved quantitative measurements of protein abundance, mobilities and interactions are needed, but current methods have limited capabilities to study dynamic parameters. Here we describe a microscope based on light-sheet illumination2 that allows massively parallel fluorescence correlation spectroscopy (FCS)3 measurements and use it to visualize the diffusion and interactions of proteins in mammalian cells and in isolated fly tissue. Imaging the mobility of heterochromatin protein HP1α (ref. 4) in cell nuclei we could provide high-resolution diffusion maps that reveal euchromatin areas with heterochromatin-like HP1α-chromatin interactions. We expect that FCS imaging will become a useful method for the precise characterization of cellular reaction-diffusion processes.

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Figure 1: FCS imaging using the diffraction-limited light-pad.
Figure 2: 1D- and 2D-FCS imaging of protein diffusion in Madin-Darby canine kidney (MDCK) cells.
Figure 3: 2D-FCS imaging of protein diffusion in Drosophila wing imaginal discs.
Figure 4: Spatially resolved HP1α mobility in 3T3 cells investigated by 2D-FCS imaging.


  1. Kinkhabwala, A. & Bastiaens, P.L.H. Spatial aspects of intracellular information processing. Curr. Opin. Genet. Dev. 20, 31–40 (2010).

    Article  CAS  Google Scholar 

  2. Huisken, J. & Stainier, D.Y.R. Selective plane illumination microscopy techniques in developmental biology. Development 136, 1963–1975 (2009).

    Article  CAS  Google Scholar 

  3. Elson, E.L. & Magde, D. Fluorescence correlation spectroscopy. I Conceptual basis and theory. Biopolymers 13, 1–27 (1974).

    Article  CAS  Google Scholar 

  4. Cheutin, T. et al. Maintenance of stable heterochromatin domains by dynamic HP1 binding. Science 299, 721–725 (2003).

    Article  CAS  Google Scholar 

  5. Bacia, K., Kim, S.A. & Schwille, P. Fluorescence cross-correlation spectroscopy in living cells. Nat. Methods 3, 83–89 (2006).

    Article  CAS  Google Scholar 

  6. Kim, S.A., Heinze, K.G. & Schwille, P. Fluorescence correlation spectroscopy in living cells. Nat. Methods 4, 963–973 (2007).

    Article  CAS  Google Scholar 

  7. Maeder, C.I. et al. Spatial regulation of Fus3 MAP kinase activity through a reaction-diffusion mechanism in yeast pheromone signalling. Nat. Cell Biol. 9, 1319–1326 (2007).

    Article  CAS  Google Scholar 

  8. Slaughter, B.D., Schwartz, J.W. & Li, R. Mapping dynamic protein interactions in MAP kinase signaling using live-cell fluorescence fluctuation spectroscopy and imaging. Proc. Natl. Acad. Sci. USA 104, 20320–20325 (2007).

    Article  CAS  Google Scholar 

  9. Schmidt, U. et al. Assembly and mobility of exon-exon junction complexes in living cells. RNA 15, 862–876 (2009).

    Article  CAS  Google Scholar 

  10. Kawai-Noma, S. et al. Dynamics of yeast prion aggregates in single living cells. Genes Cells 11, 1085–1096 (2006).

    Article  CAS  Google Scholar 

  11. Yu, S.R. et al. Fgf8 morphogen gradient forms by a source-sink mechanism with freely diffusing molecules. Nature 461, 533–536 (2009).

    Article  CAS  Google Scholar 

  12. Wachsmuth, M., Waldeck, W. & Langowski, J. Anomalous diffusion of fluorescent probes inside living cell nuclei investigated by spatially-resolved fluorescence correlation spectroscopy. J. Mol. Biol. 298, 677–689 (2000).

    Article  CAS  Google Scholar 

  13. Dertinger, T. et al. Two-focus fluorescence correlation spectroscopy: a new tool for accurate and absolute diffusion measurements. ChemPhysChem 8, 433–443 (2007).

    Article  CAS  Google Scholar 

  14. Digman, M.A. et al. Measuring fast dynamics in solutions and cells with a laser scanning microscope. Biophys. J. 89, 1317–1327 (2005).

    Article  CAS  Google Scholar 

  15. Huisken, J., Swoger, J., Del Bene, F., Wittbrodt, J. & Stelzer, E.H.K. Optical sectioning deep inside live embryos by selective plane illumination microscopy. Science 305, 1007–1009 (2004).

    Article  CAS  Google Scholar 

  16. Wohland, T., Shi, X., Sankaran, J. & Stelzer, E.H. Single plane illumination fluorescence correlation spectroscopy (SPIM-FCS) probes inhomogeneous three-dimensional environments. Opt. Express 18, 10627–10641 (2010).

    Article  CAS  Google Scholar 

  17. Kannan, B. et al. Spatially resolved total internal reflection fluorescence correlation microscopy using an electron multiplying charge-coupled device camera. Anal. Chem. 79, 4463–4470 (2007).

    Article  CAS  Google Scholar 

  18. Heuvelman, G., Erdel, F., Wachsmuth, M. & Rippe, K. Analysis of protein mobilities and interactions in living cells by multifocal fluorescence fluctuation microscopy. Eur. Biophys. J. 38, 813–828 (2009).

    Article  CAS  Google Scholar 

  19. Needleman, D.J., Xu, Y. & Mitchison, T.J. Pin-hole array correlation imaging: highly parallel fluorescence correlation spectroscopy. Biophys. J. 96, 5050–5059 (2009).

    Article  CAS  Google Scholar 

  20. Sakaue-Sawano, A. et al. Visualizing spatiotemporal dynamics of multicellular cell-cycle progression. Cell 132, 487–498 (2008).

    Article  CAS  Google Scholar 

  21. Gurdon, J.B. & Bourillot, P.Y. Morphogen gradient interpretation. Nature 413, 797–803 (2001).

    Article  CAS  Google Scholar 

  22. Dinant, C. & Luijsterburg, M.S. The emerging role of HP1 in the DNA damage response. Mol. Cell Biol. 29, 6335–6340 (2009).

    Article  CAS  Google Scholar 

  23. Kwon, S.H. & Workman, J.L. The heterochromatin protein 1 (HP1) family: put away a bias toward HP1. Mol. Cells 26, 217–227 (2008).

    CAS  PubMed  Google Scholar 

  24. Grewal, S.I. & Moazed, D. Heterochromatin and epigenetic control of gene expression. Science 301, 798–802 (2003).

    Article  CAS  Google Scholar 

  25. Hediger, F. & Gasser, S.M. Heterochromatin protein 1: don't judge the book by its cover! Curr. Opin. Genet. Dev. 16, 143–150 (2006).

    Article  CAS  Google Scholar 

  26. Müller, K.P. et al. Multiscale analysis of dynamics and interactions of heterochromatin protein 1 by fluorescence fluctuation microscopy. Biophys. J. 97, 2876–2885 (2009).

    Article  Google Scholar 

  27. Schotta, G. et al. A silencing pathway to induce H3–K9 and H4–K20 trimethylation at constitutive heterochromatin. Genes Dev. 18, 1251–1262 (2004).

    Article  CAS  Google Scholar 

  28. Grewal, S.I. & Jia, S. Heterochromatin revisited. Nat. Rev. Genet. 8, 35–46 (2007).

    Article  CAS  Google Scholar 

  29. Holekamp, T.F., Turaga, D. & Holy, T.E. Fast three-dimensional fluorescence imaging of activity in neural populations by objective-coupled planar illumination microscopy. Neuron 57, 661–672 (2008).

    Article  CAS  Google Scholar 

  30. Keller, P.J. et al. Fast, high-contrast imaging of animal development with scanned light-sheet-based structured-illumination microscopy. Nat. Methods 7, 637–642 (2010).

    Article  CAS  Google Scholar 

  31. Planchon, T.A. et al. Rapid three-dimensional isotropic imaging of living cells using Bessel beam plane illumination. Nat. Methods 8, 417–423 (2011).

    Article  CAS  Google Scholar 

  32. Ritter, J.G., Veith, R., Veenendaal, A., Siebrasse, J.P. & Kubitscheck, U. Light-sheet microscopy for single molecule tracking in living tissue. PLoS ONE 5, e11639 (2010).

    Article  Google Scholar 

  33. Bestvater, F. et al. EMCCD-based spectrally resolved fluorescence correlation spectroscopy. Opt. Express 18, 23818–23828 (2010).

    Article  CAS  Google Scholar 

  34. Im, K.B. et al. Two-photon spectral imaging with high temporal and spectral resolution. Opt. Express 18, 26905–26914 (2010).

    Article  CAS  Google Scholar 

  35. Gregor, I., Patra, D. & Enderlein, J. Optical saturation in fluorescence correlation spectroscopy under continuous-wave and pulsed excitation. ChemPhysChem 6, 164–170 (2005).

    Article  CAS  Google Scholar 

  36. Gröner, N., Capoulade, J., Cremer, C. & Wachsmuth, M. Measuring and imaging diffusion with multiple scan speed image correlation spectroscopy. Opt. Express 18, 21225–21237 (2010).

    Article  Google Scholar 

  37. Huh, W.K. et al. Global analysis of protein localization in budding yeast. Nature 425, 686–691 (2003).

    Article  CAS  Google Scholar 

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We thank the mechanical and the electronics workshop of the European Molecular Biology Laboratory (EMBL) for custom hardware, M. Meurer for yeast cell culture, D. Holzer for mammalian cell culture, T. Weimbs for providing the MDCK II cells, A. Ephrussi for providing the flies expressing Ubi-GFP-NLS and K. Rippe for providing 3T3 cells expressing HP1α-EGFP. We would like to thank Leica Microsystems as well as R. Pepperkok, J. Ellenberg and the Advanced Light Microscopy Facility of EMBL for support. A. Aulehla, P. Keller and A. Khmelinskii are kindly acknowledged for helpful comments, as are many other colleagues for discussions. L.H. was supported by the center for modeling and simulation in the biosciences (BioMS). We are grateful for financial support from EMBL and from the EpiSys project within the BMBF SysTec program (grant no. 0315502C to M.W.).

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Authors and Affiliations



M.K. and M.W. conceived the research. J.C. implemented the light-pad microscope. J.C. and M.W. conducted the yeast and mammalian work. J.C., M.W. and L.H. conducted the Drosophila wing disc work. J.C., M.W. and M.K. analyzed the data and wrote the manuscript. All authors commented on the manuscript.

Corresponding authors

Correspondence to Malte Wachsmuth or Michael Knop.

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Competing interests

J.C., M.K. and M.W. are named inventors on a patent application on technologies described in this manuscript.

Supplementary information

Supplementary Text and Figures

Supplementary Table 1, Supplementary Results and Supplementary Figures 1–8 (PDF 2661 kb)

Supplementary Video S1

z-stack of images of yeast cells expressing Pma1-GFP acquired with the light-pad microscope (MOV 784 kb)

Supplementary Video S2

1D-FCS recording of 20 nm fluorescent beads diffusing in water (MOV 5291 kb)

Supplementary Video S3

2D-FCS recording of 20 nm fluorescent beads diffusing in water (MOV 1460 kb)

Supplementary Video S4

20 nm fluorescent beads diffusing in water recorded with the imaging camera showing Brownian motion and convective flow (MOV 1684 kb)

Supplementary Video S5

3D reconstruction of a Drosophila larva wing imaginal disc expressing GFP-NLS acquired with the light-pad microscope (MOV 1734 kb)

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Capoulade, J., Wachsmuth, M., Hufnagel, L. et al. Quantitative fluorescence imaging of protein diffusion and interaction in living cells. Nat Biotechnol 29, 835–839 (2011).

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