Enhancing the biocompatibility of rhodamine fluorescent probes by a neighbouring group effect

Abstract

Fluorescence microscopy is an essential tool for understanding dynamic processes in living cells and organisms. However, many fluorescent probes for labelling cellular structures suffer from unspecific interactions and low cell permeability. Herein, we demonstrate that the neighbouring group effect which results from positioning an amide group next to a carboxyl group in the benzene ring of rhodamines dramatically increases cell permeability of the rhodamine-based probes through stabilizing a fluorophore in a hydrophobic spirolactone state. Based on this principle, we create probes targeting tubulin, actin and DNA. Their superb staining intensity, tuned toxicity and specificity allows long-term 3D confocal and STED nanoscopy with sub-30 nm resolution. Due to their unrestricted cell permeability and efficient accumulation on the target, the new probes produce high contrast images at low nanomolar concentrations. Superior performance is exemplified by resolving the real microtubule diameter of 23 nm and selective staining of the centrosome inside living cells for the first time.

Fig. 1. Synthesis scheme of 4-TMR-COOH, 4-580CP-COOH, 4-610CP-COOH and 4-SiR-COOH dyes and conjugates 22–42. Their photophysical properties are depicted in the colour spectrum chart and the neighbouring group effect is highlighted.

Fig. 2. Neighbouring group effect in the fluorescent probes. (a) ^DyeD₅₀ values of positional isomers of TMR-COOH and ^probeD₅₀ of TMR-LTX. (b) DFT optimized geometries of a model 4′-regioisomer fluorescent probe in spirolactone and zwitterion forms with a truncated linker and targeting moiety. (c) DFT calculated potential energy differences between the spirolactone and zwitterion of model 4′/5′/6′-regioisomer probes in 1,4-dioxane and water environment. (d) Chemical shifts of the amide proton of TMR-LTX regioisomeric probes. (e) Comparison of the retention times of TMR-LTX regioisomeric probes in HPLC analysis with an SB-C18 column under isocratic elution conditions (75 : 25 MeOH : H₂O, 25 mM HCOONH₄, pH = 3.6).

Fig. 3. Imaging performance of fluorescent probes based on rhodamine isomers. (a) Wide-field fluorescence microscopy of living primary fibroblasts stained with 100 nM TMR-LTX isomers for 1 h at 37 °C. Cells were washed once with HBSS and imaged in DMEM growth media. Inset shows zoomed-in images. Scale bars: 100 μm (large field of view), 10 μm (inset). Hoechst staining is shown in cyan and all tubulin probes are in magenta. (b) Quantification of fluorescence signal in the cytoplasm of living cells stained with tubulin probes. Data are presented as mean ± s.e.m., N = 3 independent experiments, each time n > 100 cells were quantified. (c) Cytotoxicity of tubulin fluorescent probes presented as half maximal effective concentration (EC₅₀) after 24 h incubation at 37 °C in growth media. Cytotoxicity was determined as the fraction of cells containing less than a single set of genetic material (sub G1 DNA content). Data are presented as mean ± s.e.m., N = 3 independent experiments, each time n > 100 cells were quantified. (d) Wide-field microscopy images of living primary fibroblasts. The cells were stained with 100 nM 4/5/6-580CP-Hoechst (magenta) or 100 nM 4/5/6-610CP-JAS (yellow) for 1 h at 37 °C, washed once with HBSS and imaged in DMEM media. Insets show zoomed-in images. Scale bars: 100 μm (large field of view), 10 μm (insets). Overlay with phase contrast (grey) images are shown. (e) Quantification of DNA probe fluorescence signal in the nuclei. Data are presented as mean ± s.d., N = 3 independent experiments, each n > 100 cells. (f) Quantification of 4/5/6-610CP-JAS fluorescence signal in the cytoplasm of living cells. Data are presented as mean ± s.d., N = 3 independent experiments, each n > 100 cells.

Fig. 4. Confocal and Airyscan imaging of living cells stained with rhodamine 4′-isomer probes. (a) Single microtubule and cytoplasm fluorescence signal ratio in living human fibroblasts stained with 100 nM of the indicated probe for 1 h at 37 °C and imaged without probe removal. Data are presented as mean ± s.d., N ≥ 3 independent fields of view, each time n ≥ 20 microtubules. (b) Zeiss Airyscan images of human primary fibroblasts stained with 5-SiR-Hoechst and 4-TMR-LTX for 24 h at 37 °C in growth media at indicated concentrations. Images acquired without probe removal. (c) Cell cycle of human primary fibroblasts stained with 1 nM 4-TMR-LTX (yellow) and 10 nM 5-610CP-Hoechst (magenta). Scale bar: 10 μm. Overlay with phase contrast (grey) images are shown. (d) Three-colour ZEISS Airyscan image of living HeLa cells at the metaphase stained with 3 nM 4-TMR-LTX (yellow), 20 nM 5-SiR-Hoechst (magenta) and 1000 nM 6-510R-JAS (cyan). Scale bar: 1 μm.

Fig. 5. STED nanoscopy imaging of living cells stained with rhodamine 4′-isomer probes. (a) Confocal and STED images of microtubules in human fibroblasts taken with 0.25 AU pinhole. The cells were stained with 100 nM 4-610CP-CTX for 1 h at 37 °C. Scale bar: 1 μm. (b) Fluorescence intensity profile at the rectangle in panel a. Insets show measured (mean ± s.d., N = 20) and predicted diameter from the cryo-electron microscopy model of tubulin (orange) bound to paclitaxel (blue). (c) Apparent microtubule FWHM measured by different microscopy methods. Human fibroblasts stained with 100 nM probes for 1 h at 37 °C. * – diameter measured between two peaks of the fitted intensity profile. Data presented as mean ± s.d., N ≥ 3 independent fields of view, each time n ≥ 10 microtubules. (d) Nine-fold symmetry of centriole resolved in the deconvolved STED DyMIN image of U-2 OS cell stained with 1 μM 4-610CP-CTX for 1 h at 37 °C. White dashed lines mark a second centriole. Scale bar: 1 μm. (e) Deconvolved STED image of human female fibroblast nucleus stained with 100 nM 4-580CP-Hoechst showing the inactivated X chromosome (Xi). Insets – zoomed-in confocal and STED images of the Xi region. Scale bars: 500 nm (insets), 1 μm (main image). (f) Two-colour STED no-wash image of human fibroblasts stained with 100 nM 4-610CP-JAS and 10 nM 4-TMR-LTX for 1 h at 37 °C. Scale bar: 10 μm.