Fixation-resistant photoactivatable fluorescent proteins for CLEM

Nature Methods volume 12, pages215–218(2015)Cite this article

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A Corrigendum to this article was published on 30 June 2015

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Abstract

Fluorescent proteins facilitate a variety of imaging paradigms in live and fixed samples. However, they lose their fluorescence after heavy fixation, hindering applications such as correlative light and electron microscopy (CLEM). Here we report engineered variants of the photoconvertible Eos fluorescent protein that fluoresce and photoconvert normally in heavily fixed (0.5–1% OsO4), plastic resin–embedded samples, enabling correlative super-resolution fluorescence imaging and high-quality electron microscopy.

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Figure 1: Osmium resistance of the mEos4 probes.
Figure 2: Correlative PALM and TEM imaging using the consecutive-section approach.
Figure 3: Correlative PALM and TEM/SEM imaging using the same-section approach.

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  • 04 May 2015

    In the version of this article initially published, scale bars that had been erroneously inserted into Figure 1c during image processing were covered with black boxes. The error has been corrected, through removal of both the boxes and the scale bars beneath them, for the HTML and PDF versions of the article.

References

  1. 1

    Shaner, N.C., Patterson, G.H. & Davidson, M.W. J. Cell Sci. 120, 4247–4260 (2007).

    CAS  Article  Google Scholar 

  2. 2

    Lippincott-Schwartz, J. & Patterson, G.H. Trends Cell Biol. 19, 555–565 (2009).

    CAS  Article  Google Scholar 

  3. 3

    Huang, B. Curr. Opin. Chem. Biol. 14, 10–14 (2010).

    CAS  Article  Google Scholar 

  4. 4

    Betzig, E. et al. Science 313, 1642–1645 (2006).

    CAS  Article  Google Scholar 

  5. 5

    Hess, S.T., Girirajan, T.P. & Mason, M.D. Biophys. J. 91, 4258–4272 (2006).

    CAS  PubMed  PubMed Central  Google Scholar 

  6. 6

    Wiedenmann, J. et al. Proc. Natl. Acad. Sci. USA 101, 15905–15910 (2004).

    CAS  Article  Google Scholar 

  7. 7

    Nienhaus, G.U. et al. Photochem. Photobiol. 82, 351–358 (2006).

    CAS  Article  Google Scholar 

  8. 8

    McKinney, S.A., Murphy, C.S., Hazelwood, K.L., Davidson, M.W. & Looger, L.L. Nat. Methods 6, 131–133 (2009).

    CAS  Article  Google Scholar 

  9. 9

    Zhang, M. et al. Nat. Methods 9, 727–729 (2012).

    CAS  Article  Google Scholar 

  10. 10

    Shroff, H., Galbraith, C.G., Galbraith, J.A. & Betzig, E. Nat. Methods 5, 417–423 (2008).

    CAS  Article  Google Scholar 

  11. 11

    Watanabe, S. et al. Nat. Methods 8, 80–84 (2011).

    CAS  Article  Google Scholar 

  12. 12

    Tokuyasu, K.T. J. Cell Biol. 57, 551–565 (1973).

    CAS  Article  Google Scholar 

  13. 13

    Kopek, B.G., Shtengel, G., Xu, C.S., Clayton, D.A. & Hess, H.F. Proc. Natl. Acad. Sci. USA 109, 6136–6141 (2012).

    CAS  Article  Google Scholar 

  14. 14

    Chang, Y.W. et al. Nat. Methods 11, 737–739 (2014).

    CAS  Article  Google Scholar 

  15. 15

    Collins, J.S. & Goldsmith, T.H. J. Histochem. Cytochem. 29, 411–414 (1981).

    CAS  Article  Google Scholar 

  16. 16

    Kopek, B.G., Shtengel, G., Grimm, J.B., Clayton, D.A. & Hess, H.F. PLoS ONE 8, e77209 (2013).

    CAS  Article  Google Scholar 

  17. 17

    Li, J., Wang, Y., Chiu, S.L. & Cline, H.T. Front. Neural Circuits 4, 6 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  18. 18

    Martell, J.D. et al. Nat. Biotechnol. 30, 1143–1148 (2012).

    CAS  Article  Google Scholar 

  19. 19

    Shu, X. et al. PLoS Biol. 9, e1001041 (2011).

    CAS  Article  Google Scholar 

  20. 20

    Robertson, C.E. Methods Cell Biol. 79, 169–191 (2007).

    CAS  Article  Google Scholar 

  21. 21

    Kozak, M. J. Mol. Biol. 196, 947–950 (1987).

    CAS  Article  Google Scholar 

  22. 22

    Creighton, T.E. Proteins: Structures and Molecular Properties 2nd edn. (W.H. Freeman, 1993).

  23. 23

    Nienhaus, K., Nienhaus, G.U., Wiedenmann, J. & Nar, H. Proc. Natl. Acad. Sci. USA 102, 9156–9159 (2005).

    CAS  Article  Google Scholar 

  24. 24

    Habuchi, S., Tsutsui, H., Kochaniak, A.B., Miyawaki, A. & van Oijen, A.M. PLoS ONE 3, e3944 (2008).

    Article  Google Scholar 

  25. 25

    Kunkel, T.A. Proc. Natl. Acad. Sci. USA 82, 488–492 (1985).

    CAS  Article  Google Scholar 

  26. 26

    Studier, F.W. Protein Expr. Purif. 41, 207–234 (2005).

    CAS  Article  Google Scholar 

  27. 27

    Costantini, L.M., Fossati, M., Francolini, M. & Snapp, E.L. Traffic 13, 643–649 (2012).

    CAS  Article  Google Scholar 

  28. 28

    Brown, T.A., Fetter, R.D., Tkachuk, A.N. & Clayton, D.A. Methods 51, 458–463 (2010).

    CAS  Article  Google Scholar 

  29. 29

    Hayat, M.A. Principles and Techniques of Electron Microscopy: Biological Applications 4th edn. (Cambridge Univ. Press, 2000).

  30. 30

    Clancy, B. & Cauller, L.J. J. Neurosci. Methods 83, 97–102 (1998).

    CAS  Article  Google Scholar 

  31. 31

    Mortensen, K.I., Churchman, L.S., Spudich, J.A. & Flyvbjerg, H. Nat. Methods 7, 377–381 (2010).

    CAS  Article  Google Scholar 

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Acknowledgements

We thank D. Murphy and R. Fetter for helpful discussions. E. Betzig generously allowed use of his PALM microscope early in the project. S. Winfrey, H. White and A. Tkachuk assisted with cell culture. B. Kopek gave advice and technical support on PALM and CLEM. P. Rivlin gave advice on EM. P. Hulamm, L. Shao and T.-L. Chew provided technical support for imaging. T. Gallagher, M. Ramirez, P. Nguyen and K. McGowan aided in molecular biology. J. Marvin, J. Akerboom, M. Verdecia and E. Schreiter provided advice on biochemical characterization techniques. S. McKinney was helpful in the design of the anisotropy assay. Several members of the Davidson laboratory (FSU) assisted with characterization of the protein fusions: P. Cranfill, B. Sell, L. Case, J. Shirley, S. Gilbert, K. Hendrickson, R. Labaddan and R. Clarke. M.G.P.-S. thanks M. Reddy for guidance and mentoring in biochemistry and molecular biology. This work was supported by the Howard Hughes Medical Institute.

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Affiliations

  1. Howard Hughes Medical Institute, Janelia Farm Research Campus, Ashburn, Virginia, USA

    Maria G Paez-Segala, Mei G Sun, Gleb Shtengel, Sarada Viswanathan, John J Macklin, Ronak Patel, Harald F Hess, Yalin Wang & Loren L Looger

  2. National High Magnetic Field Laboratory, Florida State University, Tallahassee, Florida, USA

    Michelle A Baird, John R Allen, Elizabeth S Howe & Michael W Davidson

  3. Department of Biological Science, Florida State University, Tallahassee, Florida, USA

    Michelle A Baird, John R Allen, Elizabeth S Howe & Michael W Davidson

  4. National Heart, Lung, and Blood Institute, Bethesda, Maryland, USA

    Grzegorz Piszczek

Authors
  1. Maria G Paez-Segala
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  2. Mei G Sun
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  3. Gleb Shtengel
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  4. Sarada Viswanathan
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  5. Michelle A Baird
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  6. John J Macklin
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  7. Ronak Patel
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  8. John R Allen
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  9. Elizabeth S Howe
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  10. Grzegorz Piszczek
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  11. Harald F Hess
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  12. Michael W Davidson
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  13. Yalin Wang
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  14. Loren L Looger
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Contributions

M.G.P.-S. and L.L.L. conceived of the project and designed mutations. M.G.P.-S. made mutants, designed and implemented the in vitro screens, performed cell culture and light microscopy, and primary-fixed samples for EM. M.G.S. and Y.W. prepared samples for PALM/EM and performed TEM. G.S. and H.F.H. performed PALM imaging and quantification, SEM and CLEM data registration. S.V. assisted in mutation discovery. M.A.B., J.R.A., E.S.H. and M.W.D. constructed and screened FP fusions. J.J.M. and R.P. performed photophysical characterization of variants. G.P. performed AUC characterization. M.G.P.-S. and L.L.L. managed the project. M.G.P.-S., M.G.S. and L.L.L. wrote the manuscript, with the help of the other authors.

Corresponding author

Correspondence to Loren L Looger.

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The authors declare no competing financial interests.

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Supplementary Figures 1–10, Supplementary Tables 1 and 2 and Supplementary Methods (PDF 47142 kb)

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Paez-Segala, M., Sun, M., Shtengel, G. et al. Fixation-resistant photoactivatable fluorescent proteins for CLEM. Nat Methods 12, 215–218 (2015). https://doi.org/10.1038/nmeth.3225

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