Skip to main content

Thank you for visiting nature.com. 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 optimize and functionalize red-shifted rhodamine dyes

Nature Methods volume 17, pages 815–821 (2020)Cite this article

Subjects

Abstract

Expanding the palette of fluorescent dyes is vital to push the frontier of biological imaging. Although rhodamine dyes remain the premier type of small-molecule fluorophore owing to their bioavailability and brightness, variants excited with far-red or near-infrared light suffer from poor performance due to their propensity to adopt a lipophilic, nonfluorescent form. We report a framework for rationalizing rhodamine behavior in biological environments and a general chemical modification for rhodamines that optimizes long-wavelength variants and enables facile functionalization with different chemical groups. This strategy yields red-shifted ‘Janelia Fluor’ (JF) dyes useful for biological imaging experiments in cells and in vivo.

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

Relevant articles

Open Access articles citing this article.

Access options

Rent or buy this article

Get just this article for as long as you need it

$39.95

Learn more

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: General rubric relating lactone–zwitterion equilibrium to rhodamine dye performance and optimizing short-wavelength dyes by decreasing KL–Z.
Fig. 2: Optimizing long-wavelength rhodamine dyes by increasing KL–Z and subsequent derivatization.
Fig. 3: Fine-tuning of NIR rhodamines.

Data availability

The data that support the findings of this study are provided in the Source Data files or available from the corresponding author upon request. Source data are provided with this paper.

References

  1. Lavis, L. D. Chemistry is dead. Long live chemistry! Biochemistry 56, 5165–5170 (2017).

    CAS  PubMed  Google Scholar 

  2. Abdelfattah, A. S. et al. Bright and photostable chemigenetic indicators for extended in vivo voltage imaging. Science 365, 699–704 (2019).

    CAS  PubMed  Google Scholar 

  3. Grimm, J. B. et al. A general method to improve fluorophores for live-cell and single-molecule microscopy. Nat. Methods 12, 244–250 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  4. Grimm, J. B. et al. A general method to fine-tune fluorophores for live-cell and in vivo imaging. Nat. Methods 14, 987–994 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  5. Hinckley, D. A. & Seybold, P. G. A spectroscopic/thermodynamic study of the rhodamine B lactone–zwitterion equilibrium. Spectrochim. Acta A. 44, 1053–1059 (1988).

    Google Scholar 

  6. Grimm, J. B., Brown, T. A., Tkachuk, A. N. & Lavis, L. D. General synthetic method for Si-fluoresceins and Si-rhodamines. ACS Cent. Sci. 3, 975–985 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  7. Zheng, Q. et al. Rational design of fluorogenic and spontaneously blinking labels for super-resolution imaging. ACS Cent. Sci. 5, 1602–1613 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  8. 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).

    CAS  PubMed  Google Scholar 

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

    PubMed  Google Scholar 

  10. Wang, L. et al. A general strategy to develop cell permeable and fluorogenic probes for multicolour nanoscopy. Nat. Chem. Biol. 12, 165–172 (2020).

    CAS  Google Scholar 

  11. Chi, W., Qi, Q., Lee, R., Xu, Z. & Liu, X. A unified push–pull model for understanding the ring-opening mechanism of rhodamine dyes. J. Phys. Chem. C. 124, 3793–3801 (2020).

    CAS  Google Scholar 

  12. Arden-Jacob, J., Frantzeskos, J., Kemnitzer, N. U., Zilles, A. & Drexhage, K. H. New fluorescent markers for the red region. Spectrochim. Acta A. 57, 2271–2283 (2001).

    CAS  Google Scholar 

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

    CAS  PubMed  PubMed Central  Google Scholar 

  14. Koide, Y., Urano, Y., Hanaoka, K., Terai, T. & Nagano, T. Evolution of Group 14 rhodamines as platforms for near-infrared fluorescence probes utilizing photoinduced electron transfer. ACS Chem. Biol. 6, 600–608 (2011).

    CAS  PubMed  Google Scholar 

  15. Ioffe, I. S. & Zelenin, K. N. Synthesis of azafluorescein. Zh. Obshch. Khim. 34, 2811 (1964).

    CAS  Google Scholar 

  16. Van Duuren, B. L., Goldschmidt, B. M. & Seltzman, H. H. The synthesis and aggregation of 9-phenyl- and 9-s-butyl-3,6-bisdimethylaminoacridine in solution. The dealkylation of 9-t-butylacridans. J. Chem. Soc. B. 1967, 814–819 (1967).

    Google Scholar 

  17. Fischer, C. & Sparr, C. Direct transformation of esters into heterocyclic fluorophores. Angew. Chem. Int. Ed. 57, 2436–2440 (2018).

    CAS  Google Scholar 

  18. Gannon, M. K. 2nd et al. Rhodamine inhibitors of P-glycoprotein: an amide/thioamide ‘switch’ for ATPase activity. J. Med. Chem. 52, 3328–3341 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  19. Zhou, X., Lai, R., Beck, J. R., Li, H. & Stains, C. I. Nebraska Red: a phosphinate-based near-infrared fluorophore scaffold for chemical biology applications. Chem. Commun. 52, 12290–12293 (2016).

    CAS  Google Scholar 

  20. Zhou, X., Fang, Y., Lesiak, L. & Stains, C. I. A phosphinate-containing fluorophore capable of selectively inducing apoptosis in cancer cells. Chem. Bio. Chem. 20, 1712–1716 (2019).

    CAS  PubMed  Google Scholar 

  21. Grzybowski, M. et al. A highly photostable near-infrared labeling agent based on a phospha-rhodamine for long-term and deep imaging. Angew. Chem. Int. Ed. 57, 10137–10141 (2018).

    CAS  Google Scholar 

  22. Sauer, M., Nasufovic, V., Arndt, H.-D. & Vilotijevic, I. Robust synthesis of NIR-emissive P-rhodamine fluorophores. Org. Biomol. Chem. 18, 1567–1571 (2020).

    CAS  PubMed  Google Scholar 

  23. Liu, J. et al. Sulfone-rhodamines: a new class of near-infrared fluorescent dyes for bioimaging. ACS Appl. Mater. Interfaces 8, 22953–22962 (2016).

    CAS  PubMed  Google Scholar 

  24. Hansch, C., Leo, A. & Taft, R. W. A survey of Hammett substituent constants and resonance and field parameters. Chem. Rev. 91, 165–195 (1991).

    CAS  Google Scholar 

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

    CAS  PubMed  Google Scholar 

  26. Sun, X. et al. Development of SNAP-tag fluorogenic probes for wash-free fluorescence imaging. Chem. Bio. Chem. 12, 2217–2226 (2011).

    CAS  PubMed  Google Scholar 

  27. Stagge, F., Mitronova, G. Y., Belov, V. N., Wurm, C. A. & Jakobs, S. SNAP-, CLIP- and Halo-tag labelling of budding yeast cells. PLoS ONE 8, e78745–e78745 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  28. Presman, D. M. et al. Quantifying transcription factor binding dynamics at the single-molecule level in live cells. Methods 123, 76–88 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  29. Erdmann, R. S. et al. Labeling strategies matter for super-resolution microscopy: A comparison between HaloTags and SNAP-tags. Cell Chem. Biol. 26, 584–592.e586 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  30. Correa, I. R. et al. Substrates for improved live-cell fluorescence labeling of SNAP-tag. Curr. Pharm. Des. 19, 5414–5420 (2013).

    CAS  PubMed  Google Scholar 

  31. Liu, Z. et al. 3D imaging of Sox2 enhancer clusters in embryonic stem cells. eLife 3, e04236 (2014).

    PubMed  PubMed Central  Google Scholar 

  32. Liu, J. et al. Rational design and synthesis of a novel class of highly fluorescent rhodamine dyes that have strong absorption at long wavelengths. Tetrahedron Lett. 44, 4355–4359 (2003).

    CAS  Google Scholar 

  33. Gee, K. R. et al. Novel derivatization of protein thiols with fluorinated fluoresceins. Tetrahedron Lett. 37, 7905–7908 (1996).

    CAS  Google Scholar 

  34. Mitronova, G. Y. et al. Functionalization of the meso-phenyl ring of rhodamine dyes through SNAr with sulfur nucleophiles: synthesis, biophysical characterizations, and comprehensive NMR analysis. Eur. J. Org. Chem. 2015, 337–349 (2015).

    CAS  Google Scholar 

  35. Kolmakov, K. et al. Far-red emitting fluorescent dyes for optical nanoscopy: fluorinated silicon-rhodamines (SiRF dyes) and phosphorylated oxazines. Chemistry 21, 13344–13356 (2015).

    CAS  PubMed  Google Scholar 

  36. Shen, Z., Lu, Z., Chhatbar, P. Y., O’Herron, P. & Kara, P. An artery-specific fluorescent dye for studying neurovascular coupling. Nat. Methods 9, 273–276 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  37. Patterson, D. M., Nazarova, L. A. & Prescher, J. A. Finding the right (bioorthogonal) chemistry. ACS Chem. Biol. 9, 592–605 (2014).

    CAS  PubMed  Google Scholar 

  38. Nemoto, H., Kubota, Y. & Yamamoto, Y. Development of a new acyl anion equivalent for the preparation of masked activated esters, and their use to prepare a dipeptide. J. Org. Chem. 55, 4515–4516 (1990).

    CAS  Google Scholar 

  39. Wysocki, L. M. et al. Facile and general synthesis of photoactivatable xanthene dyes. Angew. Chem. Int. Ed. 50, 11206–11209 (2011).

    CAS  Google Scholar 

  40. Dwight, S. J. & Levin, S. Scalable regioselective synthesis of rhodamine dyes. Org. Lett. 18, 5316–5319 (2016).

    CAS  PubMed  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).

    CAS  PubMed  Google Scholar 

  42. Lambert, T. J. FPbase: a community-editable fluorescent protein database. Nat. Methods 16, 277–278 (2019).

    CAS  PubMed  Google Scholar 

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

    PubMed  PubMed Central  Google Scholar 

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

    CAS  PubMed  PubMed Central  Google Scholar 

  45. Schindelin, J. et al. Fiji: an open-source platform for biological-image analysis. Nat. Methods 9, 676–682 (2012).

    CAS  Google Scholar 

  46. 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).

    PubMed  Google Scholar 

Download references

Acknowledgements

We thank S. Sternson for initial discussions on umpolung reagents; K. Svoboda for advice on in vivo labeling experiments; C. Deo and E. Schreiter for purified HaloTag protein and contributive discussions; B. Mensh and P. Kumar for a critical reading of the manuscript; R. Tjian (University of California, Berkeley) for the mouse embryonic stem cell line and the Janelia Cell and Tissue Culture, Anatomy and Histology and Vivarium teams for assistance with biological experiments. This work was supported by the Howard Hughes Medical Institute.

Author information

Authors and Affiliations

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

    Jonathan B. Grimm, Ariana N. Tkachuk, Liangqi Xie, Heejun Choi, Boaz Mohar, Natalie Falco, Kathy Schaefer, Ronak Patel, Qinsi Zheng, Zhe Liu, Jennifer Lippincott-Schwartz, Timothy A. Brown & Luke D. Lavis

Authors
  1. Jonathan B. Grimm
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Ariana N. Tkachuk
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Liangqi Xie
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Heejun Choi
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Boaz Mohar
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Natalie Falco
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Kathy Schaefer
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Ronak Patel
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Qinsi Zheng
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Zhe Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Jennifer Lippincott-Schwartz
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Timothy A. Brown
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. Luke D. Lavis
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

L.D.L. and J.B.G. conceived the project. J.B.G. contributed organic synthesis and one-photon spectroscopy measurements. A.N.T., L.X. and H.C. contributed cultured cell imaging experiments. B.M. contributed in vivo labeling and tissue imaging experiments. N.F. and Q.Z. contributed organic synthesis. L.X. and K.S. contributed flow cytometry experiments. R.P. contributed two-photon spectroscopy measurements. Z.L., J.L.-S. and T.A.B. directed the project. L.D.L. directed the project and wrote the paper with input from the other authors.

Corresponding author

Correspondence to Luke D. Lavis.

Ethics declarations

Competing interests

US Patent 9,933,417 and patent applications US 2019/0367736 and US 2019/0106573 describing azetidine-containing fluorophores and variant compositions (with inventors J.B.G. and L.D.L.) are assigned to HHMI.

Additional information

Peer review information Rita Strack was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 Utility of JF479–HaloTag (11HTL) ligand in cellular imaging experiments.

a–b, Fluorescence excitation (ex) and emission (em) spectra of 1 (a) and 11 (b). c, Two-photon absorption spectra of 1, 11, and reference dye fluorescein. d, Structure of JF503–HaloTag ligand (12HTL). e, Nuclear fluorescence vs. time upon addition of ligands 11HTL or 12HTL (200 nM) to live cells expressing HaloTag–histone H2B; error bars indicate SE; n = 100 nuclei. f–i, Confocal imaging experiments of fixed U2OS cells expressing either HaloTag histone–H2B fusion protein (f,h; nucleus) or HaloTag–PDGFR transmembrane domain (TMD) fusion protein (g,i; plasma membrane) labeled with JF479–HaloTag ligand (11HTL; 100 nM, 1 h, 2× wash; f,g) or JF503–HaloTag ligand (12HTL; 100 nM, 1 h, 2× wash; h,i); scale bars: 21 μm; these imaging experiments were duplicated with similar results.

Source data

Extended Data Fig. 2 Comparison of JF479–HaloTag ligand (11HTL) and JF503–HaloTag ligand (12HTL) in two-color experiments with JF525–cpSNAP-tag ligand (9STL).

a, Structure of JF525–cpSNAP-tag ligand (9STL). b, Fluorescence excitation spectra of JF479–HaloTag ligand (11HTL) or JF503–HaloTag ligand (12HTL) bound to HaloTag protein. Dashed lines highlight 488 nm or 532 nm excitation. c–e, Enlarged confocal images and line-scans from Fig. 1i showing live U2OS cells expressing HaloTag–histone H2B labeled with 11HTL (500 nM, 3.5 h, 3× wash) and TOMM20–SNAP-tag labeled with 9STL (100 nM, 30 min, 3× wash) excited with 532 nm; this imaging experiment was duplicated with similar results. c, Confocal image from Fig. 1i with blue line indicating line-scan position; d, Line-scan profile; e, Over-exposed image showing low nuclear signal. f–h, Enlarged confocal images and line-scan from Fig. 1i showing U2OS cells expressing HaloTag–histone H2B labeled with 12HTL (500 nM, 3.5 h, 3× wash) and TOMM20–SNAP-tag labeled with 9STL (100 nM, 30 min, 3× wash) excited with 532 nm; this imaging experiment was duplicated with similar results. f, Confocal image from Fig. 1i with green line indicating line-scan position; g, Line-scan profile; h, Over-exposed image showing high nuclear signal. Scale bars for all images: 10 μm.

Source data

Extended Data Fig. 3 Utility of JF552–cpSNAP-tag ligand (13STL) in cellular imaging experiments.

a, Representative flow cytometry plot showing forward light scatter (FSC) vs. side light scatter (SSC) demonstrating gating strategy to separate cells from debris; experiment was duplicated with similar results. b, Representative flow cytometry plot showing SSC vs. fluorescence of 13STL:SNAP-tag measured using the Y585-PE channel (561 nm laser excitation, 585 nm with a 42 nm bandpass emission) to demonstrate gating strategy to separate fluorescent and nonfluorescent cells; experiment was duplicated with similar results. c, Representative flow cytometry plots showing the change in % fluorescent cells as a function of incubation time with 13STL (10 nM); top row: wild-type (WT) embryonic stem (ES) cells; bottom row: SNAP-tag–histone H2B (ST) expressing ES cells; experiment was duplicated with similar results. d, Plot of fluorescent mouse ES cells (%) vs. time determined by the flow cytometry experiment exemplified in c. WT cells or ST cells were incubated with 2STL or 13STL (10 nM) for different times and % fluorescent cells were measured; error bars show SE; experiments using 2STL n = 3; experiments using 13STL n = 4 except for t = 7.5 min where n = 2 and t = 210 min where n = 3. e, Violin plot of photon counts from a single-particle tracking (SPT) experiment using U2OS cells expressing SNAP-tag–histone H2B and labeled with 2STL or 13STL (2 nM, 30 min, 3× wash); lines indicate median and quartiles; n = 19008 single-molecule events for experiment using 2STL and n = 9511 single-molecule events for experiment using 13STL. f, Histogram of track lengths from SPT experiment using cells expressing SNAP-tag–histone H2B and labeled with 2STL or 13STL (2 nM, 30 min, 3× wash); n = 10822 single-molecule events for experiment using 2STL and n = 9387 single-molecule events for experiment using 13STL. g, Structure of JF549–HaloTag ligand (2HTL). h, Image of individual SPT traces in live U2OS cells expressing SNAP-tag–histone H2B and labeled with 2STL (2 nM, 30 min, 3× wash); dashed line indicates outline of nucleus; arrows highlight nonspecific staining in cytosol; scale bar: 2 μm. (i) 3D kymograph showing data from h detailing single-particle track position and length as a function of time. Diffusion coefficient values (D) are calculated from single-particle tracking data and color-coded; experiment was duplicated with similar results.

Source data

Extended Data Fig. 4 Synthesis and spectral properties of 15–20.

a, Synthesis of Janelia Fluor dyes 15–20. b–g, Fluorescence excitation (ex) and emission (em) spectra of JF669 (15; b), JF690 (16; c), JF722 (17; d), JF724 (18; e), JF571 (19; f), and JF593 (20; g).

Source data

Extended Data Fig. 5 Derivatization of JF669.

a, Reaction of azide 21 with strained alkynes 25 or 26 to form triazole adducts 27 or 28. b, Synthesis of amine 22 via reaction of JF669 (15) with NH3, reduction of azide 21, or Curtius rearrangement starting from ester 29 showing consistent regioselectivity of SNAr reactions. c, Reaction of 15 with amine-containing chelator groups 30 and 31 to form far-red K+ indicator 32 and far-red Zn2+ indicator 33. d, Fluorescence emission spectra of 32 in the absence or presence of 100 mM K+. e, Fluorescence emission spectra of 30 in the absence or presence of 10 μM Zn2+.

Source data

Extended Data Fig. 6 Synthesis and properties of new HaloTag and SNAP-tag ligands based on 15–20 and 37–38.

a, Expanded synthetic scheme of HaloTag ligands 15HTL–20HTL and 37HTL–38HTL starting with nucleophilic aromatic substitution (SNAr) of 15–20 and 37–38 with masked acyl cyanide 34. b, Two-color montage image of fixed coronal slices with zoom-in regions from a mouse expressing HaloTag–GFP in neurons transduced by IV administration of the viral vector PHP-eB-Syn-HaloTag–GFP followed by IV administration of 15HTL (100 nmol), perfusion, and slicing; GFP signal in green and 15HTL signal in magenta; scale bar = 3 mm; experiment was duplicated with similar results. c, Structures of JF669–HaloTag ligand (15HTL), JF646–HaloTag ligand (5HTL), JF593–HaloTag ligand (20HTL), and JF570–HaloTag ligand (3HTL). d–e, Confocal image and line-scan from Fig. 2l showing live U2OS cells expressing Sec61β–HaloTag labeled with 2HTL (30 nM, 30 min, 3× wash) and SNAP-tag–histone variant H2A.Z labeled with 15STL (30 nM, 30 min, 3× wash); costained with Hoechst 33342 (1 μM, 30 min, 3× wash); d, Confocal image with white line indicating line-scan position; e, Line-scan profile; scale bar: 5 μm; experiment was duplicated with similar results. f, Two-photon absorption spectra of the HaloTag conjugates of HaloTag ligands 15HTL–20HTL, and 37HTL–38HTL. g, Plot of fluorescence from cells expressing HaloTag–H2B labeled with 2HTL (200 nM) or 19HTL (200 nM) over 30 bleach cycles; error bars indicate SE; n = 3 independent cellular samples. h, Plot of fluorescence from fixed U2OS cells expressing HaloTag–H2B labeled with 3HTL (200 nM) or 20HTL (200 nM) over 30 bleach cycles; error bars indicate SE; n = 3 independent cellular samples.

Source data

Extended Data Fig. 7 Further fine-tuning of JF571 (19) and JF722 (17).

a, Structure of JF571 (19) and JF559 (37). b–c, Full plot of KL–Z vs. λabs (b) and zoom-in (c) showing decreased KL–Z for dye 37. d, Fluorescence excitation (ex) and emission (em) spectra of JF559 (37). e, Structure of JF559–HaloTag ligand (37HTL). f, Widefield imaging experiment of U2OS cells expressing HaloTag–histone H2B labeled with 37HTL (100 nM, 30 min, 3× wash); scale bar: 51 μm; experiment was duplicated with similar results. g, Fluorescence excitation (ex) and emission (em) spectra of JF711 (38). h, Nuclear fluorescence vs. time upon addition of ligands 17HTL (200 nM) or 38HTL (200 nM) to live cells expressing HaloTag–histone H2B; error bars indicate SE; n = 100 nuclei except for: t = 0.5 h with 38HTL where n = 86 nuclei; t = 1 h with 38HTL where n = 94 nuclei; t = 2 h with 38HTL where n = 96 nuclei; t = 0.5 h with 17HTL where n = 94 nuclei. i, Plot of fluorescence from cells expressing HaloTag–H2B labeled with 17HTL (200 nM) or 38HTL (200 nM) over 30 bleach cycles; error bars indicate SE;; n = 3 independent cellular samples.

Source data

Supplementary information

Supplementary Information

Supplementary Note detailing chemical syntheses and characterization.

Reporting Summary

Source data

Source Data Fig. 1

Raw numerical data.

Source Data Fig. 2

Raw numerical data.

Source Data Fig. 3

Raw numerical data.

Source Data Extended Data Fig. 1

Raw numerical data.

Source Data Extended Data Fig. 2

Raw numerical data.

Source Data Extended Data Fig. 3

Raw numerical data.

Source Data Extended Data Fig. 4

Raw numerical data.

Source Data Extended Data Fig. 5

Raw numerical data.

Source Data Extended Data Fig. 6

Raw numerical data.

Source Data Extended Data Fig. 7

Raw numerical data.

Rights and permissions

Reprints and Permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Grimm, J.B., Tkachuk, A.N., Xie, L. et al. A general method to optimize and functionalize red-shifted rhodamine dyes. Nat Methods 17, 815–821 (2020). https://doi.org/10.1038/s41592-020-0909-6

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41592-020-0909-6

This article is cited by

Search

Advanced search

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