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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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.).
J.C., M.K. and M.W. are named inventors on a patent application on technologies described in this manuscript.
Supplementary Table 1, Supplementary Results and Supplementary Figures 1–8 (PDF 2661 kb)
z-stack of images of yeast cells expressing Pma1-GFP acquired with the light-pad microscope (MOV 784 kb)
1D-FCS recording of 20 nm fluorescent beads diffusing in water (MOV 5291 kb)
2D-FCS recording of 20 nm fluorescent beads diffusing in water (MOV 1460 kb)
20 nm fluorescent beads diffusing in water recorded with the imaging camera showing Brownian motion and convective flow (MOV 1684 kb)
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). https://doi.org/10.1038/nbt.1928
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