Article | Published:

A general method to improve fluorophores for live-cell and single-molecule microscopy

Nature Methods volume 12, pages 244250 (2015) | Download Citation

Abstract

Specific labeling of biomolecules with bright fluorophores is the keystone of fluorescence microscopy. Genetically encoded self-labeling tag proteins can be coupled to synthetic dyes inside living cells, resulting in brighter reporters than fluorescent proteins. Intracellular labeling using these techniques requires cell-permeable fluorescent ligands, however, limiting utility to a small number of classic fluorophores. Here we describe a simple structural modification that improves the brightness and photostability of dyes while preserving spectral properties and cell permeability. Inspired by molecular modeling, we replaced the N,N-dimethylamino substituents in tetramethylrhodamine with four-membered azetidine rings. This addition of two carbon atoms doubles the quantum efficiency and improves the photon yield of the dye in applications ranging from in vitro single-molecule measurements to super-resolution imaging. The novel substitution is generalizable, yielding a palette of chemical dyes with improved quantum efficiencies that spans the UV and visible range.

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Acknowledgements

We thank S. Eddy and E. Betzig for contributive discussions, M. Dahan (Curie Institut) and X. Darzacq (Berkeley) for the Snap-tag–TetR plasmid, M. Tadross (Janelia) for the purified HaloTag protein and W. Hu (Janelia) for cloning and purifying the recombinant HaloTag-MS2 protein. This work was supported by the Howard Hughes Medical Institute. Salary for R.H.S. is funded by US National Institutes of Health grants GM57071, NS83085 and EB13571.

Author information

Affiliations

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

    • Jonathan B Grimm
    • , Brian P English
    • , Jiji Chen
    • , Joel P Slaughter
    • , Zhengjian Zhang
    • , Andrey Revyakin
    • , Ronak Patel
    • , John J Macklin
    • , Davide Normanno
    • , Robert H Singer
    • , Timothée Lionnet
    •  & Luke D Lavis
  2. Department of Biochemistry, University of Leicester, Leicester, UK.

    • Andrey Revyakin
  3. Laboratoire Physico-Chimie Curie, Institut Curie, Paris, France.

    • Davide Normanno
  4. Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, New York, USA.

    • Robert H Singer

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Contributions

J.B.G. conceived of the project and performed organic synthesis. B.P.E. and J.C. designed and performed cellular microscopy experiments and analyzed data. J.P.S. performed organic synthesis. Z.Z. prepared bioconjugates, performed in vitro single-molecule microscopy and analyzed data. A.R. performed in vitro single-molecule microscopy and analyzed data. R.P. and J.J.M. performed two-photon spectroscopy and fluorescence lifetime measurements. D.N. designed and validated the Snap-tag–TetR plasmid. R.H.S. interpreted data. T.L. designed experiments and performed data analysis. L.D.L. conceived of the project, performed one-photon spectroscopic measurements and wrote the manuscript with input from the other authors.

Competing interests

J.B.G., J.C., J.P.S., Z.Z., A.R., T.L. and L.D.L. have filed patent applications on azetidine-containing fluorophores, whose value may be affected by this publication.

Corresponding authors

Correspondence to Timothée Lionnet or Luke D Lavis.

Integrated supplementary information

Supplementary information

PDF files

  1. 1.

    Supplementary Text and Figures

    Supplementary Figures 1–4 and Supplementary Note

Videos

  1. 1.

    Single-molecule microscopy using JF549

    Imaging of individual molecules of JF549-labeled HaloTag–H2B in a live HeLa cell.

  2. 2.

    Two-color single-molecule microscopy using JF549 and JF646

    Imaging of individual molecules of JF549-labeled SnapTag–TetR in a live HeLa cell (red) overlayed with a super-resolution dSTORM image of JF646-labeled HaloTag–H2B (green).

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DOI

https://doi.org/10.1038/nmeth.3256

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