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

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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Figure 1: Development and utility of JF549.
Figure 2: Utility of JF646 in cellular imaging.
Figure 3: Utility of azetidinyl coumarins in cellular imaging.

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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

Authors and Affiliations

Authors

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.

Corresponding authors

Correspondence to Timothée Lionnet or Luke D Lavis.

Ethics declarations

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.

Integrated supplementary information

Supplementary Figure 1 Additional characterization of tetraalkylrhodamine dyes.

(a) Normalized absorption (abs) and fluorescence emission spectra (fl) for dyes 1, 2, and 47. (b) Peak molecular brightness (kcps/molecule) of dyes 2, 47 at λex 830 nm (black; 12.5 mW) and 1020 nm (grey; 20 mW). (c) Plot of normalized absorbance versus dielectric constant (ɛr) for dyes 2 and 4.

Supplementary Figure 2 Additional super-resolution microscopy results using tetramethylrhodamine and JF549.

(a) dSTORM and wide-field (inset) fluorescence microscopy image of the nucleus of a fixed U2OS cell expressing HaloTag–H2B and labeled with TMR ligand 10. The dSTORM image is comprised of 10,000 consecutive frames and the 42,463 detected particles are displayed according to their localization full-width at half-maximum. The mean localization error was 19.2 nm, the median localization error was 17.0 nm; scale bar = 5 μm. (b) Normalized distributions of the localization errors for imaging experiments using the JF549 ligand 9 (red, Fig. 1i) and the TMR ligand 10 (black, a). (c) dSTORM fluorescence microscopy image of the nucleus of a live HeLa cell expressing HaloTag–H2B and labeled with JF549 ligand 9. The dSTORM image is comprised of 10,000 consecutive frames and the 269,922 detected particles are displayed according to their localization full-width at half-maximum; scale bar = 5 μm.

Supplementary Figure 3 Normalized absorption (abs) and fluorescence emission spectra (fl) for fluorophores 11–26.

Supplementary Figure 4 Additional characterization and cellular imaging using SiTMR and JF646.

(a) dSTORM fluorescence microscopy image of the nucleus of a fixed U2OS cell expressing HaloTag–H2B and labeled with SiTMR–HaloTag ligand 28. The dSTORM image is comprised of 5,000 consecutive frames and the 227,185 detected particles are displayed according to their localization FWHM. The mean localization error was 11.9 nm, the median localization error was 9.0 nm; scale bar = 5 μm. (b) Normalized distributions of the localization errors for imaging experiments using the JF646 ligand 27 (red, Fig. 2b) and the SiTMR ligand 28 (black, a). (c,d) Microscopy images of a live HeLa cell expressing HaloTag–tubulin and labeled with JF646 ligand 27. (c) Wide-field fluorescence image. (d) dSTORM image is comprised of 10,000 consecutive frames and the 664,866 detected particles are displayed according to their localization full-width at half-maximum. The mean localization error was 9.23 nm; the median localization error was 7.14 nm. (e) Plot of line scan intensity in the wide-field image (blue) and dSTORM image (gold) as a function of line length. (f) Additional examples of wide-field fluorescence microscopy images of live unwashed HeLa cells expressing HaloTag–H2B and incubated with 100 nM of either JF646–HaloTag ligand 27 (top row) or SiTMR–HaloTag ligand 28 (bottom row); scale bars = 10 μm. (g) Rendering of single-molecule trajectories of SnapTag–TetR–JF549 conjugate from ligand 29 (different colors; n = 4,124) overlaid on a dSTORM image of HaloTag–H2B labeled with JF646 ligand 27 (grayscale) in the nucleus of a live U2OS cell. The dSTORM image is comprised of 10,000 consecutive frames and the 283,501 detected particles are displayed according to their localization FWHM; scale bar = 5 μm. (h). Normalized distributions of the apparent diffusion coefficients (Dapp) of SnapTag–TetR that colocalize with HaloTag–H2B (black) or do not colocalize with HaloTag–H2B (red).

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–4 and Supplementary Note (PDF 8563 kb)

Single-molecule microscopy using JF549

Imaging of individual molecules of JF549-labeled HaloTag–H2B in a live HeLa cell. (MOV 5215 kb)

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). (MOV 22071 kb)

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Grimm, J., English, B., Chen, J. et al. A general method to improve fluorophores for live-cell and single-molecule microscopy. Nat Methods 12, 244–250 (2015). https://doi.org/10.1038/nmeth.3256

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