Extended Data Fig. 7. Further fine-tuning of JF₅₇₁ (19) and JF₇₂₂ (17).

(a) Structure of JF₅₇₁ (19) and JF₅₅₉ (37). (b–c) Full plot of K_L–Z vs. λ_abs (b) and zoom-in (c) showing decreased K_L–Z for dye 37. (d) Fluorescence excitation (ex) and emission (em) spectra of JF₅₅₉ (37). (e) Structure of JF₅₅₉–HaloTag ligand (37_HTL). (f) Widefield imaging experiment of U2OS cells expressing HaloTag–histone H2B labeled with 37_HTL (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 17_HTL (200 nM) or 38_HTL (200 nM) to live cells expressing HaloTag–histone H2B; error bars indicate SE; n = 100 nuclei except for: t = 0.5 h with 38_HTL where n = 86 nuclei; t = 1 h with 38_HTL where n = 94 nuclei; t = 2 h with 38_HTL where n = 96 nuclei; t = 0.5 h with 17_HTL where n = 94 nuclei. (i) Plot of fluorescence from cells expressing HaloTag–H2B labeled with 17_HTL (200 nM) or 38_HTL (200 nM) over 30 bleach cycles; error bars indicate SE; ; n = 3 independent cellular samples.