Abstract

Small-molecule fluorophores are important tools for advanced imaging experiments. We previously reported a general method to improve small, cell-permeable fluorophores which resulted in the azetidine-containing 'Janelia Fluor' (JF) dyes. Here, we refine and extend the utility of these dyes by synthesizing photoactivatable derivatives that are compatible with live-cell labeling strategies. Once activated, these derived compounds retain the superior brightness and photostability of the JF dyes, enabling improved single-particle tracking and facile localization microscopy experiments.

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Acknowledgements

We thank W. Legant and E. Betzig for contributive discussions; H. White, D. Walpita, K. Schaefer, and P. Nguyen for assistance with molecular biology and cell culture; and A. Berro, A. Abdelfattah, and E. Schreiter (all at Janelia) for the purified HaloTag and SNAP-tag proteins. This work was supported by the Howard Hughes Medical Institute.

Author information

Author notes

    • Jonathan B Grimm
    •  & Brian P English

    These authors contributed equally to this work.

Affiliations

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

    • Jonathan B Grimm
    • , Brian P English
    • , Heejun Choi
    • , Anand K Muthusamy
    • , Brian P Mehl
    • , Peng Dong
    • , Timothy A Brown
    • , Jennifer Lippincott-Schwartz
    • , Zhe Liu
    • , Timothée Lionnet
    •  & Luke D Lavis

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Contributions

J.B.G., B.P.E., Z.L., T.L., and L.D.L. conceived the project. J.B.G. contributed organic synthesis and photochemistry experiments; B.P.E., H.C., B.P.M., P.D., and T.A.B. contributed cell biological experiments and data analysis; A.K.M. contributed organic synthesis; J.L.-S., Z.L., T.L., and L.D.L. directed the project and contributed data analysis. L.D.L. wrote the paper with input from the other authors.

Competing interests

J.B.G., B.P.E., A.K.M., Z.L., T.L., and L.D.L. have filed patent applications (e.g., PCT/US2015/023953) 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 and 2, Supplementary Tables 1 and 2, and Supplementary Note.

Videos

  1. 1.

    One-photon activation of PA-JF549 in live cells

    One-photon activation (405 nm) of an ES cell expressing HaloTag-Sox2 labeled with PA-JF549-HaloTag ligand.

  2. 2.

    Two-photon activation of PA-JF549 in live cells

    Two-photon activation (800 nm; spelling “HHMI”) in a HeLa cell expressing histone H2B-HaloTag labeled with PA-JF549-HaloTag ligand followed by full-field one-photon activation (405 nm).

  3. 3.

    Activation of PA-JF549 with and without activation light

    A fixed U2OS cell expressing ensconsin-HaloTag labeled with PA-JF549-HaloTag ligand and imaged in PALM mode with constant excitation light (561 nm) without activation light (405 nm, 1000 frames; “OFF”) and with activation light (405 nm, 1000 frames; “ON”).

  4. 4.

    mEos3.2 vs. PA-JF dyes in sptPALM experiments

    Comparison of sptPALM in ES cells expressing mEos3.2–Sox2 or HaloTag–Sox2 labeled with JF549 or JF646.

  5. 5.

    PA-JF549 vs. TMR in single-particle tracking experiments

    Side-by-side comparison of 3D single-particle tracking (spt) experiments in an ES cell expressing HaloTag–Sox2 labeled with TMR HaloTag ligand (left) in spt-dSTORM mode or PA-JF549 (right) in sptPALM mode.

  6. 6.

    3D PALM imaging using PA-JF549

    3D PALM imaging of cell expressing Sec61β-HaloTag fusions and labeled with PA-JF549-HaloTag ligand.

  7. 7.

    One-photon activation of PA-JF646 in live cells

    One-photon activation (405 nm) of an ES cell expressing GFP-HP1 (green) and HaloTag-Sox2 labeled with PA-JF646-HaloTag ligand (magenta).

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DOI

https://doi.org/10.1038/nmeth.4034

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