A general method to optimize and functionalize red-shifted rhodamine dyes

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.

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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.

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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.

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Authors

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.

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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.

ab, 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. fi, 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. ce, 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. fh, 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 1520. bg, 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 15HTL20HTL and 37HTL38HTL starting with nucleophilic aromatic substitution (SNAr) of 1520 and 3738 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). de, 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 15HTL20HTL, and 37HTL38HTL. 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). bc, 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

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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

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