A general method to improve fluorophores for live-cell and single-molecule microscopy

Abstract

Specific labeling of biomolecules with bright fluorophores is the keystone of fluorescence microscopy. Genetically encoded self-labeling tag proteins can be coupled to synthetic dyes inside living cells, resulting in brighter reporters than fluorescent proteins. Intracellular labeling using these techniques requires cell-permeable fluorescent ligands, however, limiting utility to a small number of classic fluorophores. Here we describe a simple structural modification that improves the brightness and photostability of dyes while preserving spectral properties and cell permeability. Inspired by molecular modeling, we replaced the N,N-dimethylamino substituents in tetramethylrhodamine with four-membered azetidine rings. This addition of two carbon atoms doubles the quantum efficiency and improves the photon yield of the dye in applications ranging from in vitro single-molecule measurements to super-resolution imaging. The novel substitution is generalizable, yielding a palette of chemical dyes with improved quantum efficiencies that spans the UV and visible range.

Figure 1. Development and utility of JF₅₄₉

(a) Spectroscopic data for rhodamines 1–7. (b) Jabłoński diagram showing the process of twisted internal charge transfer (TICT). (c) Synthesis of rhodamines 3–7 from fluorescein (8) using Pd-catalyzed cross-coupling. (d) Two-photon excitation spectra of fluorophores 2, 4–7. (e) Normalized absorption (abs) and fluorescence emission (fl) spectra for tetramethylrhodamine (2) and JF₅₄₉ (4). (f) Chemical structure of JF₅₄₉–HaloTag ligand 9 and TMR–HaloTag ligand 10. (g) Confocal maximum projection image of nucleus from a live, washed HeLa cell expressing HaloTag–H2B and incubated with JF₅₄₉–HaloTag ligand 9; scale bar = 5 μm. (h) Whisker plot comparing brightness and track length of HaloTag–H2B molecules labeled with ligand 9 or 10 (n > 4,000); cross indicates mean; whiskers span 10–90 percentile. (i) dSTORM fluorescence microscopy image of a fixed U2OS cell expressing HaloTag–H2B and labeled with JF₅₄₉ ligand 9. The dSTORM image is comprised of 10,000 consecutive frames and the 44,937 detected particles are displayed according to their localization FWHM. The mean localization error was 17.2 nm, the median localization error was 14.1 nm; scale bar = 5 μm.

Figure 2. Utility of JF₆₄₆ in cellular imaging

(a) Chemical structures of JF₆₄₆–HaloTag ligand 27 and SiTMR–HaloTag ligand 28. (b) dSTORM fluorescence microscopy image of fixed U2OS cells expressing HaloTag–H2B and labeled with 27. The dSTORM image is comprised of 5,000 consecutive frames and the 263,415 detected particles are displayed according to their localization FWHM. The mean localization error was 11.1 nm, the median localization error was 8.4 nm; scale bar = 5 μm. (c, d) Absorbance spectra of ligands 28 (5 μM; c) and 27 (5 μM; d) in the absence (−HT) and presence (+HT) of excess HaloTag protein. (e, f) Wide-field fluorescence microscopy image of a live HeLa cell transfected with H2B–HaloTag, incubated with 28 (100 nM; e) or 27 (100 nM; f), and imaged without intermediate washing steps; dashed line indicates cellular boundary; scale bars: 5 μm. (g) Plot of line scan intensity in e (green) as a function of line length. (h) Plot of line scan intensity in f (magenta) as a function of line length. (i) Chemical structure of JF₅₄₉–SnapTag ligand 29. (j) Overlay of the dSTORM image of H2B and regions of fast TetR diffusivity (2–10 μm² s⁻¹; yellow) and slow TetR diffusivity (<2 μm² s⁻¹; blue).

Figure 3. Utility of azetidinyl coumarins in cellular imaging

(a) Chemical structures of commercial coumarin SnapTag ligand 30 and azetidine-containing ligand 31. (b, d) Wide-field fluorescence microscopy images of live HeLa cells expressing SnapTag–H2B and labeled with DRAQ5 and commercial SnapTag ligand 30. (b) Fluorescence of DRAQ5 nuclear staining. (d) Fluorescence of coumarin 30-labeled SnapTag–H2B. (c, e) Wide-field fluorescence microscopy image of live HeLa cells expressing SnapTag–H2B and labeled with DRAQ5 and novel azetidinyl-coumarin SnapTag ligand 31. (c) Fluorescence of DRAQ5 nuclear staining. (e) Fluorescence of coumarin 31-labeled SnapTag–H2B. Scale bars for all images: 50 μm. (f) Quantification of the average nuclear fluorescence above background coumarin label in cells when labeled with ligand 30 (black) or 31 (magenta; error bars show s.e.m.).