A unique approach toward near-infrared fluorescent probes for bioimaging with remarkably enhanced contrast

Abstract

Near-infrared (NIR) fluorescent probes are attractive molecular tools for bioimaging because of their low autofluorescence interference, deep tissue penetration, and minimal damage to sample. However, most previously reported NIR probes exhibit small Stokes shift, typically less than 30 nm, and low fluorescence quantum yield, strictly limited contrast and spatial resolution for bioimaging. Herein, by expanding the π-conjugated system of rhodamine B, while, at the same time, keeping its rigid and planar structure, we reported an efficient NIR dye, HN7, with large stokes shift of 73 nm and fluorescence quantum yield as high as 0.72 in ethanol, values superior to those of such traditional cyanine NIR dyes as Cy5. Using HN7, living cells, tissues and mice were imaged, and the results showed significantly enhanced contrast, improved spatial resolution, and satisfactory tissue imaging depth when compared to Cy5. Moreover, the nonfluorescent spirocyclic structure of rhodamine B is an inherent component of HN7; therefore, our strategy provided a universal platform for the design of efficient NIR turn-on bioimaging probes for various targets. As a proof-of-concept, two different NIR probes, HN7-N2 and HN7-S for NO and Hg²⁺, respectively, were designed, synthesized, and successfully applied for the imaging of NO and Hg²⁺ in living cells, tissues and mice, respectively, demonstrating the potential bioimaging applications of the new probes. In sum, this new type of dye may present new avenues for the development of efficient NIR fluorescent probes for contrast-enhanced imaging in biological applications.

Scheme 1. Synthesis of HN1–7, HN7-N2 and HN7-S; structures of classical rhodamine B and Cy5. Conditions: (a) H₂SO₄, Δ, then 70% HClO₄; (b) NaH, Δ; (c) DCC/DMAP or POCl₃/TEA; (d) Lawesson's reagent.

Fig. 1. Absorption (a) and emission (b) spectra of HN6 (navy-blue triangle), HN7 (purple triangle) in pH 7.4 PBS buffer. The spectra of rhodamine B (wine hexagon) and Cy5 (olive-green star) are also shown for comparison. (c) Color and fluorescence images of rhodamine B, HN1–7, and Cy5 in pH 7.4 PBS buffer (3.0 μM, containing 10% EtOH).

Fig. 2. (a) Photostability of HN2 (black), HN4 (red), HN6 (blue) and HN7 (magenta) in pH 7.4 PBS buffer (containing 10% EtOH). The samples were continuously irradiated by a Xe lamp (150 W) at 5 nm slit width at the maximal absorption wavelength of HN2, HN4, HN6, and HN7. (b) Chemical stability of HN2, HN4, HN6, and HN7 in pH 7.4 PBS buffer (containing 10% EtOH). The samples were reacted with various ROS (100 μM) for 120 min (black: HOCl, red: H₂O₂, blue: TBHP, olive-green: HO·, cyan: tBuO·).

Fig. 3. (a) The proposed structures and the putative transformations of HN7 and HN7-N2 at various pH values. (b) pH-dependence of the normalized fluorescence intensity of HN7 (red circle) and HN7-N2 (black square).

Fig. 4. (a) DFT optimized structure of HN7. In the ball-and-stick representation, carbon, nitrogen, oxygen, and hydrogen atoms are colored in gray, blue, red, and white, respectively. (b) Molecular orbital plots (LUMO and HOMO) and HOMO/LUMO energy gaps of HN7.

Fig. 5. Confocal fluorescence images of HeLa cells incubated with various dyes at 37 °C for 60 min (from left to right: 0, 1, 3, 10 μM): (a–d) excitation at 543 nm for red emission of rhodamine B (560–630 nm); (e–h) excitation at 543 nm for red emission of HN7 (650–720 nm); (i–l) excitation at 635 nm for red emission of HN7 (650–720 nm); (m–p) excitation at 543 nm for red emission of Cy5 (650–720 nm); (q–t) excitation at 635 nm for red emission of HN7 (650–720 nm). Scale bar = 30 μm. (u) Relative fluorescence intensity obtained from the selected ellipse region of (a–t), respectively, averaged and plotted as a ratio to blank (a, e, i, m, q).

Fig. 6. Depth fluorescence images of Cy5 ((a), 5 μM) and HN7 ((b), 5 μM) in tissues. The changes of fluorescence intensity with scan depth were determined by spectral confocal microscopy (Olympus, FV1000) in the z-scan mode (from 0 to 150 μm; step size: 1 μm). The images were collected at 650–720 nm (red channel). Scale bar = 120 μm.

Fig. 7. Fluorescent images of mice (pseudocolor). Thirty minutes after dye injection, mice were imaged using a Caliper VIS Lumina XR small animal optical in vivo imaging system with an excitation filter 605 nm and a Cy5.5 emission filter. (a) The blank group; (b) the Cy5-incubated group; (c) the HN7-incubated group; (d) quantification of fluorescence emission intensity from the selected circle region of (a–c) was averaged and plotted as a ratio to blank.

Scheme 2. (a) NO-induced ring opening of HN7-N2. (b) Hg²⁺-induced ring opening of HN7-S.

Fig. 8. (a) Fluorescence spectra of HN7-N2 (5 μM) in the presence of various concentrations of NO (from diethylamine NONOate sodium salt, 0–100 μM) in PBS buffer (0.01 M, pH 7.4, containing 10% EtOH) with excitation at 600 nm. Inset: change in color (i) and fluorescence (ii) in the absence of NO (left) and in the presence of 100 μM NO (right). (b) Fluorescence spectra of HN7-S (5 μM) in the presence of various concentrations of Hg²⁺ (0–10 μM) in PBS buffer (0.01 M, pH 7.4, containing 10% EtOH) with excitation at 600 nm. Inset: change in color (i) and fluorescence (ii) in the absence of Hg²⁺ (left) and in the presence of 10 μM Hg²⁺ (right).

Fig. 9. Confocal fluorescence imaging of NO in HeLa cells with HN7-N2 (10 μM) for 30 min and then NO donor, diethylamine NONOate (from left to right: 0, 10, 20, 50 μM, respectively) for 30 min. Excitation at 543 nm for red emission of HN7-N2 (650–720 nm).

Fig. 10. Depth fluorescence images of 10 μM HN7-N2 for NO detection in tissues were obtained with spectral confocal microscopy (Olympus, FV1000). The changes of fluorescence intensity with scan depth were determined by spectral confocal microscopy (Olympus, FV1000) in the z-scan mode (from 0 to 150 μm; step size: 1 μm). The images were collected at 650–720 nm (red channel). The slice was cultured with HN7-N2 (5 μM) for 60 min and then NO (50 μM) for another 60 min.

Fig. 11. Fluorescent images of mice (pseudocolor). The mice were imaged using a Caliper VIS Lumina XR small animal optical in vivo imaging system with an excitation filter 605 nm and a Cy5.5 emission filter. Left: imaging of control group after intraperitoneal injection of HN7-N2 probe (20 nanomoles) for 30 min; right: intraperitoneal injection of HN7-N2 probe (20 nanomoles) for 30 min, followed by intraperitoneal injection of NO donor (50 nanomoles). The mice were imaged 30 min after NO donor injection.