Bright photoactivatable fluorophores for single-molecule imaging

Abstract

Small-molecule fluorophores are important tools for advanced imaging experiments. We previously reported a general method to improve small, cell-permeable fluorophores which resulted in the azetidine-containing 'Janelia Fluor' (JF) dyes. Here, we refine and extend the utility of these dyes by synthesizing photoactivatable derivatives that are compatible with live-cell labeling strategies. Once activated, these derived compounds retain the superior brightness and photostability of the JF dyes, enabling improved single-particle tracking and facile localization microscopy experiments.

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Figure 1: Synthesis, characterization, and utility of photoactivatable Janelia Fluor 549 (PA-JF549).
Figure 2: Multicolor imaging using photoactivatable Janelia Fluor 646 (PA-JF646).

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Acknowledgements

We thank W. Legant and E. Betzig for contributive discussions; H. White, D. Walpita, K. Schaefer, and P. Nguyen for assistance with molecular biology and cell culture; and A. Berro, A. Abdelfattah, and E. Schreiter (all at Janelia) for the purified HaloTag and SNAP-tag proteins. This work was supported by the Howard Hughes Medical Institute.

Author information

Affiliations

Authors

Contributions

J.B.G., B.P.E., Z.L., T.L., and L.D.L. conceived the project. J.B.G. contributed organic synthesis and photochemistry experiments; B.P.E., H.C., B.P.M., P.D., and T.A.B. contributed cell biological experiments and data analysis; A.K.M. contributed organic synthesis; J.L.-S., Z.L., T.L., and L.D.L. directed the project and contributed data analysis. L.D.L. wrote the paper with input from the other authors.

Corresponding authors

Correspondence to Timothée Lionnet or Luke D Lavis.

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Competing interests

J.B.G., B.P.E., A.K.M., Z.L., T.L., and L.D.L. have filed patent applications (e.g., PCT/US2015/023953) whose value may be affected by this publication.

Integrated supplementary information

Supplementary Figure 1 Properties and performance of photoactivatable Janelia Fluor 549 (PA-JF549).

(a) Absorbance spectrum of PA‑JF549-HaloTag ligand (6) bound to HaloTag protein before (–hv, black line) and after photoactivation (365 nm; +hv, magenta lines). (b) Structure of JF549-HaloTag ligand (8) and TMR‑HaloTag ligand (9). (c) Comparison of the absolute absorbance spectrum of 8 (5 μM) incubated with HaloTag protein (black) with the absorbance spectrum of 6 (5 μM) incubated with (magenta) or without (dashed magenta) HaloTag protein and then exhaustively photoactivated with 365 nm light. (d) Absorbance spectrum of PA‑JF549-SNAP-tag ligand (7) bound to HaloTag protein before (–hv, black line) and after photoactivation (365 nm; +hv, magenta lines). (e) Structure of JF549-SNAP-tag ligand (10). (f) Comparison of the absolute absorbance spectrum of 10 (5 μM) incubated with SNAP-tag protein with the absorbance spectrum of 7 (5 μM) incubated with (magenta) or without (dashed magenta) SNAP-tag protein and then exhaustively photoactivated with 365 nm light. (g) Activation rates of histone H2B–HaloTag labeled with PA‑JF549-HaloTag ligand (6) or histone H2B–mEos3.2 in live U2OS cells under identical imaging conditions (n=5, shading shows ± s.e.m.) overlaid with exponential fit (black). (h) Plot comparing the spontaneous activation of TOMM20–HaloTag labeled with PA-JF549-HaloTag ligand (6; magenta) or TOMM20–mEos3.2 (black) in fixed U2OS cells in the absence of 405 nm activation light; solid lines show linear fits. (i) Histogram of detected photons/localization using TOMM20–HaloTag labeled with PA-JF549-HaloTag ligand (6) or TOMM20–mEos3.2 in fixed U2OS cells. (j) Cartoon showing experimental workflow of spt-dSTORM experiment (top) and sptPALM experiment (bottom). ES cells expressing HaloTag–Sox2 were labeled to saturation with PA-JF549-HaloTag ligand 6 or TMR-HaloTag ligand 8 and imaged on a multifocus microscope, which allows simultaneous imaging of 9 focal planes across an axial depth of ~4 μm. (k) Plot of the number of single molecule trajectories measured per frame in ES cell expressing HaloTag–Sox2 labeled with PA-JF549 ligand 6 (sptPALM mode, magenta) or the commercial TMR-HaloTag ligand (9; spt-dSTORM mode, black); n = 5 cells for each ligand; s.d. shown in gray. (lm) Image of cumulative single-particle tracks imaged for frames 2000–2500 (between dashed lines in k; only trajectories observed in >5 successive frames shown); lower left: number of trajectories measured; lower right: scale bars: 2 μm. (l) Cell labeled with PA-JF549 ligand 6 (1544 trajectories). (m) Cell labeled with standard TMR ligand 9 (191 trajectories). (no) Statistics from 3D tracking experiments (shown in jm) in ES cells expressing HaloTag–Sox2 and labeled with 6 or 9. (n) Histogram of detected photons/particle/frame using labels 6 (magenta) or 9 (black). (o) Histogram of particle localization/frame using labels 6 (magenta) or 9 (black). (p) PALM image of fixed U2OS cells expressing TOMM20–mEos3.2 fusions. Scale-bar: 2 μm. The 195,422 detected molecules are displayed according to their localization full-width at half-maximum. The median calculated localization error was 20.2 nm. (q) Zoom-in of PALM image of fixed U2OS cell expressing clathrin–HaloTag fusions and labeled with ligand 6. The full-chip image is composed of 1,048,575 drift-corrected localizations with a median localization error of 10.8 nm. The data shows the expected ring structure of clathrin; each subpanel is 1 μm2. (r) PALM image of U2OS cell expressing ensconsin–HaloTag fusions and labeled with ligand 6. The image is composed of 445,242 localizations with a median calculated localization error of 28.7 nm. Scale-bar: 5 μm. (s) PALM image of U2OS cell expressing Sec61β–HaloTag fusions and labeled with ligand 6. The image is composed of 29,356 localizations with a median calculated localization error of 20.9 nm. Scale bar: 5 μm. (tv) Analysis of blinking behavior of PA-JF549 during PALM of ensconsin–HaloTag shown in r. (t) Histogram of the duration of individual blinking events (magenta), overlaid with the fit to an exponential distribution (black). (u) Histogram of the duration of intervals between blinking events (magenta), overlaid with the fit to an exponential distribution (black). (v) Histogram of the number of blinking events per molecule (magenta), overlaid with the fit to a geometric distribution (black). Source data

Supplementary Figure 2 Synthesis, properties, and performance of photoactivatable Janelia Fluor 646 (PA-JF646).

(a) Synthesis and photochemistry of PA-JF646. Treatment of JF646 (11) with oxalyl chloride and TMS diazomethane yields PA-JF646 (12). Photoactivation (365 nm) yields only a trace amount of both the phenylacetic derivative (13; <1%) and methyl-substituted JF549 (14; 4%); the major product is the indanone 15 (24%; isolated yields). (b) Structure of JF646-HaloTag ligand 18 and JF646-SNAP-tag ligand 19. (c) Absolute absorbance spectrum of PA‑JF646-HaloTag ligand (16) bound to HaloTag protein before (–hv, black line) and after photoactivation (365 nm; +hv, magenta lines). (d) Comparison of the absolute absorbance spectrum of 18 (5 μM) incubated with HaloTag protein (black) with the absorbance spectrum of 16 (5 μM) incubated with (magenta) or without (dashed magenta) HaloTag protein and then exhaustively photoactivated with 365 nm light. (e) Comparison of the absolute absorbance spectrum of 19 (5 μM) incubated with SNAP-tag protein (black) with the absorbance spectrum of 17 (5 μM) incubated with (magenta) or without (dashed magenta) SNAP-tag protein and then exhaustively photoactivated with 365 nm light. (f) Activation rate of histone H2B–HaloTag labeled with PA-JF646-HaloTag ligand (6) in live U2OS cells (identical photoactivation conditions and equivalent excitation power to Supplementary Fig. 1g; n=5, shading shows ± s.e.m.) overlaid with exponential fit (black). (g) Plot comparing the spontaneous activation of TOMM20–HaloTag labeled with PA-JF646-HaloTag ligand (16; magenta) or TOMM20–mEos3.2 (black) in fixed U2OS cells in the absence of 405 nm activation light; solid lines show linear fits. (hi) Evaluation of background staining of HaloTag ligands 6 and 16. COS7 cells expressing no fusion protein (left panels) or a histone H2B–HaloTag fusion (right panels) were fixed, stained with HaloTag ligand (100 nM; 30 min) and Hoechst 33342 (5 μg/mL), photoactivated (405 nm), and then imaged using the same settings. Scale bars: 50 μm. (h) PA-JF549-HaloTag ligand 6. (i) PA-JF646-HaloTag ligand 16. (jk) Two live ES cells expressing histone H2B–SNAP-tag labeled with PA-JF646-SNAP-tag ligand (17) and HaloTag–Sox2 labeled with PA-JF549-HaloTag ligand (6). Upper images show localization microscopy image (PALM), center images show cumulative single-particle trajectories, and lower images show apparent diffusion coefficient map. Scale bar: 5 μm. (j) Images from histone H2B–SNAP-tag labeled with 17. (k) Images from HaloTag–Sox2 labeled with 6. (l) PALM image of U2OS cell expressing vimentin–HaloTag fusions and labeled with ligand 16. The image is composed of 151,808 localizations with a median calculated localization error of 20.4 nm. Scale-bar: 5 μm. (m) PALM image of U2OS cell expressing TOMM20–HaloTag fusions and labeled with ligand 16. The PALM image is composed of 203,015 detected molecules. The median calculated localization error was 13.8 nm. Scale-bar: 2 μm. (n) Histogram of calculated localization precision when performing PALM of TOMM20 using the 16–HaloTag fusion (magenta, median = 13.8 nm) or mEos3.2 fusion (black, median = 20.2 nm). Source data

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1 and 2, Supplementary Tables 1 and 2, and Supplementary Note. (PDF 2805 kb)

One-photon activation of PA-JF549 in live cells

One-photon activation (405 nm) of an ES cell expressing HaloTag-Sox2 labeled with PA-JF549-HaloTag ligand. (MOV 5657 kb)

Two-photon activation of PA-JF549 in live cells

Two-photon activation (800 nm; spelling “HHMI”) in a HeLa cell expressing histone H2B-HaloTag labeled with PA-JF549-HaloTag ligand followed by full-field one-photon activation (405 nm). (MOV 2264 kb)

Activation of PA-JF549 with and without activation light

A fixed U2OS cell expressing ensconsin-HaloTag labeled with PA-JF549-HaloTag ligand and imaged in PALM mode with constant excitation light (561 nm) without activation light (405 nm, 1000 frames; “OFF”) and with activation light (405 nm, 1000 frames; “ON”). (MOV 4236 kb)

mEos3.2 vs. PA-JF dyes in sptPALM experiments

Comparison of sptPALM in ES cells expressing mEos3.2–Sox2 or HaloTag–Sox2 labeled with JF549 or JF646. (MOV 6383 kb)

PA-JF549 vs. TMR in single-particle tracking experiments

Side-by-side comparison of 3D single-particle tracking (spt) experiments in an ES cell expressing HaloTag–Sox2 labeled with TMR HaloTag ligand (left) in spt-dSTORM mode or PA-JF549 (right) in sptPALM mode. (MOV 4329 kb)

3D PALM imaging using PA-JF549

3D PALM imaging of cell expressing Sec61β-HaloTag fusions and labeled with PA-JF549-HaloTag ligand. (MOV 1164 kb)

One-photon activation of PA-JF646 in live cells

One-photon activation (405 nm) of an ES cell expressing GFP-HP1 (green) and HaloTag-Sox2 labeled with PA-JF646-HaloTag ligand (magenta). (MOV 5770 kb)

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Grimm, J., English, B., Choi, H. et al. Bright photoactivatable fluorophores for single-molecule imaging. Nat Methods 13, 985–988 (2016). https://doi.org/10.1038/nmeth.4034

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