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A general method to fine-tune fluorophores for live-cell and in vivo imaging

Nature Methods volume 14, pages 987–994 (2017)Cite this article

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Abstract

Pushing the frontier of fluorescence microscopy requires the design of enhanced fluorophores with finely tuned properties. We recently discovered that incorporation of four-membered azetidine rings into classic fluorophore structures elicits substantial increases in brightness and photostability, resulting in the Janelia Fluor (JF) series of dyes. We refined and extended this strategy, finding that incorporation of 3-substituted azetidine groups allows rational tuning of the spectral and chemical properties of rhodamine dyes with unprecedented precision. This strategy allowed us to establish principles for fine-tuning the properties of fluorophores and to develop a palette of new fluorescent and fluorogenic labels with excitation ranging from blue to the far-red. Our results demonstrate the versatility of these new dyes in cells, tissues and animals.

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Figure 1: Fine-tuning rhodamine dyes.
Figure 2: Rational fine-tuning of other dyes.
Figure 3: Labeling in tissue and in vivo.

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Acknowledgements

We thank A. Berro and E. Schreiter (Janelia) for purified HaloTag protein, and H. Choi (Janelia) for the Sec61β-HaloTag plasmid, contributive discussions and critical reading of the manuscript. This work was supported by the Howard Hughes Medical Institute.

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Authors and Affiliations

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

    Jonathan B Grimm, Anand K Muthusamy, Yajie Liang, Timothy A Brown, William C Lemon, Ronak Patel, Rongwen Lu, John J Macklin, Philipp J Keller, Na Ji & Luke D Lavis

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  1. Jonathan B Grimm
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Contributions

L.D.L. and J.B.G. conceived the project. J.B.G. contributed organic synthesis and one-photon spectroscopy measurements. A.K.M. contributed organic synthesis and computational chemistry experiments. Y.L., R.L. and N.J. contributed mouse imaging experiments. T.A.B. contributed cultured cell imaging experiments. W.C.L. and P.J.K. contributed larval explant imaging experiments. R.P. and J.J.M. contributed two-photon spectroscopy measurements. L.D.L. contributed one-photon spectroscopy measurements and wrote the manuscript with input from the other authors.

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Correspondence to Luke D Lavis.

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The authors declare competing interests: J.B.G. and L.D.L. have filed patent applications whose value may be affected by this publication.

Integrated supplementary information

Supplementary Figure 1 Spectral data for fluorophores 1–12, 16, 21, and 25.

Normalized absorbance (abs), fluorescence excitation (flex), and fluorescence emission (flem) spectra for rhodamines (1, 5–12), rhodols (4, 16) carborhodamines (2, 21), and Si‑rhodamines (3, 25) in 10 mM HEPES, pH 7.3; the flex spectra are delineated by a dashed line. Note: the normalized absorbance spectra of fluorophores 3, 21, and 25 exhibit higher noise due to low visible absorption in aqueous buffer.

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Supplementary Figure 2 Labeling cells with Janelia Fluor dyes.

(a) Chemical structure of JF525–SNAP-tag (15). (b) Image of COS7 cells expressing SNAP-tag–histone H2B and stained with ligand 15. (c) HaloTag and SNAP-tag ligands have no effect on COS7 cell viability at concentrations used for labeling: HaloTag ligands 13, 14, 17, 23, 26, 27 were incubated with cells for 1 h; SNAP-tag ligands 15, 20, 24, 29 were incubated for 3 h; error bars show ± s.d.; n = 3. (d) Chemical structures of known 488 nm-excited HaloTag ligands 18 and 19. (e) Plot of average cellular fluorescence vs. incubation time for live cells loaded with ligands 17–19. (f) Chemical structure of JF503–SNAP-tag ligand (20). (g) Image of COS7 cells expressing SNAP-tag–histone H2B and stained with ligand 20. (h) Structure of JF585–SNAP-tag ligand (24). (i) Image of COS7 cells expressing histone H2B–SNAP-tag and stained with ligand 24. (j) Multicolor image of U2OS cells expressing Sec61β–HaloTag fusion (stable) and TOMM20–SNAP-tag (transient) labeled with JF503–SNAP-tag ligand 20 (mitochondria, green), JF585-HaloTag ligand 23 (ER, orange), and JF646–Hoechst33 (nucleus, red). (k) Chemical structure of SiTMR–HaloTag ligand 28. (l) Images of COS7 cells expressing HaloTag–histone H2B fusion and labeled with 250 nM of HaloTag ligand 28 for 1 h and imaged directly without washing. The number indicates mean signal (nuclear) to background (cytosol) ratio (S/B) in three fields of view (n = 152 areas). This image was taken with identical microscope settings to those used with ligands 26 and 27 (Fig. 2m,n). (m) Chemical structure of JF635–SNAP-tag ligand (29). (n) Image of COS7 cells expressing SNAP-tag–histone H2B and stained with ligand 29. (o) Multicolor image of U2OS cells expressing Sec61β–HaloTag fusion (stable) and histone H2B–SNAP-tag (transient) labeled with JF525–SNAP-tag ligand 15 (nucleus, yellow) and JF635–HaloTag ligand 27 (ER, red). Scale bars for all images: 15 μm.

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Supplementary Figure 3 In vivo imaging using the Janelia Fluor dyes

(a–b) Comparison of Basin cell and pan-neronal labeling in tissue. (a) SiMView light-sheet microscopy image of the ventral nerve cord region of Drosophila larval explant expressing HaloTag protein in Basin neurons and stained with JF635–HaloTag ligand (27; same imaging data set as Fig. 3a). Lower panel shows image of the anteroposterior (AP) cross-section of the indicated volume. (b) SiMView light-sheet microscopy image of ventral nerve cord region of Drosophila larval explant expressing GCaMP6s protein pan-neuronally (Gal4/UAS system; 57C10-Gal4 driver line). Lower panel shows image of the AP cross-section of the indicated volume. Scale bars for a and b: 50 μm. (c) SiMView light-sheet microscopy image (same as Fig. 3a) with inset showing a single imaging slice from the 3D projection through neuronal cell bodies. (d) Representative images from the labeling time course for JF585–HaloTag ligand (23) in vivo. Bright field image showing cranial window and epi-fluorescence images of green (GCaMP6s; t = 0) and red (JF585, t = 0 and 6 h. Scale bar: 0.5 mm. (e) Plot of GCaMP6s green fluorescence vs. JF585 red fluorescence for 2-photon imaging experiments. Found: Pearson linear correlation coefficient (ρ) = 0.768; n = 106 regions of interest (ROIs).

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In vivo labelling of layer 5 cortical neurons using JF585–HaloTag ligand

Layer 5 neurons expressing GCaMP6s and HaloTag were labeled with JF585–HaloTag ligand (23) through intraperitoneal (IP) injection and imaged with two-photon fluorescence microscopy. JF585 was excited at 1100 nm and the stack (307 μm × 307 μm × 530 μm) was acquired from 50 to 580 μm below dura mater at 2 μm step in Z. 3D movie was made by the ImageJ 3D view plugin (unit is in μm).

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Grimm, J., Muthusamy, A., Liang, Y. et al. A general method to fine-tune fluorophores for live-cell and in vivo imaging. Nat Methods 14, 987–994 (2017). https://doi.org/10.1038/nmeth.4403

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