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).
References
Kao, H. & Verkman, A. Tracking of single fluorescent particles in three dimensions: use of cylindrical optics to encode particle position. Biophys. J. 67, 1291–1300 (1994).
Huang, B., Wang, W., Bates, M. & Zhuang, X. Three-dimensional super-resolution imaging by stochastic optical reconstruction microscopy. Science 319, 810–813 (2008).
Pavani, S. R. P. & Piestun, R. Three dimensional tracking of fluorescent microparticles using a photon-limited double-helix response system. Opt. Express 16, 22048 (2008).
Shechtman, Y., Weiss, L. E., Backer, A. S., Sahl, S. J. & Moerner, W. E. Precise three-dimensional scan-free multiple-particle tracking over large axial ranges with tetrapod point spread functions. Nano Lett. 15, 4194–4199 (2015).
Aristov, A., Lelandais, B., Rensen, E. & Zimmer, C. ZOLA-3D allows flexible 3D localization microscopy over an adjustable axial range. Nat. Commun. 9, 2409 (2018).
Shechtman, Y., Weiss, L. E., Backer, A. S., Lee, M. Y. & Moerner, W. E. Multicolour localization microscopy by point-spread-function engineering. Nat. Photonics 10, 590–594 (2016).
Dong, B. et al. Super-resolution spectroscopic microscopy via photon localization. Nat. Commun. 7, 12290 (2016).
Hershko, E., Weiss, L. E., Michaeli, T. & Shechtman, Y. Multicolor localization microscopy and point-spread-function engineering by deep learning. Opt. Express 27, 6158 (2019).
Erdelyi, M. et al. Correcting chromatic offset in multicolor super-resolution localization microscopy. Opt. Express 21, 10978–88 (2013).
Niekamp, S. et al. Nanometer-accuracy distance measurements between fluorophores at the single-molecule level. Proc. Natl. Acad. Sci. USA 116, 4275–4284 (2019).
Gómez-García, P. A., Garbacik, E. T., Otterstrom, J. J., Garcia-Parajo, M. F. & Lakadamyali, M. Excitation-multiplexed multicolor superresolution imaging with fm-STORM and fm-DNA-PAINT. Proc. Natl. Acad. Sci. USA 115, 12991–12996 (2018).
Broeken, J., Rieger, B. & Stallinga, S. Simultaneous measurement of position and color of single fluorescent emitters using diffractive optics. Opt. Lett. 39, 3352 (2014).
Martens, K. J. A. et al. Enabling spectrally resolved single-molecule localization microscopy at high emitter densities. Nano Lett. 22, 8618–8625 (2022).
Dertinger, T., Colyer, R., Iyer, G., Weiss, S. & Enderlein, J. Fast, background-free, 3D super-resolution optical fluctuation imaging (SOFI). Proc. Natl. Acad. Sci. USA 106, 22287–22292 (2009).
Vandenberg, W., Leutenegger, M., Duwé, S. & Dedecker, P. An extended quantitative model for super-resolution optical fluctuation imaging (SOFI). Opt. Express 27, 25749 (2019).
Purohit, A. et al. Spatio-temporal correlation super-resolution optical fluctuation imaging. Europhys. Lett. 125, 20005 (2019).
Siraganian, R. P., de Castro, R. O., Barbu, E. A. & Zhang, J. Mast cell signaling: the role of protein tyrosine kinase syk, its activation and screening methods for new pathway participants. FEBS Lett. 584, 4933–4940 (2010).
Andrews, N. L. et al. Small, mobile fcepsilonri receptor aggregates are signaling competent. Immunity 31, 469–479 (2009).
Schnitzbauer, J., Strauss, M. T., Schlichthaerle, T., Schueder, F. & Jungmann, R. Super-resolution microscopy with dna-paint. Nat. Protoc. 12, 1198–1228 (2017).
Huppa, J. B. et al. TCR–peptide–MHC interactions in situ show accelerated kinetics and increased affinity. Nature 463, 963–7 (2010).
Liu, F. T. et al. Monoclonal dinitrophenyl-specific murine IgE antibody: preparation, isolation, and characterization. J. Immunol. 124, 2728–2737 (1980).
Schwartz, S. L. et al. Differential mast cell outcomes are sensitive to FCμRI-Syk binding kinetics. Mol. Biol. Cell 28, 3397–3414 (2017).
Ovesny, M., Křížek, P., Borkovec, J., Svindrych, Z. & Hagen, G. M. ThunderSTORM: a comprehensive ImageJ plug-in for PALM and STORM data analysis and super-resolution imaging. Bioinformatics 30, 2389–2390 (2014).
Dedecker, P., Duwé, S., Neely, R. K. & Zhang, J. Localizer: fast, accurate, open-source, and modular software package for superresolution microscopy. J. Biomed. Opt. 17, 126008 (2012).
Van den Eynde, R. et al. Data and code for ‘Simultaneous multicolor fluorescence imaging using PSF splitting’. Zenodo https://doi.org/10.5281/zenodo.11196841 (2024).
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).
Author information
Author notes
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
Authors
- Robin Van den Eynde
View author publications
You can also search for this author in PubMedGoogle Scholar
- Fabian Hertel
View author publications
You can also search for this author in PubMedGoogle Scholar
- Sergey Abakumov
View author publications
You can also search for this author in PubMedGoogle Scholar
- Bartosz Krajnik
View author publications
You can also search for this author in PubMedGoogle Scholar
- Siewert Hugelier
View author publications
You can also search for this author in PubMedGoogle Scholar
- Alexander Auer
View author publications
You can also search for this author in PubMedGoogle Scholar
- Joschka Hellmeier
View author publications
You can also search for this author in PubMedGoogle Scholar
- Thomas Schlichthaerle
View author publications
You can also search for this author in PubMedGoogle Scholar
- Rachel M. Grattan
View author publications
You can also search for this author in PubMedGoogle Scholar
- Diane S. Lidke
View author publications
You can also search for this author in PubMedGoogle Scholar
- Ralf Jungmann
View author publications
You can also search for this author in PubMedGoogle Scholar
- Marcel Leutenegger
View author publications
You can also search for this author in PubMedGoogle Scholar
- Wim Vandenberg
View author publications
You can also search for this author in PubMedGoogle Scholar
- Peter Dedecker
View author publications
You can also search for this author in PubMedGoogle Scholar
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.
Ethics declarations
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.
Peer review
Peer review information
Nature Methods thanks the anonymous reviewers for their contribution to the peer review of this work. Primary Handling Editor: Rita Strack, in collaboration with the Nature Methods team. Peer reviewer reports are available.
Additional information
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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.
Rights and permissions
Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.
About this article
Cite this article
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
Received:
Accepted:
Published:
DOI: https://doi.org/10.1038/s41592-024-02383-7
