Fixation-resistant photoactivatable fluorescent proteins for CLEM

A Corrigendum to this article was published on 30 June 2015

This article has been updated

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.

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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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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.

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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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