Skip to main content

Thank you for visiting You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

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


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.

This is a preview of subscription content, access via your institution

Relevant articles

Open Access articles citing this article.

Access options

Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

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


  1. Kremers, G.-J., Gilbert, S.G., Cranfill, P.J., Davidson, M.W. & Piston, D.W. Fluorescent proteins at a glance. J. Cell Sci. 124, 157–160 (2011).

    Article  CAS  Google Scholar 

  2. Xia, T., Li, N. & Fang, X. Single-molecule fluorescence imaging in living cells. Annu. Rev. Phys. Chem. 64, 459–480 (2013).

    Article  CAS  Google Scholar 

  3. Griffin, B.A., Adams, S.R. & Tsien, R.Y. Specific covalent labeling of recombinant protein molecules inside live cells. Science 281, 269–272 (1998).

    Article  CAS  Google Scholar 

  4. Keppler, A. et al. A general method for the covalent labeling of fusion proteins with small molecules in vivo. Nat. Biotechnol. 21, 86–89 (2003).

    Article  CAS  Google Scholar 

  5. Los, G.V. et al. HaloTag: a novel protein labeling technology for cell imaging and protein analysis. ACS Chem. Biol. 3, 373–382 (2008).

    Article  CAS  Google Scholar 

  6. Encell, L.P. et al. Development of a dehalogenase-based protein fusion tag capable of rapid, selective and covalent attachment to customizable ligands. Curr. Chem. Genomics 6, 55–71 (2012).

    Article  Google Scholar 

  7. Wombacher, R. et al. Live-cell super-resolution imaging with trimethoprim conjugates. Nat. Methods 7, 717–719 (2010).

    Article  CAS  Google Scholar 

  8. Hori, Y., Ueno, H., Mizukami, S. & Kikuchi, K. Photoactive yellow protein-based protein labeling system with turn-on fluorescence intensity. J. Am. Chem. Soc. 131, 16610–16611 (2009).

    Article  CAS  Google Scholar 

  9. Uttamapinant, C. et al. A fluorophore ligase for site-specific protein labeling inside living cells. Proc. Natl. Acad. Sci. USA 107, 10914–10919 (2010).

    Article  CAS  Google Scholar 

  10. Testa, I. et al. Multicolor fluorescence nanoscopy in fixed and living cells by exciting conventional fluorophores with a single wavelength. Biophys. J. 99, 2686–2694 (2010).

    Article  CAS  Google Scholar 

  11. Zhao, Z.W. et al. Spatial organization of RNA polymerase II inside a mammalian cell nucleus revealed by reflected light-sheet superresolution microscopy. Proc. Natl. Acad. Sci. USA 111, 681–686 (2014).

    Article  CAS  Google Scholar 

  12. Abrahamsson, S. et al. Fast multicolor 3D imaging using aberration-corrected multifocus microscopy. Nat. Methods 10, 60–63 (2013).

    Article  CAS  Google Scholar 

  13. Chen, J. et al. Single-molecule dynamics of enhanceosome assembly in embryonic stem cells. Cell 156, 1274–1285 (2014).

    Article  CAS  Google Scholar 

  14. Lavis, L.D. & Raines, R.T. Bright ideas for chemical biology. ACS Chem. Biol. 3, 142–155 (2008).

    Article  CAS  Google Scholar 

  15. Beija, M., Afonso, C.A.M. & Martinho, J.M.G. Synthesis and applications of rhodamine derivatives as fluorescent probes. Chem. Soc. Rev. 38, 2410–2433 (2009).

    Article  CAS  Google Scholar 

  16. Lavis, L.D. & Raines, R.T. Bright building blocks for chemical biology. ACS Chem. Biol. 9, 855–866 (2014).

    Article  CAS  Google Scholar 

  17. Gautier, A. et al. An engineered protein tag for multiprotein labeling in living cells. Chem. Biol. 15, 128–136 (2008).

    Article  CAS  Google Scholar 

  18. Lukinavičius, G. et al. A near-infrared fluorophore for live-cell super-resolution microscopy of cellular proteins. Nat. Chem. 5, 132–139 (2013).

    Article  Google Scholar 

  19. Mujumdar, R.B., Ernst, L.A., Mujumdar, S.R., Lewis, C.J. & Waggoner, A.S. Cyanine dye labeling reagents: sulfoindocyanine succinimidyl esters. Bioconjug. Chem. 4, 105–111 (1993).

    Article  CAS  Google Scholar 

  20. Panchuk-Voloshina, N. et al. Alexa dyes, a series of new fluorescent dyes that yield exceptionally bright, photostable conjugates. J. Histochem. Cytochem. 47, 1179–1188 (1999).

    Article  CAS  Google Scholar 

  21. Haugland, R.P., Spence, M.T.Z., Johnson, I.D. & Basey, A. The Handbook: A Guide to Fluorescent Probes and Labeling Technologies 10th edn. (Molecular Probes, 2005).

  22. Zhang, Z., Revyakin, A., Grimm, J.B., Lavis, L.D. & Tjian, R. Single-molecule tracking of the transcription cycle by sub-second RNA detection. eLife 3, e01775 (2014).

    Article  Google Scholar 

  23. Bosch, P.J. et al. Evaluation of fluorophores to label SNAP-tag fused proteins for multicolor single-molecule tracking microscopy in live cells. Biophys. J. 107, 803–814 (2014).

    Article  CAS  Google Scholar 

  24. Grimm, J.B. et al. Carbofluoresceins and carborhodamines as scaffolds for high-contrast fluorogenic probes. ACS Chem. Biol. 8, 1303–1310 (2013).

    Article  CAS  Google Scholar 

  25. Vogel, M., Rettig, W., Sens, R. & Drexhage, K.H. Structural relaxation of rhodamine dyes with different N-substitution patterns: a study of fluorescence decay times and quantum yields. Chem. Phys. Lett. 147, 452–460 (1988).

    Article  CAS  Google Scholar 

  26. Grabowski, Z.R., Rotkiewicz, K. & Rettig, W. Structural changes accompanying intramolecular electron transfer: focus on twisted intramolecular charge-transfer states and structures. Chem. Rev. 103, 3899–4032 (2003).

    Article  Google Scholar 

  27. Song, X., Johnson, A. & Foley, J. 7-Azabicyclo[2.2.1]heptane as a unique and effective dialkylamino auxochrome moiety: demonstration in a fluorescent rhodamine dye. J. Am. Chem. Soc. 130, 17652–17653 (2008).

    Article  CAS  Google Scholar 

  28. Grimm, J.B. & Lavis, L.D. Synthesis of rhodamines from fluoresceins using Pd-catalyzed C-N cross-coupling. Org. Lett. 13, 6354–6357 (2011).

    Article  CAS  Google Scholar 

  29. Heilemann, M. et al. Subdiffraction-resolution fluorescence imaging with conventional fluorescent probes. Angew. Chem. Int. Ed. Engl. 47, 6172–6176 (2008).

    Article  CAS  Google Scholar 

  30. Dempsey, G.T., Vaughan, J.C., Chen, K.H., Bates, M. & Zhuang, X. Evaluation of fluorophores for optimal performance in localization-based super-resolution imaging. Nat. Methods 8, 1027–1036 (2011).

    Article  CAS  Google Scholar 

  31. Ha, T. & Tinnefeld, P. Photophysics of fluorescence probes for single molecule biophysics and super-resolution imaging. Annu. Rev. Phys. Chem. 63, 595–617 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  32. Lukinavičius, G. et al. Fluorogenic probes for live-cell imaging of the cytoskeleton. Nat. Methods 11, 731–733 (2014).

    Article  Google Scholar 

  33. Loving, G. & Imperiali, B. A versatile amino acid analogue of the solvatochromic fluorophore 4-N,N-dimethylamino-1,8-naphthalimide: a powerful tool for the study of dynamic protein interactions. J. Am. Chem. Soc. 130, 13630–13638 (2008).

    Article  CAS  Google Scholar 

  34. Kubota, Y. & Steiner, R.F. Fluorescence decay and quantum yield characteristics of acridine orange and proflavine bound to DNA. Biophys. Chem. 6, 279–289 (1977).

    Article  CAS  Google Scholar 

  35. Lee, L.G., Berry, G.M. & Chen, C.-H. Vita Blue: a new 633-nm excitable fluorescent dye for cell analysis. Cytometry 10, 151–164 (1989).

    Article  Google Scholar 

  36. Bancaud, A. et al. Molecular crowding affects diffusion and binding of nuclear proteins in heterochromatin and reveals the fractal organization of chromatin. EMBO J. 28, 3785–3798 (2009).

    Article  CAS  Google Scholar 

  37. Bancaud, A., Lavelle, C., Huet, S. & Ellenberg, J. A fractal model for nuclear organization: Current evidence and biological implications. Nucleic Acids Res. 40, 8783–8792 (2012).

    Article  CAS  Google Scholar 

  38. Speight, L.C. et al. Efficient synthesis and in vivo incorporation of acridon-2-ylalanine, a fluorescent amino acid for lifetime and Förster resonance energy transfer/luminescence resonance energy transfer studies. J. Am. Chem. Soc. 135, 18806–18814 (2013).

    Article  CAS  Google Scholar 

  39. Mitronova, G.Y. et al. New fluorinated rhodamines for optical microscopy and nanoscopy. Chemistry 16, 4477–4488 (2010).

    Article  CAS  Google Scholar 

  40. Altman, R.B. et al. Cyanine fluorophore derivatives with enhanced photostability. Nat. Methods 9, 68–71 (2012).

    Article  CAS  Google Scholar 

  41. Suzuki, K. et al. Reevaluation of absolute luminescence quantum yields of standard solutions using a spectrometer with an integrating sphere and a back-thinned CCD detector. Phys. Chem. Chem. Phys. 11, 9850–9860 (2009).

    Article  CAS  Google Scholar 

  42. Critchfield, F.E., Gibson, J.A. Jr. & Hall, J.L. Dielectric constant for the dioxane–water system from 20 to 35°. J. Am. Chem. Soc. 75, 1991–1992 (1953).

    Article  CAS  Google Scholar 

  43. Mütze, J. et al. Excitation spectra and brightness optimization of two-photon excited probes. Biophys. J. 102, 934–944 (2012).

    Article  Google Scholar 

  44. Akerboom, J. et al. Optimization of a GCaMP calcium indicator for neural activity imaging. J. Neurosci. 32, 13819–13840 (2012).

    Article  CAS  Google Scholar 

  45. Magde, D., Rojas, G.E. & Seybold, P.G. Solvent dependence of the fluorescence lifetimes of xanthene dyes. Photochem. Photobiol. 70, 737–744 (1999).

    Article  CAS  Google Scholar 

  46. Sergé, A., Bertaux, N., Rigneault, H. & Marguet, D. Multiple-target tracing (MTT) algorithm probes molecular dynamics at cell surface. Protoc. Exch. doi:10.1038/nprot.2008.128 (2008).

    Article  CAS  Google Scholar 

  47. Sergé, A., Bertaux, N., Rigneault, H. & Marguet, D. Dynamic multiple-target tracing to probe spatiotemporal cartography of cell membranes. Nat. Methods 5, 687–694 (2008).

    Article  Google Scholar 

  48. Mortensen, K.I., Churchman, L.S., Spudich, J.A. & Flyvbjerg, H. Optimized localization analysis for single-molecule tracking and super-resolution microscopy. Nat. Methods 7, 377–381 (2010).

    Article  CAS  Google Scholar 

  49. Dedecker, P., Duwé, S., Neely, R.K. & Zhang, J. Localizer: fast, accurate, open-source, and modular software package for superresolution microscopy. J. Biomed. Opt. 17, 126008 (2012).

    Article  Google Scholar 

  50. Guizar-Sicairos, M., Thurman, S.T. & Fienup, J.R. Efficient subpixel image registration algorithms. Opt. Lett. 33, 156–158 (2008).

    Article  Google Scholar 

Download references


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



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)

Source data

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

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).

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI:

This article is cited by


Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing