Data availability
The data used in the manuscript are available via Zenodo at https://doi.org/10.5281/zenodo.11196841 (ref. 25).
Code availability
The code used in the manuscript is available via Zenodo at https://doi.org/10.5281/zenodo.11196841 (ref. 25).
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Acknowledgements
We thank A. Bouwens (KU Leuven) for fruitful discussions regarding the Circulator design and T. Van Thillo (KU Leuven) for the preparation of alignment samples. We thank Y. Shechtman and E. Nehme (Technion) for discussions and insights regarding the use of machine learning for the data analysis. R.V.d.E. and S.H. thank the Research Foundation-Flanders (FWO Vlaanderen) for a doctoral and postdoctoral fellowship, respectively. We thank D. Rinaldi for the generation of the fluorescent IgE. This work was supported by the European Research Council through grant 714688 NanoCellActivity (to P.D.), the Research Foundation-Flanders through grants G090819N and G010723N (to P.D.) and the National Institutes of Health via grant R35GM126934 (to D.S.L.). B.K. thanks the Polish National Science Center (SONATA 16, grant no. 2020/39/D/ST5/03359).
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Fabian Hertel
Present address: Department of Nuclear Medicine, University Hospital RWTH Aachen University, Aachen, Germany
Authors and Affiliations
Department of Chemistry, KU Leuven, Leuven, Belgium
Robin Van den Eynde,Fabian Hertel,Sergey Abakumov,Bartosz Krajnik,Siewert Hugelier,Wim Vandenberg&Peter Dedecker
Department of Experimental Physics, Wroclaw University of Science and Technology, Wroclaw, Poland
Bartosz Krajnik
Max Planck Institute of Biochemistry, Martinsried, Germany
Alexander Auer,Joschka Hellmeier,Thomas Schlichthaerle&Ralf Jungmann
Faculty of Physics and Center for Nanoscience, Ludwig Maximilian University, Munich, Germany
Alexander Auer,Joschka Hellmeier,Thomas Schlichthaerle&Ralf Jungmann
Department of Pathology and Comprehensive Cancer Center, University of New Mexico, Albuquerque, NM, USA
Rachel M. Grattan&Diane S. Lidke
Max Planck Institute of Biophysical Chemistry, Göttingen, Germany
Marcel Leutenegger
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Contributions
W.V. and P.D. designed research. P.D. supervised research. M.L. invented the optical layout of the Circulator. B.K., R.V.d.E. and W.V. constructed the Circulator. R.V.d.E., F.H. and W.V. performed experiments and analyzed data with assistance from S.H. W.V., S.A. and P.D. developed new analysis tools and algorithms and analyzed data. A.A., T.S. and J.H. created samples under the supervision of R.J. and performed initial experiments. D.S.L. and R.M.G. provided samples and interpretation for the SPT experiments. P.D. wrote the manuscript with input from W.V. and R.V.d.E.
Corresponding authors
Correspondence to Wim Vandenberg or Peter Dedecker.
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Competing interests
M.L., P.D. and W.V. hold a patent on the Circulator (PCT/EP2020/051264). The other authors declare no competing interests.
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Extended data
Extended Data Fig. 1 SOFI with the Circulator.
(a) Overall concept of SOFI-based imaging using the Circulator. (b) Fluorescence image obtained by calculating the average of all acquired fluorescence images. (c) SOFI images obtained from a single acquisition performed on cells in which microtubules, vimentin and clathrin were labeled with ATTO643, Abberior Star 488 and Cy3B respectively. Image acquisition time: 100 seconds. This was a representative measurement from 4 biological repeats.
Extended Data Fig. 2 SPT with the Circulator.
(a) A schematic description of DNA origami rafts on top of a lipid bilayer. The rafts are free to diffuse at the bilayer interface and are attached via cholesterol anchors. Each raft is stained with fluorophores of a single color. (b) Example SPT traces obtained for the freely diffusing origami rafts, as well as MSD plots for each color and the calculated diffusion coefficients. Data was acquired from 5 technical repeats and resulted in 948 green, 908 orange and 755 red tracks. (c) Conceptual illustration of IgE crosslinking at the cellular membrane, the different PSF shapes this may create, as well as an example acquired fluorescence image. (d) Fraction of molecules that appear as double- or multilobe PSFs before- and after-stimulation with DNP-BSA. The unpaired two-sided t-test with 8 degrees of freedom (DOF) returned a t-value of 13.72, corresponding to a p-value of 7.6 ⋯ 10−7, showing a significant difference between two populations. (e) Step-size PDFs and CDFs for labeled IgE molecules pre- and post-addition of DNP-BSA. Multilobe fluorophores are not shown in the resting state due to insufficient statistics, which is consistent with a lack of aggregates before DNP-BSA addition. Data was acquired from 4 and 6 cells pre- and post-stimulation (biological repeats) respectively, where pre-stimulation served as the control group. In total, this yielded 2298 (2326) green, 2513 (2386) orange, 2755 (2639) red, and 41 (213) multicolor tracks pre- and (post-) stimulation.
Supplementary information
Supplementary Information
Supplementary Figs. 1–10 and discussion (Notes 1–6).
Supplementary Movie 1
A short capture (100 frames) of the recorded movie for the simultaneous three-color DNA-PAINT (SMLM) experiment depicted in Fig. 2.
Supplementary Movie 2
A short capture (100 frames) of the recorded movie for the simultaneous three-color DNA-PAINT (SOFI) experiment depicted in Extended Data Fig. 1.
Supplementary Movie 3
A short capture (100 frames) of the recorded movie for the simultaneous three-color SPT (DNA origami) experiment depicted in Extended Data Fig. 2b.
Supplementary Movie 4
A short capture (100 frames) of the recorded movie for the simultaneous three-color SPT (IgE) experiment depicted in Extended Data Fig. 2d,e.
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Van den Eynde, R., Hertel, F., Abakumov, S. et al. Simultaneous multicolor fluorescence imaging using PSF splitting. Nat Methods (2024). https://doi.org/10.1038/s41592-024-02383-7
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DOI: https://doi.org/10.1038/s41592-024-02383-7
