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

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.

Figure 1. Fine-tuning rhodamine dyes

(a) Comparing coarse-tuning of λ_abs for dyes 1–4 and fine-tuning observed for azetidinyl rhodamines 5–12. (b) Correlation between calculated (DFT) and experimental λ_abs values for dyes 1, 5–12; dashed line shows ideal fit. (c) Correlation of experimental λ_abs vs. inductive Hammett constants (σ_I) for dyes 1, 5, 8–12. For the geminal disubstituted compounds 5 and 12 the σ_I of the substituent was doubled. Solid line shows linear regression (R² = 0.97). (d) Fine-tuning of the lactone–zwitterion equilibrium constant (K_L–Z) for dyes 1, 5, 9–12. (e) Normalized absorption vs. dielectric constant (ε_r) for dyes 1, 5, and 9–12; error bars show ± s.e.m; n = 4. (f) Absolute absorbance of 1, 5, 9–12 (5 μM) in 1:1 dioxane:H₂O. (g) Correlation of K_L–Z vs. inductive Hammett constants (σ_I) for dyes 1, 5, 9–12. For the geminal disubstituted compounds 5 and 12 the σ_I of the substituent was doubled. Solid line shows linear regression (R² = 0.91). (h) Chemical structure of JF₅₂₅–HaloTag ligand 13 and JF₅₄₉–HaloTag ligand 14. (i) Image of live, washed COS7 cells expressing histone H2B–HaloTag fusions and labeled with ligand 13. Scale bar: 35 μm. (j) Plot of percent labeling of histone H2B–HaloTag fusions in live cells vs. incubation time for ligands 13 (100 nM) and 14 (100 nm); error bars show ± s.e.m; n = 113–248 (see Methods).

Figure 2. Rational fine-tuning of other dyes

(a) Tuning of JF₅₁₉ (4) to yield JF₅₀₃ (16). (b) Structure of JF₅₀₃–HaloTag ligand 17. (c) Image of live, washed COS7 cells expressing histone H2B–HaloTag fusions and labeled with ligand 17. Scale bar: 35 μm. (d) Comparison of the photostability of cells labeled with 17 and cells labeled with 488 nm-excited dyes 18 and 19 (Supplementary Fig. 2d); the initial photobleaching measurements are fitted to a linear regression. (e) Tuning of JF₆₀₈ (2) to yield JF₅₈₅ (21). (f) Structure of HaloTag ligands derived from JF₆₀₈ (22) and JF₅₈₅ (23). (g) Absorbance of HaloTag ligands 22 and 23 in the presence (+HT) or absence (–HT) of excess HaloTag protein; n = 2. (h,i) Representative images of COS7 cells expressing HaloTag–histone H2B fusion and labeled with 250 nM of HaloTag ligands 22 and 23 for 1 h and imaged directly without washing. The image for each dye pair was taken with identical microscope settings, λ_ex = 594 nm. Numbers indicate mean signal (nuclear) to background (cytosol) ratio (S/B) in three fields of view. (h) JF₆₀₈ ligand 22 (S/B from n = 224 areas). (i) JF₅₈₅ ligand 23 (S/B from n = 235 areas). (j) Tuning of JF₆₄₆ (3) to yield JF₆₃₅ (25). (k) Structure of HaloTag ligands derived from JF₆₄₆ (26) and JF₆₃₅ (27) (l) Absorbance of HaloTag ligands 26 and 27 in the presence (+HT) or absence (−HT) of excess HaloTag protein (n = 2). (m,n) Representative images of COS7 cells expressing HaloTag–histone H2B fusion and labeled with 250 nM of HaloTag ligands 26 and 27 for 1 h and imaged directly without washing. The image for each dye pair was taken with identical microscope settings, λ_ex = 647 nm. Numbers indicate mean signal (nuclear) to background (cytosol) ratio (S/B) in three fields of view. (m) JF₆₄₆ ligand 26 (S/B from n = 175 areas). (n) JF₆₃₅ ligand 27 (S/B from n = 278 areas). Scale bars for h,i,m,n: 15 μm.

Figure 3. Labeling in tissue and in vivo

(a) SiMView light-sheet microscopy image (3D projection) of the central nervous system of a third instar Drosophila larva expressing myristoylated HaloTag protein in ‘Basin’ neurons (BNs) and stained with JF₆₃₅–HaloTag ligand (27); LBL: left brain lobe; VNC: ventral nerve cord; RBL: right brain lobe. Scale bar: 100 μm. (b) Zoom in of boxed area in panel a showing individual BN cell bodies. Scale bar: 20 μm. (c) Two-photon fluorescence excitation spectra of HaloTag conjugates (1 μM) from HaloTag ligands 13, 14, 17, 23, 26, and 27 in 10 mM HEPES buffer (pH 7.3). The two-photon excitation spectra for mCherry is shown for reference. (d) Ratio of JF₅₈₅ fluorescence to GCaMP6s epifluorescence at different time points after a single injection of JF₅₈₅–HaloTag ligand (23, 100 nmol) either intravenous (IV) or intraperitoneal (IP) into mice expressing HaloTag protein in either layer 4 (L4) or layer 5 (L5) cortical neurons; n = 3 fields of view; error bars show ± s.e.m. (e) Two-photon microscopy images of neurons in layer 5 of the visual cortex coexpressing GCaMP6s (green) and JF₅₈₅-labeled HaloTag (magenta) after IV injection of ligand 23 (t = 5 h). Scale bar: 100 μm. Yellow circles in the merged image indicate individual neurons as regions of interest (ROIs). (f) Raster plot of spontaneous neuronal activity in different ROIs (n = 61) before and after labeling with JF₅₈₅–HaloTag ligand (23). (g) Plot of average spontaneous neural activity in each ROI before and after labeling with JF₅₈₅–HaloTag ligand (23); central line shows mean; error bars show ± s.d.; no significant difference is observed between time points (one-way ANOVA: p = 0.95).