Super-resolution fluorescence imaging, particularly Stimulated Emission Depletion (STED) microscopy, requires fluorescent probes with exceptional properties: high brightness, excellent photostability, and a long Stokes shift. Current probes often compromise on one or more of these properties. The high-powered depletion laser in STED microscopy causes significant photobleaching, further limiting the effectiveness of many fluorophores. Ideal probes would also possess various excitation/emission wavelengths for multicolor imaging and be cell-permeable for live-cell studies. Rhodamine and its derivatives are commonly used in STED microscopy, but their photostability and Stokes shift often limit their application. Previous strategies to improve rhodamine properties have focused on either inhibiting the twisted-intramolecular-charge-transfer (TICT) state, which reduces brightness and photostability, or inducing vibronic structure to increase Stokes shift, often at the cost of brightness. This study aims to overcome these limitations by developing a synergistic strategy that combines both TICT inhibition and vibronic structure generation to achieve simultaneous improvements in brightness, photostability, and Stokes shift.

Existing literature highlights several strategies to enhance the properties of rhodamine dyes. Inhibiting TICT, a non-radiative decay pathway, improves brightness and photostability by reducing or preventing the rotation of the C-N bond. This has been achieved through modifications like replacing dialkylamino groups with other moieties (e.g., 7-azabicyclo[2.2.1]heptane, azetidine). Increasing the energy barrier of TICT formation via electron-withdrawing groups (EWGs) also enhances brightness. Strategies to improve Stokes shift have focused on generating vibronic structures, although these often result in reduced brightness in aqueous solutions. However, no single strategy has successfully enhanced all three properties (brightness, photostability, and Stokes shift) simultaneously. This gap in the literature highlights the need for a new approach, and that is the focus of this paper.

The researchers designed a synergistic strategy combining TICT inhibition and vibronic structure generation. They synthesized a series of asymmetric rhodamines (YL dyes) by introducing various EWGs into the quinoxaline moiety of the fluorophore scaffold. This was achieved through condensation reactions and subsequent modifications. The photophysical properties (brightness, Stokes shift, photostability, and quantum yield) of these new dyes were characterized using UV-Vis absorption and fluorescence spectroscopy. Density Functional Theory (DFT) calculations were performed to understand the electronic structure and energy landscape of the dyes, examining TICT formation and vibronic structures. The cell permeability and performance of the dyes in live-cell imaging were evaluated using confocal microscopy. The best-performing dye, YL578, was further conjugated to a HaloTag ligand for specific protein labeling and tested in STED microscopy, comparing its performance to existing photostable probes (580CP-Halo, CPY-Halo, JF608-Halo). The generalizability of the strategy was tested by applying it to other fluorophores (rhodol, pyronin, coumarin, and Boranil), assessing their improved properties. A probe for sensing alkaline phosphatase (ALP) was developed as a proof-of-concept application of the enhanced properties.

The introduction of the 2-(2,2,2-trifluoroethyl)octahydropyrrolo[1,2-a]pyrazine moiety to rhodamines resulted in the development of YL578, demonstrating a 2.4-fold increase in quantum yield (0.74) and a twofold enhancement in brightness (εx = 6.6 × 104 L mol−1 cm−1) compared to RhB. YL578 also exhibited a significantly longer Stokes shift (56 nm) than RhB (27 nm). In STED microscopy using a 775 nm depletion laser, a YL578-derived probe (YL578-Halo) showed superior photostability, providing three times more frames than existing photostable probes (580CP-Halo, CPY-Halo, JF608-Halo) with better resolution. YL578-Halo enabled 3D STED imaging of mitochondria. The strategy was shown to be widely applicable, improving brightness, photostability, and Stokes shift across different fluorophore scaffolds (rhodol, pyronin, coumarin, and Boranil). The largest Stokes shift observed was 136 nm for one of the modified Boranil dyes. A fluorogenic probe (11-ALP) based on the modified rhodol showed significantly improved performance for detecting alkaline phosphatase, both in vitro and in live cells. DFT calculations supported the synergistic effect of TICT inhibition and vibronic structure generation in improving the overall properties.

This study successfully demonstrates a novel strategy for simultaneously enhancing the brightness, photostability, and Stokes shift of fluorescent dyes. The key to this improvement lies in the synergistic action of inhibiting the TICT state and introducing a vibronic structure. The superior performance of YL578 and its derivatives in STED microscopy highlights the potential of this strategy for advancing super-resolution imaging. The successful application of this strategy to various fluorophore scaffolds suggests a broad applicability and potential for developing a wide range of improved probes for diverse bioimaging applications. The development of the ALP sensor further underscores the practical utility of this approach.

This research presents a novel synergistic strategy for developing brighter, more photostable, and long-Stokes-shift fluorescent dyes. The successful application of this strategy to various fluorophore scaffolds, resulting in significant improvements in key properties, is a major contribution. Future research could focus on exploring additional modifications to further optimize these properties, expanding the range of applicable fluorophore scaffolds, and developing probes for various applications (e.g., in vivo imaging, disease diagnostics).

While the study demonstrates significant improvements in dye properties, further investigation is needed to fully assess the long-term photostability of the new probes in complex biological environments. The current study primarily focuses on in vitro and in cellulo experiments; in vivo applications require further testing. The generality of the approach, while promising, may need refinement for specific fluorophore scaffolds to achieve optimal results.