Rational Design of Fluorogenic and Spontaneously Blinking Labels for Super-Resolution Imaging

Abstract

Rhodamine dyes exist in equilibrium between a fluorescent zwitterion and a nonfluorescent lactone. Tuning this equilibrium toward the nonfluorescent lactone form can improve cell-permeability and allow creation of "fluorogenic" compounds-ligands that shift to the fluorescent zwitterion upon binding a biomolecular target. An archetype fluorogenic dye is the far-red tetramethyl-Si-rhodamine (SiR), which has been used to create exceptionally useful labels for advanced microscopy. Here, we develop a quantitative framework for the development of new fluorogenic dyes, determining that the lactone-zwitterion equilibrium constant (K _L-Z) is sufficient to predict fluorogenicity. This rubric emerged from our analysis of known fluorophores and yielded new fluorescent and fluorogenic labels with improved performance in cellular imaging experiments. We then designed a novel fluorophore-Janelia Fluor 526 (JF₅₂₆)-with SiR-like properties but shorter fluorescence excitation and emission wavelengths. JF₅₂₆ is a versatile scaffold for fluorogenic probes including ligands for self-labeling tags, stains for endogenous structures, and spontaneously blinking labels for super-resolution immunofluorescence. JF₅₂₆ constitutes a new label for advanced microscopy experiments, and our quantitative framework will enable the rational design of other fluorogenic probes for bioimaging.

Figure 1

Fluorogenicity of rhodamines. (a) Lactone–zwitterion equilibrium of SiR (1). (b) Mechanism of improved cell-permeability and fluorogenicity of 1. (c) Structures of Janelia Fluor dyes 2–7. (d) Absorption at λ_abs vs SDS concentration for 1 and 2 in 20 mM Na₂HPO₄, pH 7.0; error bars show ± s.e.m.; shading indicates [SDS] above the critical micelle concentration (c.m.c.). (e) Change in fluorescence over the basal fluorescence (ΔF/F₀) of HaloTag ligands 1_HTL–10_HTL upon labeling purified HaloTag protein vs the K_L–Z of the corresponding free dyes 1–10; solid line indicates a linear fit (R² = 0.90); shading indicates ΔF/F₀ = 5–10 and K_L–Z = 10^–2–10^–3.

Figure 2

Synthesis and testing of SiR₁₁₀. (a) Synthesis of SiR₁₁₀ (8). (b) Synthesis of SiR₁₁₀–HaloTag ligand (8_HTL). (c) Absorption spectra of 8_HTL (5 μM) in the absence (black) or presence (orange) of excess HaloTag protein. (d) Confocal image of U2OS cells expressing histone H2B–HaloTag fusion protein and labeled with 8_HTL. Scale bar: 20 μm. (e) Relative photostability of 8_HTL and JF₅₈₅–HaloTag ligand (5_HTL) in live cells.

Scheme 1. Syntheses of JF₅₅₂ and JF₅₄₉ Derivatives: (a) Synthesis of 9, (b) Synthesis of 9_HTL, and (c) Synthesis of 6_TMP and 9_TMP

Figure 3

JF₅₅₂ ligands show improved cell-permeability. Overlay of fluorescence and bright-field images of yeast cells expressing a histone H2A.Z–HaloTag fusion protein and labeled with 6_HTL (a) or 9_HTL (b). Overlay of fluorescence and bright-field images of U2OS cells expressing histone H2B–eDHFR fusion protein and labeled with 6_TMP (c) or 9_HTL (d). Scale bars for all images: 5 μm.

Figure 4

Synthesis and no-wash imaging of JF₅₂₆ ligands. Synthesis of JF₅₂₆ (a) and JF₅₂₆ (b) ligands. (c) Structures of JF₅₂₅ and JF₅₂₆–HaloTag and SNAP-tag ligands. Confocal images of COS7 cells expressing a histone H2B–HaloTag fusion protein and labeled with 500 nM JF₅₂₅–HaloTag ligand (7_HTL, d) or JF₅₂₆–HaloTag ligand (10_HTL, e). Confocal images of COS7 cells expressing histone H2B–SNAP-tag fusion protein and labeled with 1 μM JF₅₂₅–SNAP-tag ligand (7_STL, f) or JF₅₂₆–SNAP-tag ligand (10_STL, g). Scale bars for all images: 5 μm.

Figure 5

Extending the repertoire of JF₅₂₆ ligands. (a) Structures of JF₅₂₆ ligands. (b) Confocal image of live U2OS cells stained with JF₅₂₆–Hoechst (10_HST). (c) Confocal image of mouse primary hippocampal neurons stained with JF₅₂₆–Taxol (10_TXL) and JF₆₄₆–Hoechst (2_HST). (d) Confocal image of U2OS cells expressing histone-H2B–HaloTag fusion protein and labeled with JF₅₂₆–pepstatin A (10_PEP), JF₅₈₅–HaloTag ligand (5_HTL), and “SiR–tubulin” (1_TXL). All images were acquired without washing. Scale bars: 5 μm.

Figure 6

Advanced microscopy imaging using JF₅₂₆. (a) Confocal and SIM images of mouse primary hippocampal neurons stained with 10_PEP and JF₆₄₆–Hoechst (2_HST). (b) Confocal and STED microscopy images of U2OS cells stained with 10_TXL. (c) Three-color live-cell STED image of U2OS cells expressing Sec61β–SNAP-tag labeled with JF₆₄₆–SNAP-tag ligand (2_STL), TOMM20–HaloTag labeled with JF₅₈₅–HaloTag ligand (5_HTL), and microtubules stained with 10_TXL. (d) Lattice light-sheet microscopy image of U2OS cells stained with 10_PEP and 2_HST. Scale bars for all images: 5 μm.

Figure 7

Localization microscopy with HM-JF₅₂₆. (a) Blinking behavior of HM-SiR (32). (b) Synthesis of HM-JF₅₂₆ NHS (37). Immunofluorescence images of tubulin labeled with a 37–antibody conjugate: (c) SMLM image, (d) diffraction-limited image. (e) Transverse profiles of fluorescence intensity corresponding to boxed regions in parts c and d. Immunofluorescence images of TOMM20 labeled with a 37–antibody conjugate: (f) SMLM image; (g) diffraction-limited image. (h) Transverse profiles of fluorescence intensity corresponding to boxed regions in parts f and g. Solid lines in parts e and h indicate Gaussian fits; numbers indicate the full width at half-maximum (fwhm) determined by the Gaussian fits of the SMLM (green) and diffraction-limited imaging (black). Scale bars for all images: 5 μm.