Chemical Approaches to Optimize the Properties of Organic Fluorophores for Imaging and Sensing.

Organic fluorophores are indispensable tools in cells, tissue and in vivo imaging, and have enabled much progress in the wide range of biological and biomedical fields. However, many available dyes suffer from insufficient performances, such as short absorption and emission wavelength, low brightness, poor stability, small Stokes shift, and unsuitable permeability, restricting their application in advanced imaging technology and complex imaging. Over the past two decades, many efforts have been made to improve these performances of fluorophores. Starting with the luminescence principle of fluorophores, this review clarifies the mechanisms of the insufficient performance for traditional fluorophores to a certain extent, systematically summarizes the modified approaches of optimizing properties, highlights the typical applications of the improved fluorophores in imaging and sensing, and indicates existing problems and challenges in this area. This progress not only proves the significance of improving fluorophores properties, but also provide a theoretical guidance for the development of high-performance fluorophores.

"Zero" Intrinsic Fluorescence Sensing-Platforms Enable Ultrasensitive Whole Blood Diagnosis and In Vivo Imaging.

Abnormal physiological processes and diseases can lead to content or activity fluctuations of biocomponents in organelles and whole blood. However, precise monitoring of these abnormalities remains extremely challenging due to the insufficient sensitivity and accuracy of available fluorescence probes, which can be attributed to the background fluorescence arising from two sources, 1) biocomponent autofluorescence (BCAF) and 2) probe intrinsic fluorescence (PIF). To overcome these obstacles, we have re-engineered far-red to NIR II rhodol derivatives that possess weak BCAF interference. And a series of "zero" PIF sensing-platforms were created by systematically regulating the open-loop/spirocyclic forms. Leveraging these advancements, we devised various ultra-sensitive NIR indicators, achieving substantial fluorescence boosts (190 to 1300-fold). Among these indicators, 8-LAP demonstrated accurate tracking and quantifying of leucine aminopeptidase (LAP) in whole blood at various stages of tumor metastasis. Furthermore, coupling 8-LAP with an endoplasmic reticulum-targeting element enabled the detection of ERAP1 activity in HCT116 cells with p53 abnormalities. This delicate design of eliminating PIF provides insights into enhancing the sensitivity and accuracy of existing fluorescence probes toward the detection and imaging of biocomponents in abnormal physiological processes and diseases.

Family of hNQO1 Activatable Near-Infrared Fluoro-Photoacoustic Probes for Diagnosis of Wound Infection and Ulcerative Colitis.

Bacterial infections can easily occur when patients mishandle wounds or eat moldy food. The prompt diagnosis of a bacterial infection could effectively reduce the risk of possible anatomical damage. However, non-invasive early detection of bacterial infections is difficult to achieve due to the lack of favorable tools. Here, we designed two hNQO1 fluorescent probes (RX2 and RX3) to visualize bacterial infection after deep learning on the pathogenesis of bacterial infection. RX2 and RX3 enable early detection of bacterial infection and are verified to be, respectively, suitable for fluorescence imaging (FLI) and photoacoustic imaging (PAI) by comparing the signal-to-background ratio of both probes in a mouse model of myositis caused by Escherichia coli infection. In view of the difference in penetration depth between the two imaging modalities, we further applied RX2 for FLI of E. coli-infected wounds and RX3 for PAI of E. coli-infected inflammatory bowel disease, suggesting the great potential of both probes for early diagnosis of bacterial infections.

Tuning the Cellular Uptake and Retention of Rhodamine Dyes by Molecular Engineering for High-Contrast Imaging of Cancer Cells.

Probes allowing high-contrast discrimination of cancer cells and effective retention are powerful tools for the early diagnosis and treatment of cancer. However, conventional small-molecule probes often show limited performance in both aspects. Herein, we report an ingenious molecular engineering strategy for tuning the cellular uptake and retention of rhodamine dyes. Introduction of polar aminoethyl leads to the increased brightness and reduced cellular uptake of dyes, and this change can be reversed by amino acetylation. Moreover, these modifications allow cancer cells to take up more dyes than normal cells (16-fold) through active transport. Specifically, we further improve the signal contrast (56-fold) between cancer and normal cells by constructing activatable probes and confirm that the released fluorophore can remain in cancer cells with extended time, enabling long-term and specific tumor imaging.

Faster and More Specific: Excited-State Intramolecular Proton Transfer-Based Dyes for High-Fidelity Dynamic Imaging of Lipid Droplets within Cells and Tissues.

Lipid droplets (LDs), a type of dynamic organelle residing at the center of cellular lipid storage, have been identified to play important roles in multiple biological processes, metabolic disorders, and diseases. The highly dynamic characters of LDs were found to correspond to their physiological and pathological functions. Hence, the fluorescent probes which enable dynamic tracking of LDs should be very helpful for better understanding the mechanisms of LDs involved biological processes and diseases. Herein we present, to the best of our knowledge, the first class of excited-state intramolecular proton transfer (ESIPT) fluorescence dyes (Flp-(11-13, 19)) for dynamic imaging of LDs based on 3-hydroxyflavone (3HF) derivatives. Flp-(11-13, 19) display strong fluorescence from yellow to NIR in lipid but exhibit almost nonfluorescence in aqueous solution. Besides, they also show large Stokes shifts (>150 nm), narrow absorption and emission peaks, and good oil-water separation efficiency, which makes them specifically target and stain LDs with very low background noisy in both living cells and fixed cells. They stain intracellular LDs quite quickly (within 30 s) with very low dosage (as low as 500 nM). Benefitting from these advantages, Flp-(11-13, 19) are applied successfully in tracking the dynamic nature of LDs and accumulation of LDs in both aqueous solution and living cells, 3D imaging of LDs for visualization of their repartition within the cells, and visualizing LDs in tissues of diseases mice models including adipose, skeletal muscle, and fatty liver tissues, underscoring the potential utility of these dyes in both LDs biology research and medical diagnosis of LDs involved diseases.

A synergistic strategy to develop photostable and bright dyes with long Stokes shift for nanoscopy.

The quality and application of super-resolution fluorescence imaging greatly lie in the dyes' properties, including photostability, brightness, and Stokes shift. Here we report a synergistic strategy to simultaneously improve such properties of regular fluorophores. Introduction of quinoxaline motif with fine-tuned electron density to conventional rhodamines generates new dyes with vibration structure and inhibited twisted-intramolecular-charge-transfer (TICT) formation synchronously, thus increasing the brightness and photostability while enlarging Stokes shift. The new fluorophore YL578 exhibits around twofold greater brightness and Stokes shift than its parental fluorophore, Rhodamine B. Importantly, in Stimulated Emission Depletion (STED) microscopy, YL578 derived probe possesses a superior photostability and thus renders threefold more frames than carbopyronine based probes (CPY-Halo and 580CP-Halo), known as photostable fluorophores for STED imaging. Furthermore, the strategy is well generalized to offer a new class of bright and photostable fluorescent probes with long Stokes shift (up to 136 nm) for bioimaging and biosensing.

Visualization of Sulfane Sulfur in Plants with a Near-Infrared Fluorescent Probe.

Sulfane sulfur species are an important type of reactive sulfur species. These compounds have unique reactivity to attach reversibly to other sulfur atoms and exhibit regulatory effects in diverse biological systems. Recent studies have suggested that sulfane sulfurs are involved in signal transduction processes of hydrogen sulfide (H2S). The development of probes for selective, rapid, and sensitive detection of sulfane sulfur is of great significance for studying their physiological and pathological roles in biological systems, especially in plant systems for which physiological research has lagged behind. However, so far there is still a lack of sufficient chemical tools for directly tracking and measuring sulfane sulfur in biological systems, and in particular, the detection of sulfane sulfur in living plant tissues is still challenging. Herein, we report a near-infrared fluorescent probe, SSNIP, for the selective imaging of sulfane sulfur. SSNIP is capable of detecting sulfane sulfur at physiological concentrations in both aqueous buffer and living human cells. Then, with SSNIP, we demonstrate the fluorescent monitoring of endogenous sulfane sulfur in plant tissues such as Arabidopsis thaliana roots for the first time. Furthermore, the application of SSNIP in evaluating the level of sulfane sulfur in Arabidopsis thaliana roots at different growth stages is performed. The results show that the level of sulfane sulfur in Arabidopsis thaliana roots correlates well with their growth stages, which suggests that sulfane sulfurs might act as actual signaling molecules to promote plant growth and root elongation. In addition, it reveals potential applications for the biological and pathological studies of sulfane sulfur, especially in plant physiology.