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Caveat fluorophore: an insiders’ guide to small-molecule fluorescent labels

Nature Methods volume 19, pages 149–158 (2022)Cite this article

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Abstract

The last three decades have brought a revolution in fluorescence microscopy. The development of new microscopes, fluorescent labels and analysis techniques has pushed the frontiers of biological imaging forward, moving from fixed to live cells, from diffraction-limited to super-resolution imaging and from simple cell culture systems to experiments in vivo. The large and ever-evolving collection of tools can be daunting for biologists, who must invest substantial time and effort in adopting new technologies to answer their specific questions. This is particularly relevant when working with small-molecule fluorescent labels, where users must navigate the jargon, idiosyncrasies and caveats of chemistry. Here, we present an overview of chemical dyes used in biology and provide frank advice from a chemist’s perspective.

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Fig. 1: Fluorescence basics.
Fig. 2: Labeling strategies.
Fig. 3: Fluorophores across the visible spectrum.
Fig. 4: Caveats of small-molecule fluorescent dyes.
Fig. 5: Photobleaching of fluorescent dyes.
Fig. 6: Synthesis of Alexa Fluor 488.

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References

  1. Coons, A. H., Creech, H., Jones, R. & Berliner, E. The demonstration of pneumococcal antigen in tissues by the use of fluorescent antibody. J. Immunol. 45, 159–170 (1942).

    Article  CAS  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  5. Brightman, R. Perkin and the dyestuffs industry in Britain. Nature 177, 815 (1956).

    Article  Google Scholar 

  6. Schäfer, F. P. (ed) Dye Lasers 2nd edn (Springer, 1977).

  7. Loudet, A. & Burgess, K. BODIPY dyes and their derivatives: syntheses and spectroscopic properties. Chem. Rev. 107, 4891–4932 (2007).

    Article  CAS  PubMed  Google Scholar 

  8. Smith, L. M. et al. Fluorescence detection in automated DNA sequence analysis. Nature 321, 674–679 (1986).

    Article  CAS  PubMed  Google Scholar 

  9. Lee, L. G. et al. DNA sequencing with dye-labeled terminators and T7 DNA polymerase: effect of dyes and dNTPs on incorporation of dye-terminators and probability analysis of termination fragments. Nucleic Acids Res. 20, 2471–2483 (1992).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  Google Scholar 

  11. 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  PubMed  Google Scholar 

  12. Rodriguez, E. A. et al. The growing and glowing toolbox of fluorescent and photoactive proteins. Trends Biochem. Sci. 42, 111–129 (2017).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  14. Minta, A., Kao, J. P. & Tsien, R. Y. Fluorescent indicators for cytosolic calcium based on rhodamine and fluorescein chromophores. J. Biol. Chem. 264, 8171–8178 (1989).

    Article  CAS  PubMed  Google Scholar 

  15. Chan, J., Dodani, S. C. & Chang, C. J. Reaction-based small-molecule fluorescent probes for chemoselective bioimaging. Nat. Chem. 4, 973–984 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Huang, Y.-L., Walker, A. S. & Miller, E. W. A photostable silicon rhodamine platform for optical voltage sensing. J. Am. Chem. Soc. 137, 10767–10776 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Bai, X., Ng, K. K., Hu, J. J., Ye, S. & Yang, D. Small-molecule-based fluorescent sensors for selective detection of reactive oxygen species in biological systems. Annu. Rev. Biochem. 88, 605–633 (2019).

    Article  CAS  PubMed  Google Scholar 

  18. Wu, L. et al. Forster resonance energy transfer (FRET)-based small-molecule sensors and imaging agents. Chem. Soc. Rev. 49, 5110–5139 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Greenwald, E. C., Mehta, S. & Zhang, J. Genetically encoded fluorescent biosensors illuminate the spatiotemporal regulation of signaling networks. Chem. Rev. 118, 11707–11794 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Sabatini, B. L. & Tian, L. Imaging neurotransmitter and neuromodulator dynamics in vivo with genetically encoded indicators. Neuron 108, 17–32 (2020).

    Article  CAS  PubMed  Google Scholar 

  21. Zhang, Z., Cheng, X., Zhao, Y. & Yang, Y. Lighting up live-cell and in vivo central carbon metabolism with genetically encoded fluorescent sensors. Annu. Rev. Anal. Chem. 13, 293–314 (2020).

    Article  CAS  Google Scholar 

  22. Tebo, A. G. et al. Orthogonal fluorescent chemogenetic reporters for multicolor imaging. Nat. Chem. Biol. 17, 30–38 (2021).

    Article  CAS  PubMed  Google Scholar 

  23. Deo, C. et al. The HaloTag as a general scaffold for far-red tunable chemigenetic indicators. Nat. Chem. Biol. 17, 718–723 (2021).

    Article  CAS  PubMed  Google Scholar 

  24. Kasha, M. Characterization of electronic transitions in complex molecules. Faraday Discuss. 9, 14–19 (1950).

    Article  Google Scholar 

  25. Valeur, B. & Berberan-Santos, M. N. Molecular Fluorescence: Principles and Applications 2nd edn (Wiley-VCH, 2012).

    Book  Google Scholar 

  26. Zheng, Q. & Lavis, L. D. Development of photostable fluorophores for molecular imaging. Curr. Opin. Chem. Biol. 39, 32–38 (2017).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Stokes, G. G. On the change of refrangibility of light. Philos. Trans. R. Soc. Lond. 142, 463–562 (1852).

    Google Scholar 

  29. Hermann, J. P. & Ducuing, J. Dispersion of the two-photon cross section in rhodamine dyes. Opt. Commun. 6, 101–105 (1972).

    Article  CAS  Google Scholar 

  30. Crivat, G. & Taraska, J. W. Imaging proteins inside cells with fluorescent tags. Trends Biotechnol. 30, 8–16 (2012).

    Article  CAS  PubMed  Google Scholar 

  31. Bruchez, M. P. Dark dyes–bright complexes: fluorogenic protein labeling. Curr. Opin. Chem. Biol. 27, 18–23 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. 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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Koniev, O. & Wagner, A. Developments and recent advancements in the field of endogenous amino acid selective bond forming reactions for bioconjugation. Chem. Soc. Rev. 44, 5495–5551 (2015).

    Article  CAS  PubMed  Google Scholar 

  34. 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).

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

    Article  PubMed  Google Scholar 

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

    Article  PubMed  Google Scholar 

  37. Lukinavičius, G. et al. SiR-Hoechst is a far-red DNA stain for live-cell nanoscopy. Nat. Commun. 6, 8497 (2015).

    Article  PubMed  Google Scholar 

  38. Lukinavičius, G. et al. Fluorogenic probes for multicolor imaging in living cells. J. Am. Chem. Soc. 138, 9365–9368 (2016).

    Article  PubMed  Google Scholar 

  39. Legant, W. R. et al. High-density three-dimensional localization microscopy across large volumes. Nat. Methods 13, 359–365 (2016).

    Article  PubMed  PubMed Central  Google Scholar 

  40. 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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  44. Chin, J. W. Expanding and reprogramming the genetic code. Nature 550, 53–60 (2017).

    Article  CAS  PubMed  Google Scholar 

  45. Himo, F. et al. Copper(I)-catalyzed synthesis of azoles. DFT study predicts unprecedented reactivity and intermediates. J. Am. Chem. Soc. 127, 210–216 (2005).

    Article  CAS  PubMed  Google Scholar 

  46. Beatty, K. E., Xie, F., Wang, Q. & Tirrell, D. A. Selective dye-labeling of newly synthesized proteins in bacterial cells. J. Am. Chem. Soc. 127, 14150–14151 (2005).

    Article  CAS  PubMed  Google Scholar 

  47. Baskin, J. M. et al. Copper-free click chemistry for dynamic in vivo imaging. Proc. Natl Acad. Sci. USA 104, 16793–16797 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Chang, P. V. et al. Copper-free click chemistry in living animals. Proc. Natl Acad. Sci. USA 107, 1821–1826 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Blackman, M. L., Royzen, M. & Fox, J. M. Tetrazine ligation: fast bioconjugation based on inverse-electron-demand Diels–Alder reactivity. J. Am. Chem. Soc. 130, 13518–13519 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Devaraj, N. K., Weissleder, R. & Hilderbrand, S. A. Tetrazine-based cycloadditions: application to pretargeted live cell imaging. Bioconjug. Chem. 19, 2297–2299 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Lang, K. et al. Genetic encoding of bicyclononynes and trans-cyclooctenes for site-specific protein labeling in vitro and in live mammalian cells via rapid fluorogenic Diels–Alder reactions. J. Am. Chem. Soc. 134, 10317–10320 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Seitchik, J. L. et al. Genetically encoded tetrazine amino acid directs rapid site-specific in vivo bioorthogonal ligation with trans-cyclooctenes. J. Am. Chem. Soc. 134, 2898–2901 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Keppler, A. et al. A general method for the covalent labeling of fusion proteins with small molecules in vivo. Nat. Biotechnol. 21, 86–89 (2002).

    Article  PubMed  Google Scholar 

  54. 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  PubMed  Google Scholar 

  55. Szent-Gyorgyi, C. et al. Fluorogen-activating single-chain antibodies for imaging cell surface proteins. Nat. Biotechnol. 26, 235–240 (2008).

    Article  CAS  PubMed  Google Scholar 

  56. Miller, L. W., Cai, Y., Sheetz, M. P. & Cornish, V. W. In vivo protein labeling with trimethoprim conjugates: a flexible chemical tag. Nat. Methods 2, 255–257 (2005).

    Article  CAS  PubMed  Google Scholar 

  57. You, M. & Jaffrey, S. R. Structure and mechanism of RNA mimics of green fluorescent protein. Annu. Rev. Biophys. 44, 187–206 (2015).

    Article  CAS  PubMed  Google Scholar 

  58. Crissman, H. A. & Hirons, G. T. Staining of DNA in live and fixed cells. Methods Cell. Biol. 41, 195–209 (1994).

    Article  CAS  PubMed  Google Scholar 

  59. Kapuscinski, J. DAPI: A DNA-specific fluorescent probe. Biotech Histochem. 70, 220–233 (1995).

    Article  CAS  PubMed  Google Scholar 

  60. Nakamura, A. et al. Hoechst tagging: a modular strategy to design synthetic fluorescent probes for live-cell nucleus imaging. Chem. Commun. (Camb.) 50, 6149–6152 (2014).

    Article  CAS  Google Scholar 

  61. Whitaker, J. E. et al. Cascade Blue derivatives: water soluble, reactive, blue emission dyes evaluated as fluorescent labels and tracers. Anal. Biochem. 198, 119–130 (1991).

    Article  CAS  PubMed  Google Scholar 

  62. Subach, O. M., Cranfill, P. J., Davidson, M. W. & Verkhusha, V. V. An enhanced monomeric blue fluorescent protein with the high chemical stability of the chromophore. PLoS One 6, e28674 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  63. Baeyer, A. Ueber eine Neue Klasse von Farbstoffen. Ber. Dtsch. Chem. Ges. 4, 555–558 (1871).

    Article  Google Scholar 

  64. Shaner, N. C. et al. A bright monomeric green fluorescent protein derived from Branchiostoma lanceolatum. Nat. Methods 10, 407–409 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  65. Aaron, C. & Barker, C. C. Steric effects in di- and tri-arylmethane dyes. Part X. Electronic absorption spectra of bridged derivatives of malachite green and crystal violet. J. Chem. Soc. B Phys. Org. 1971, 319–324 (1971).

    Article  Google Scholar 

  66. 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).

    Article  CAS  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  68. Cooper, M. et al. Cy3B: improving the performance of cyanine dyes. J. Fluoresc. 14, 145–150 (2004).

    Article  CAS  PubMed  Google Scholar 

  69. Bindels, D. S. et al. mScarlet: a bright monomeric red fluorescent protein for cellular imaging. Nat. Methods 14, 53–56 (2017).

    Article  CAS  PubMed  Google Scholar 

  70. Mao, F., Leung, W.-Y. & Haugland, R. P. Sulfonated xanthene derivatives. US Patent 6,130,101 (2000).

  71. Zhou, M., Diwu, Z., Panchuk-Voloshina, N. & Haugland, R. P. A stable nonfluorescent derivative of resorufin for the fluorometric determination of trace hydrogen peroxide: applications in detecting the activity of phagocyte NADPH oxidase and other oxidases. Anal. Biochem. 253, 162–168 (1997).

    Article  CAS  PubMed  Google Scholar 

  72. Gao, W., Xing, B., Tsien, R. Y. & Rao, J. Novel fluorogenic substrates for imaging β-lactamase gene expression. J. Am. Chem. Soc. 125, 11146–11147 (2003).

    Article  CAS  PubMed  Google Scholar 

  73. Chu, J. et al. Non-invasive intravital imaging of cellular differentiation with a bright red-excitable fluorescent protein. Nat. Methods 11, 572–578 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  74. Vaughan, J. C., Dempsey, G. T., Sun, E. & Zhuang, X. Phosphine quenching of cyanine dyes as a versatile tool for fluorescence microscopy. J. Am. Chem. Soc. 135, 1197–1200 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  75. Michie, M. S. et al. Cyanine conformational restraint in the far-red range. J. Am. Chem. Soc. 139, 12406–12409 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  76. 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).

    Article  CAS  Google Scholar 

  77. 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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  78. Bachman, J. L., Pavlich, C. I., Boley, A. J., Marcotte, E. M. & Anslyn, E. V. Synthesis of carboxy ATTO 647N using redox cycling for xanthone access. Org. Lett. 22, 381–385 (2020).

    Article  CAS  PubMed  Google Scholar 

  79. Fu, M., Xiao, Y., Qian, X., Zhao, D. & Xu, Y. A design concept of long-wavelength fluorescent analogs of rhodamine dyes: replacement of oxygen with silicon atom. Chem. Commun. 2008, 1780–1782 (2008).

    Article  Google Scholar 

  80. 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).

    Article  CAS  PubMed  Google Scholar 

  81. Buschmann, V., Weston, K. D. & Sauer, M. Spectroscopic study and evaluation of red-absorbing fluorescent dyes. Bioconjugate Chem. 14, 195–204 (2003).

    Article  CAS  Google Scholar 

  82. Rodriguez, E. A. et al. A far-red fluorescent protein evolved from a cyanobacterial phycobiliprotein. Nat. Methods 13, 763–769 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  83. 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).

    Article  CAS  Google Scholar 

  84. Grimm, J. B. et al. A general method to optimize and functionalize red-shifted rhodamine dyes. Nat. Methods 17, 815–821 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  85. Luciano, M. P. et al. A nonaggregating heptamethine cyanine for building brighter labeled biomolecules. ACS Chem. Biol. 14, 934–940 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  86. Cosco, E. D. et al. Shortwave infrared polymethine fluorophores matched to excitation lasers enable non-invasive, multicolour in vivo imaging in real time. Nat. Chem. 12, 1123–1130 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  87. Shcherbakova, D. M., Cox Cammer, N., Huisman, T. M., Verkhusha, V. V. & Hodgson, L. Direct multiplex imaging and optogenetics of Rho GTPases enabled by near-infrared FRET. Nat. Chem. Biol. 14, 591–600 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  88. Matlashov, M. E. et al. A set of monomeric near-infrared fluorescent proteins for multicolor imaging across scales. Nat. Commun. 11, 239 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  89. Frank, W. G., Wenzel, M. S., Czerney, P. T., Desai, S. & Hermanson, G. Sulfonamide derivatives of xanthene compounds. US Patent 7,745,645 (2010).

  90. Mao, F., Leung, W.-Y., Cheung, C.-Y. & Hoover, H. E. Fluorescent dyes, fluorescent dye kits, and methods of preparing labeled molecules. US Patent 8,709,830 (2014).

  91. Thevathasan, J. V. et al. Nuclear pores as versatile reference standards for quantitative superresolution microscopy. Nat. Methods 16, 1045–1053 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  92. Bucevicius, J., Kostiuk, G., Gerasimaite, R., Gilat, T. & Lukinavicius, G. Enhancing the biocompatibility of rhodamine fluorescent probes by a neighbouring group effect. Chem. Sci. 11, 7313–7323 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  93. Verheijen, M. et al. DMSO induces drastic changes in human cellular processes and epigenetic landscape in vitro. Sci. Rep. 9, 4641 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  94. Grimm, J. et al. A general method to improve fluorophores using deuterated auxochromes. JACS Au. 1, 690–696 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  95. Butkevich, A. N., Bossi, M. L., Lukinavicius, G. & Hell, S. W. Triarylmethane fluorophores resistant to oxidative photobluing. J. Am. Chem. Soc. 141, 981–989 (2019).

    Article  CAS  PubMed  Google Scholar 

  96. Gidi, Y. et al. Unifying mechanism for thiol-induced photoswitching and photostability of cyanine dyes. J. Am. Chem. Soc. 142, 12681–12689 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  97. Zheng, Q. et al. Ultra-stable organic fluorophores for single-molecule research. Chem. Soc. Rev. 43, 1044–1056 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  98. Isselstein, M. et al. Self-healing dyes—keeping the promise? J. Phys. Chem. Lett. 11, 4462–4480 (2020).

    Article  CAS  PubMed  Google Scholar 

  99. 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  PubMed  PubMed Central  Google Scholar 

  100. Herner, A. et al. New generation of bioorthogonally applicable fluorogenic dyes with visible excitations and large Stokes shifts. Bioconjug. Chem. 25, 1370–1374 (2014).

    Article  CAS  PubMed  Google Scholar 

  101. Horvath, P., Sebej, P., Solomek, T. & Klan, P. Small-molecule fluorophores with large Stokes shifts: 9-iminopyronin analogues as clickable tags. J. Org. Chem. 80, 1299–1311 (2015).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  103. Lavis, L. D. What if we just give everything away? eLife 10, e74981 (2021).

    Article  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

We thank E. Schreiter for assistance with protein structures and E. Betzig, U. Boehm, X. Darzacq, B. English, H. Farrants, A. Hansen, Z. Liu, T. Lionnet, J. Lippincott-Schwartz, J. Schmidt and S.-H. Shu for contributive discussions. Related work in our laboratory is supported by the Howard Hughes Medical Institute. Chemical structures and spectral data were taken from references or obtained from vendor websites.

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    Jonathan B. Grimm & Luke D. Lavis

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J.B.G. and L.D.L. wrote and edited the manuscript.

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Correspondence to Luke D. Lavis.

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The authors declare the following competing financial interest: patents and patent applications describing azetidine-, fluorine- and deuterium-containing fluorophores (with inventors J.B.G. and L.D.L.) are assigned to Howard Hughes Medical Institute.

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Nature Methods thanks Lu Wang and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. 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.

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Grimm, J.B., Lavis, L.D. Caveat fluorophore: an insiders’ guide to small-molecule fluorescent labels. Nat Methods 19, 149–158 (2022). https://doi.org/10.1038/s41592-021-01338-6

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