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Rapid nonlinear image scanning microscopy

Nature Methods volume 14, pages 1087–1089 (2017)Cite this article

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Abstract

Image scanning microscopy (ISM) doubles the resolution of a conventional confocal microscope for super-resolution imaging. Here, we describe an all-optical ISM design based on rescanning microscopy for two-photon-excited fluorescence and second-harmonic generation that allows straightforward implementation into existing microscopes. The design offers improved sensitivity and high frame rates relative to those of existing systems. We demonstrate its utility using fixed and living specimens as well as collagen hydrogels.

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Figure 1: (a) Schematic of the microscope.
Figure 2: (a–c) Actin cytoskeleton in human mesenchymal stem cells.
Figure 3: Fluorescence images of D. melanogaster expressing histone2–eGFP.

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References

  1. Diaspro, A. et al. Biomed. Eng. Online 5, 36 (2006).

    Article  Google Scholar 

  2. Hoover, E.E. & Squier, J.A. Nat. Photonics 7, 93–101 (2013).

    Article  CAS  Google Scholar 

  3. Brown, E. et al. Nat. Med. 9, 796–800 (2003).

    Article  CAS  Google Scholar 

  4. Campagnola, P. Anal. Chem. 83, 3224–3231 (2011).

    Article  CAS  Google Scholar 

  5. Chen, X., Nadiarynkh, O., Plotnikov, S. & Campagnola, P.J. Nat. Protoc. 7, 654–669 (2012).

    Article  CAS  Google Scholar 

  6. Müller, C.B. & Enderlein, J. Phys. Rev. Lett. 104, 198101 (2010).

    Article  Google Scholar 

  7. Gustafsson, M.G.L. J. Microsc. 198, 82–87 (2000).

    Article  CAS  Google Scholar 

  8. York, A.G. et al. Nat. Methods 9, 749–754 (2012).

    Article  CAS  Google Scholar 

  9. Gustafsson, M.G.L. Proc. Natl. Acad. Sci. USA 102, 13081–13086 (2005).

    Article  CAS  Google Scholar 

  10. Fiolka, R., Shao, L., Rego, E.H., Davidson, M.W. & Gustafsson, M.G.L. Proc. Natl. Acad. Sci. USA 109, 5311–5315 (2012).

    Article  CAS  Google Scholar 

  11. Sheppard, C.J.R. Optik (Stuttg.) 80, 53–54 (1988).

    Google Scholar 

  12. Schulz, O. et al. Proc. Natl. Acad. Sci. USA 110, 21000–21005 (2013).

    Article  CAS  Google Scholar 

  13. De Luca, G.M.R.R. et al. Biomed. Opt. Express 4, 2644–2656 (2013).

    Article  Google Scholar 

  14. Roth, S., Sheppard, C.J.R., Wicker, K. & Heintzmann, R. Opt. Nanoscopy 2, 5 (2013).

    Article  Google Scholar 

  15. Azuma, T. & Kei, T. Opt. Express 23, 15003–15011 (2015).

    Article  CAS  Google Scholar 

  16. York, A.G. et al. Nat. Methods 10, 1122–1126 (2013).

    Article  CAS  Google Scholar 

  17. Roider, C., Ritsch-Marte, M. & Jesacher, A. Opt. Lett. 41, 3825–3828 (2016).

    Article  CAS  Google Scholar 

  18. Ingaramo, M. et al. Proc. Natl. Acad. Sci. USA 111, 5254–5259 (2014).

    Article  CAS  Google Scholar 

  19. Winter, P.W. et al. Optica 1, 181–191 (2014).

    Article  Google Scholar 

  20. Zipfel, W.R., Williams, R.M. & Webb, W.W. Nat. Biotechnol. 21, 1369–1377 (2003).

    Article  CAS  Google Scholar 

  21. Roth, S., Sheppard, C.J.R. & Heintzmann, R. Opt. Lett. 41, 2109–2112 (2016).

    Article  Google Scholar 

  22. Tinevez, J.-Y. Methods Enzymol. 506, 291–309 (2012).

    Article  CAS  Google Scholar 

  23. Donnert, G. et al. Proc. Natl. Acad. Sci. USA 103, 11440–11445 (2006).

    Article  CAS  Google Scholar 

  24. Ouzounov, D.G. et al. Nat. Methods 14, 388–390 (2017).

    Article  CAS  Google Scholar 

  25. Hortn, N.G. et al. Nat. Photonics 7, 205–209 (2013).

    Article  Google Scholar 

  26. Staunton, J.R., Doss, B.L., Lindsay, S. & Ros, R. Sci. Rep. 6, 19686 (2016).

    Article  CAS  Google Scholar 

  27. Clarkson, M. & Saint, R. DNA Cell Biol. 18, 457–462 (1999).

    Article  CAS  Google Scholar 

  28. Kanesaki, T., Edwards, C.M., Schwarz, U.S. & Grosshans, J. Integr. Biol. 3, 1112–1119 (2011).

    Article  CAS  Google Scholar 

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Acknowledgements

Part of this work (J.E., I.G.) was supported by the Cluster of Excellence and DFG Research Center Nanoscale Microscopy and Molecular Physiology of the Brain. R.R. was supported by the NSF grant 1510700. We thank F. Rehfeldt (3rd Institute of Physics-Biophysics, University of Göttingen, Göttingen, Germany) for the hMSC samples, and J. Faust for help with the Arivis image processing.

Author information

Authors and Affiliations

  1. 3rd Institute of Physics – Biophysics, University of Göttingen, Göttingen, Germany

    Ingo Gregor, Martin Spiecker & Jörg Enderlein

  2. Center for Nanoscale Microscopy and Molecular Physiology of the Brain (CNMPB), Göttingen, Germany

    Ingo Gregor & Jörg Enderlein

  3. Institute of Developmental Biochemistry, University Medical Center Göttingen, Göttingen, Germany

    Roman Petrovsky & Jörg Großhans

  4. Department of Physics, Arizona State University, Tempe, Arizona, USA

    Robert Ros

  5. Center for Biological Physics, Arizona State University, Tempe, Arizona, USA

    Robert Ros

  6. Biodesign Institute, Arizona State University, Tempe, Arizona, USA

    Robert Ros

Authors
  1. Ingo Gregor
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  2. Martin Spiecker
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  4. Jörg Großhans
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  6. Jörg Enderlein
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Contributions

I.G. developed the setup; M.S. and I.G. built the instrument and performed fluorescence measurements. R.R. prepared the collagen samples and measured the SHG images. R.P. and J.G. provided the Drosophila embryos. J.E. conceived and directed the work. All authors discussed the results. I.G., R.R., and J.E. wrote the paper and reviewed and edited the final version.

Corresponding author

Correspondence to Jörg Enderlein.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Note 1 (PDF 2087 kb)

Life Sciences Reporting Summary (PDF 130 kb)

SHG confocal

SHG signal of a collagen I gel as recorded by confocal microscopy. (MOV 19677 kb)

SHG ISM

SHG signal of a collagen I gel as recorded by image scanning microscopy. (MOV 19684 kb)

Diffusing beads

This video shows fluorescent beads (d = 100 nm) diffusing freely in water. The recording frame rate is 20 fps. The acquisition speed is sufficient to track individual beads for several frames until they diffuse out of focus. (MOV 4866 kb)

D. melanogaster embryo development 1

Time-series of 3D stacks of living Drosophila embryos. Sections are taken around the center height of the embryo and the spacing of the z-planes is 3 μm.Frame interval is 60 s. (MOV 695 kb)

D. melanogaster embryo development 2

Time-series of 3D stacks of living Drosophila embryos. Sections are taken around the center height of the embryo and the spacing of the z-planes is 3 μm.Frame interval is 60 s. (MOV 873 kb)

D. melanogaster embryo development 3

Time-series of 3D stacks of living Drosophila embryos. Sections are taken around the center height of the embryo and the spacing of the z-planes is 3 μm.Frame interval is 60 s. (MOV 1115 kb)

D. melanogaster embryo development 4

Time-series of 3D stacks of living Drosophila embryos. Sections are taken around the center height of the embryo and the spacing of the z-planes is 3 μm.Frame interval is 120 s. (MOV 755 kb)

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Gregor, I., Spiecker, M., Petrovsky, R. et al. Rapid nonlinear image scanning microscopy. Nat Methods 14, 1087–1089 (2017). https://doi.org/10.1038/nmeth.4467

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