Fluorescent Probe For Imaging Research Articles

A Dual-Cascade Activatable Near-Infrared Fluorescent Probe for Precise Intraoperative Imaging of Tumor.

Accurate intraoperative tumor delineation is critical to achieving successful surgical outcomes. However, conventional techniques typically suffer from poor specificity and low sensitivity and are time-consuming, which greatly affects intraoperative decision-making. Here, we report a cascade activatable near-infrared fluorescent (NIRF) probe IR780SS@CaP that can sequentially respond to tumor acidity and elevated glutathione levels for accurate intraoperative tumor localization. Compared with nonactivatable and single-factor activatable probes, IR780SS@CaP with a cascade strategy can minimize nonspecific activation and false positive signals in a complicated biological environment, affording a superior tumor-to-normal tissue ratio to facilitate the delineation of abdominal metastases. Small metastatic lesions that were less than 1 mm in diameter can be precisely identified by IR780SS@CaP and completely excised under NIRF imaging guidance. This study could benefit tumor diagnosis and image-guided tumor surgery by providing real-time information and reliable decision support, thus reducing the risk of both recurrence and complications to improve patient outcomes.

An ESIPT-Based Fluorescent Probe for Wash-Free Cell Membrane Imaging.

The cell membrane tracking is important for studying the membrane function and diagnosing membrane-related diseases, so the development of fluorescent molecule specifically lighting up cell membrane is highly desirable. In this work, we designed and readily prepared a fluorescent dye (BOHI) that can target cell membrane. The pH-dependent emission spectra reveal that BOHI shows fluorescence based on the excited-state intramolecular proton transfer (ESIPT) under acidic and neutral conditions, while the blue-shifted emission from the phenolate anion of BOHI was observed when pH ≥ 9. Among various solvents, the weakest fluorescence emission was observed in H2O, which is favorable to wash-free imaging. The fluorescence intensity of BOHI is greatly affected by surfactants. The anionic surfactant can induce intense green fluorescence, while very weak fluorescence was observed in the solution of cationic surfactant. Hela cells images show that BOHI can specifically targets and lights up cell membrane.

Nitrogen and Chlorine Co-Doped Carbon Dots with Yellow-Green Emission as a Ratiometric Fluorescent and Colorimetric Dual-Signal Probe for Quercetin Detection and Cell Imaging

Despite the emergence of numerous carbon dots (CDs)-based sensors, most of them are fluorescent probes. To improve the reliability of the results, using ratiometric fluorescence/UV-vis absorption dual-signal probes is an effective method. Herein, nitrogen and chlorine co-doped carbon dots (N, Cl-CDs) with yellow-green fluorescence were prepared and used as a dual-function probe for ratiometric fluorescence/UV-vis. The detection range of the fluorescence method was 0.05–78[Formula: see text][Formula: see text]M, and the limit of detection (LOD) was 1.2[Formula: see text]nM (3[Formula: see text]/s). For the colorimetric method, the linear range was 0.05–186[Formula: see text][Formula: see text]M with LOD of 10.7[Formula: see text]nM. Importantly, the N, Cl-CDs were successfully used to detect Quercetin (QT) in actual water samples. Besides, due to their good biocompatibility, the N, Cl-CDs were applied to in vivo biological imaging of oocysts and shown the potential for environmental survey and biological assay.

Chromone-based fluorescent probe for peroxynitrite imaging in Arabidopsis thaliana

As a typical reactive nitrogen species, peroxynitrite (ONOO−) plays an important role in cells and organisms. But in plants, information on the role of endogenous ONOO− remains limited. In order to explore its functions, it is necessary to probe ONOO− in biological systems, especially in plants. In this paper, we report RAW-A, a chromone-based fluorescence probe, exhibits a highly selective and sensitive response to ONOO−. It is worth noting that RAW-A has good water solubility, which is beneficial for the application of biological systems. RAW-A can respond to ONOO− even at low concentrations of ONOO−, indicating that the probe is expected to monitor ONOO− in real-time in living systems. In addition, confocal imaging experiments show that RAW-A can image endogenous and exogenous ONOO− in Arabidopsis thaliana root besides HeLa cell. It shows that RAW-A has the potential to be an imaging tool for studying the biological roles of ONOO− in plants. To our knowledge, RAW-A is the first fluorescent probe with highly selective and sensitive response to ONOO− in plants.

Construction of a human serum albumin site II-targeting supramolecular fluorescent probe for cell imaging and drug delivery

Two cationic probes (named TAB2 and TAB6) specifically for binding to site II in human serum albumin (HSA) were designed and synthesized. Both probes exhibited selective fluorescence “turn-on” responsiveness to site II in HSA, though minor differences in their cationic terminals resulted in notable variations in their albumin responsiveness. TAB2 and TAB6 also exhibited strong binding affinity toward HSA. The presence of phenylbutazone, ibuprofen, and lidocaine had minimal impact on the albumin responsiveness of these probes. Furthermore, the complex formed by TAB2/6 and water-soluble pillar[5]arene (WP5) showed enhanced HSA site II-targeting fluorescence responsiveness. More importantly, the WP5-TAB2/6 complex self-assembled into vesicular supramolecular probes in aqueous solution (pH=7.4). Those supramolecular probes were promising for HSA-mediated cell imaging and act as a pH-sensitive drug-delivery platform to enhance the anticancer efficiency of doxorubicin (DOX).

Bienzyme-Locked Activatable Fluorescent Probes for Specific Imaging of Tumor-Associated Mast Cells.

Tumor-associated mast cells (TAMCs) have been recently revealed to play a multifaceted role in the tumor microenvironment. Noninvasive optical imaging of TAMCs is thus highly desired to gain insights into their functions in cancer immunotherapy. However, due to the lack of a single enzyme that is specific to mast cells, a common probe design approach based on single-enzyme activation is not applicable. Herein, we reported a bienzyme-locked molecular probe (THCMC) based on a photoinduced electron transfer-intramolecular charge-transfer hybrid strategy for in vivo imaging of TAMCs. The bienzyme-locked activation mechanism ensures that THCMC exclusively turns on near-infrared (NIR) fluorescence only in the presence of both tryptase and chymase specifically coexpressed by mast cells. Thus, THCMC effectively distinguishes mast cells from other leukocytes, including T cells, neutrophils, and macrophages, a capability lacking in single-locked probes. Such a high specificity of THCMC allows noninvasive tracking of the fluctuation of TAMCs in the tumor of living mice during cancer immunotherapy. The results reveal that the decreased intratumoral signal of THCMC after combination immunotherapy correlates well with the reduced population of TAMCs, accurately predicting the inhibition of tumor growth. Thus, this study not only presents the first NIR fluorescent probe specific for TAMCs but also proposes a generic bienzyme-locked probe design approach for in vivo cell imaging.

BioAIEgens based on dehydroabietic acid and tetraphenylethene: How does isopropyl group of dehydroabietic acid affect their spectral performance

Based on dehydroabietic acid (DA) derived from natural rosin, two aggregation-induced emission luminogens (BioAIEgens) labeled as 12-DTPA-2pTPE and 13-DTPA-2pTPE were synthesized by introducing tetraphenylethylene (TPE) groups to dehydroabietic acid triarylamine (DTPA). Their structures were optimized, the spectral properties and AIE characteristics were investigated, and the compounds were prepared into nanoparticles (NPs) as fluorescent probes for cell imaging. The results show that the two BioAIEgens have typical AIE characteristics and large Stokes shifts (＞ 120 nm). Due to the electronic and spatial effects of the isopropyl group, the molecular configuration of 12-DTPA-2pTPE is more distorted, and the electron cloud distribution is also different from 13-DTPA-2TPE. The absorption and the emission peaks of 12-DTPA-2TPE are blue-shifted, and the fluorescence quantum yield in the solid is higher. In addition, the 13-DTPA-2TPE nanoparticles (NPs) have low cytotoxicity and good cell imaging effects in vitro.

Advancements in Small Molecule Fluorescent Probes for Superoxide Anion Detection: A Review.

Superoxide anion (O2•-), a significant reactive oxygen species (ROS) within biological systems, plays a widespread role in cellular function regulation and is closely linked to the onset and progression of numerous diseases. To unveil the pathological implications of O2•- in these diseases, the development of effective monitoring techniques within biological systems is imperative. Small molecule fluorescent probes have garnered considerable attention due to their advantages: simplicity in operation, heightened sensitivity, exceptional selectivity, and direct applicability in monitoring living cells, tissues, and animals. In the past few years, few reports have focused on small molecule fluorescence probes for the detection of O2•-. In this small review, we systematically summarize the design and application of O2•- responsive small molecule fluorescent probes. In addition, we present the limitations of the current detection of O2•- and suggest the construction of new fluorescent imaging probes to indicate O2•- in living cells and in vivo.

A lysosome-targeted fluorescent probe for fluorescence imaging of hypochlorous acid in living cells and in vivo

Lysosomes, as crucial acidic organelles in cells, play a significant role in cellular functions. The levels and distribution of hypochlorous acid (HOCl) within lysosomes can profoundly impact their biological functionality. Hence, real-time monitoring of the concentration of HOCl in lysosomes holds paramount importance for further understanding various physiological and pathological processes associated with lysosomes. In this study, we developed a bodipy-based fluorescent probe derived from pyridine and phenyl selenide for the specific detection of HOCl in aqueous solutions. Leveraging the probe's sensitive photoinduced electron transfer effect from phenyl selenide to the fluorophore, the probe exhibited satisfactory high sensitivity (with a limit of detection of 5.2 nM and a response time of 15 s) to hypochlorous acid. Further biological experiments confirmed that the introduction of the pyridine moiety enabled the probe molecule to selectively target lysosomes. Moreover, the probe successfully facilitated real-time monitoring of HOCl in cell models stimulated by N-acetylcysteine (NAC) and lipopolysaccharide (LPS), as well as in a normal zebrafish model. This provides a universal method for dynamically sensing HOCl in lysosomes.

Activatable near-infrared fluorescence and chemical exchange saturation transfer MRI multimodal imaging probe for tumor detection in vitro and in vivo

Multimodal imaging has emerged as a powerful tool in biomedical research and clinical diagnostics. Ideally, it combines multiple imaging techniques to provide complementary anatomical and molecular information in living subjects. Particularly desirable are multimodal imaging probes capable of providing differential diagnostic signals upon interaction with specific molecular targets. Hydrogen peroxide (H2O2) is a key target in this regard, since it is typically overexpressed in cancer cells. In this study, we present a small-molecule probe that not only selectively detects endogenous H2O2 through multimodal imaging, with a significant H2O2-triggered 15-fold fluorescence enhancement but also turns “on” a chemical exchange saturation transfer (CEST) magnetic resonance (MR) response with 60-fold signal enhancement at pH 7.4. Excellent selectivity against various other biologically relevant species is seen. Using this probe, we observed 3.4–4.5-fold and 2.8–5.8-fold higher H2O2 levels in cancerous cell lines and tumor tissues compared to normal cell lines and tissues, respectively. Time-dependent in vivo fluorescence and CEST imaging in a HeLa (Henrietta Lacks) tumor xenograft mouse model revealed probe-dependent tumor detection by fluorescence and CEST MRI contrast in the tumor area. These observations are attributed to the relatively high endogenous H2O2 levels produced during mitosis. This newly developed probe holds promise for advancing our understanding of H2O2-related biology and for cancer detection both in vitro and in vivo.

Homo-Dyad with Outer Hydration Layer Approach for Developing NIR-II Chromophore of High Stability and Water-Solubility as Injectable and Sprayable Optical Probe.

Dyes with extended conjugate structures are the focus of extensive design and synthesis efforts, aiming to confer unique and improved optical and electronic properties. Such advancements render these dyes applicable across a wide spectrum of uses, ranging from second-window near-infrared (NIR-II) bioimaging to organic photovoltaics. Nevertheless, the inherent benefits of long conjugation are often accompanied by persistent challenges like aggregation, fluorescence quenching, absorption blueshift, and low stability and poor water solubility. Herein, a unique structural design strategy termed "homo-dyad with outer hydration layer" is introduced to address these inherent problems, tailored for the development of imaging probes exhibiting long absorption/emission wavelengths. This approach involves bringing two heptamethine cyanines together through a flexible linker, forming a homo-dyad structure, while strategically attaching four polyethylene glycol (PEG9) chains to the terminal heterocycles. This approach imparts excellent water solubility, biocompatibility, and enhanced chemical, photo-, and spectral stability for the dyes. Utilizing this strategy, a biomarker-activatable probe (HD-FL-4PEG9-N) for NIR-II fluorescent and 3D multispectral optoacoustic tomography imaging is developed, and its effectiveness in disease visualization. It can not only serve as an injectable probe for acute kidney injury imaging due to its high water solubility, but also a sprayable probe for imaging bacterial-infected wounds.

Discovery of Natural Products Alleviating Renal Fibrosis with a Viscosity-Responsive Molecular Probe.

Renal fibrosis poses a significant threat to individuals suffering from chronic progressive kidney disease. Given the absence of effective medications for treating renal fibrosis, it becomes crucial to assess the extent of fibrosis in real time and explore the development of novel drugs with substantial therapeutic benefits. Due to the accumulation of renal tissue damage and the uncontrolled deposition of fibrotic matrix during the course of the disease, there is an increase in viscosity both intracellularly and extracellularly. Therefore, a viscosity-sensitive near-infrared fluorescence (NIRF) and photoacoustic (PA) imaging probe, BDP-KY, was developed to detect aberrant changes in viscosity during fibrosis. Furthermore, BDP-KY has been applied to screen the effective components of herbal medicine, rhubarb, resulting in the identification of potential antirenal fibrotic compounds such as emodin-8-glucoside and chrysophanol 8-O-glucoside. Ultrasound, PA, and NIRF imaging of a unilateral uretera obstruction mice model show that different concentrations of emodin-8-glucoside and chrysophanol 8-O-glucoside effectively reduce viscosity levels during the renal fibrosis process. The histological results showed a significant decrease in fibrosis factors α-smooth muscle actin and collagen deposition. Combining these findings with their pharmacokinetic characteristics, these compounds have the potential to fill the current market gap for effective antirenal fibrosis drugs. This study demonstrates the potential of BDP-KY in the evaluation of renal fibrosis, and the two identified active components from rhubarb hold great promise for the treatment of renal fibrosis.

Near-infrared imaging of head and neck squamous cell carcinoma using indocyanine green that targets the peptide.

Head and neck squamous cell carcinoma (HNSCC) has a particularly poor prognosis. Improving the surgical resection boundary, reducing local recurrence, and ultimately ameliorating the overall survival rate are the treatment goals. To obtain a complete surgical resection (R0 resection), we investigated the use of a fluorescent imaging probe that targets the integrin subtype , which is upregulated in many kinds of epithelial cancer, using animal models. expression was detected using polymerase chain reaction (PCR) and immunoprotein blotting of human tissues for malignancy. Protein expression localization was observed. and epidermal growth factor receptor (EGFR) were quantified by PCR and immunoprotein blotting, and the biosafety of targeting the probe material was examined using Cell Counting Kit-8 assays. Indocyanine green (ICG) was used as a control to determine the localization of the probe at the cellular level. In vivo animal experiments were conducted through tail vein injections to evaluate the probe's imaging effect and to confirm its targeting in tissue sections. expression was higher than EGFR expression in HNSCC, and the probe showed good targeting in in vivo and in vitro experiments with a good safety profile. The ICG- peptide probe is an exceptional and sensitive imaging tool for HNSCC that can distinguish among tumor, normal, and inflammatory tissues.

A Red-Emission Fluorescent Probe for Intracellular Biothiols and Hydrogen Sulfide Imaging in Living Cells.

This research centers on the development and synthesis of a longwave fluorescence probe, labeled as 60T, designed for the simultaneous detection of hydrogen sulfide, cysteine/homocysteine, and glutathione. The probe showcases a swift response, good linearity range, and heightened sensitivity, boasting that the detection limits of the probe for Cys, Hcy, GSH and H2S were 0.140, 0.202, 0.259 and 0.396 μM, respectively. Notably, its efficacy in monitoring thiol status changes in live MCF-7 cells is underscored by a substantial decrease in fluorescence intensity upon exposure to the thiol trapping reagent, N-ethyl maleimide (NEM). With an impressive red emission signal at 630 nm and a substantial Stokes shift of 80 nm, this probe exhibits remarkable sensitivity and selectivity for biothiols and H2S, indicating promising applications in the diagnosis and surgical navigation of relevant cancers.

Activity-Based Fluorescence Diagnostics for Cancer.

Fluorescence imaging is one of the most promising approaches to achieve intraoperative assessment of the tumor/normal tissue margins during cancer surgery. This is critical to improve the patients' prognosis, and therefore various molecular fluorescence imaging probes have been developed for the identification of cancer lesions during surgery. Among them, "activatable" fluorescence probes that react with cancer-specific biomarker enzymes to generate fluorescence signals have great potential for high-contrast cancer imaging due to their low background fluorescence and high signal amplification by enzymatic turnover. Over the past two decades, activatable fluorescence probes employing various fluorescence control mechanisms have been developed worldwide for this purpose. Furthermore, new biomarker enzymatic activities for specific types of cancers have been identified, enabling visualization of various types of cancers with high sensitivity and specificity. This Review focuses on recent advances in the design, function and characteristics of activatable fluorescence probes that target cancer-specific enzymatic activities for cancer imaging and also discusses future prospects in the field of activity-based diagnostics for cancer.

Acidity-Triggered "Sticky Spotlight": CCK2R-Targeted TME-Sensitive NIR Fluorescent Probes for Tumor Imaging In Vivo.

Cancer which causes high mortality globally threatens public health seriously. There is an urgent need to develop tumor-specific near-infrared (NIR) imaging agents to achieve precise diagnosis and guide effective treatment. In recent years, imaging probes that respond to acidic environments such as endosomes, lysosomes, or acidic tumor microenvironments (TMEs) are being developed. However, because of their nonspecific internalization by both normal and tumor cells, resulting in a poor signal-to-noise ratio in diagnosis, these pH-sensitive probes fail to be applied to in vivo tumor imaging. To address this issue, a cholecystokinin-2 receptor (CCK2R)-targeted TME-sensitive NIR fluorescent probe R2SM was synthesized by coupling pH-sensitive heptamethine cyanine with a CCK2R ligand, minigastrin analogue 11 (MG11) for in vivo imaging, in which MG11 would target overexpressed CCK2Rs in gastrointestinal stromal tumors (GISTs). Cell uptake assay demonstrated that R2SM exhibited a high affinity for CCK2R, leading to receptor-mediated internalization and making probes finally accumulated in the lysosomes of tumor cells, which suggested in the tumor tissues, the probes were distributed in the extracellular acidic TME and intracellular lysosomes. With a pKa of 6.83, R2SM can be activated at the acidic TME (pH = 6.5-6.8) and lysosomes (pH = 4.5-5.0), exhibiting an apparent pH-dependent behavior and generating more intense fluorescence in these acidic environments. In vivo imaging showed that coupling of MG11 with a pH-sensitive NIR probe facilitated the accumulation of probe and enhanced the fluorescence in CCK2R-overexpressed HT-29 tumor cells. A high signal was observed in the tumor region within 0.5 h postinjection, indicating its potential application in intraoperative imaging. Fluorescence imaging of R2SM exhibited higher tumor-to-liver and tumor-to-kidney ratios (2.1:1 and 2.3:1, respectively), compared separately with the probes that are lipophilic, pH-insensitive, or MG11-free. In vitro and in vivo studies demonstrated that the synergistic effect of tumor targeting with pH sensitivity plays a vital role in the high signal-to-noise ratio of the NIR imaging probe. Moreover, different kinds of tumor-targeting vectors could be conjugated simultaneously with the NIR dye, which would further improve the receptor affinity and targeting efficiency.

Bioinspired design of highly specific fluorescent probe for butyrylcholinesterase imaging in living cells and Alzheimer's disease model

Butyrylcholinesterase (BChE) is associated with many neurodegenerative diseases, especially Alzheimer’s disease (AD). Developing specific imaging tools for BChE is now highly pursued because BChE has substantially similar biochemical properties to acetylcholinesterase (AChE) but distinct roles in AD. Inspired by the BChE-catalyzed hydrolysis of specific substrates (benzoylcholine, tetracaine, and cocaine) containing benzoyl group in physiological condition, a bioinspired probe PM1 with high specificity towards BChE was designed. The docking models indicated that PM1 could easily access the active center of BChE rather than AChE, showing the significance of benzoyl group in specific recognition of BChE. The control probes PM2–5 with smaller recognition groups displayed lower specificity to BChE than PM1, showing the high efficiency of the bioinspired design strategy. The living cells experiments showed that PM1 signals were weakened by BChE inhibitor but not influenced by AChE inhibitor, which was in contrast to the performance of probe PM2–5, showing the specific recognition of PM1 for BChE in biological system. Moreover, PM1 has been used to study the positive correlation between BChE level and cell apoptosis for the first time, and the further study showed that Aβ deposition could induce the increase in BChE level and accompany the occurrence of cell apoptosis. Notably, PM1 was successfully traced the enhancement of BChE activity in the brain slice of AD mouse model. Such a rational approach may provide a solid basis for designing specific probes with different properties to study BChE in biosystems.

Pimonidazole-alkyne conjugate for sensitive detection of hypoxia by Cu-catalyzed click reaction.

Hypoxia is involved in various diseases, such as cancers. Pimonidazole has often been used as the gold-standard marker to visualize hypoxic regions. Pimonidazole labels hypoxic regions by forming a covalent bond with a neighboring protein under hypoxic conditions in the body, which is detected by immunohistochemistry performed on tissue sections. To date, some pimonidazole-fluorophore conjugates have been reported as fluorescent probes for hypoxia imaging that do not require immunostaining. They are superior to pimonidazole because immunostaining can produce high background signals. However, large fluorophores in the conjugates may alter the original biodistribution and reactivity. Here, we report a new hypoxia marker, Pimo-yne, as a pimonidazole-alkyne conjugate. Pimo-yne has a similar hypoxia detection capability as pimonidazole because the alkyne tag is small and can be detected by Cu-catalyzed click reaction with azide-tagged fluorescent dyes. We successfully visualized hypoxic regions in tumor tissue sections using Pimo-yne with reduced background signals. The detected regions overlapped well with those detected by pimonidazole immunohistochemistry. To further reduce the background, we employed a turn-on azide-tagged fluorescent dye.

Molecular mapping of neuronal architecture using STORM microscopy and new fluorescent probes for SMLM imaging.

Imaging neuronal architecture has been a recurrent challenge over the years, and the localization of synaptic proteins is a frequent challenge in neuroscience. To quantitatively detect and analyze the structure of synapses, we recently developed free SODA software to detect the association of pre and postsynaptic proteins. To fully take advantage of spatial distribution analysis in complex cells, such as neurons, we also selected some new dyes for plasma membrane labeling. Using Icy SODA plugin, we could detect and analyze synaptic association in both conventional and single molecule localization microscopy, giving access to a molecular map at the nanoscale level. To replace those molecular distributions within the neuronal three-dimensional (3D) shape, we used MemBright probes and 3D STORM analysis to decipher the entire 3D shape of various dendritic spine types at the single-molecule resolution level. We report here the example of synaptic proteins within neuronal mask, but these tools have a broader spectrum of interest since they can be used whatever the proteins or the cellular type. Altogether with SODA plugin, MemBright probes thus provide the perfect toolkit to decipher a nanometric molecular map of proteins within a 3D cellular context.

Recent advances in Si-rhodamine-based fluorescent probes for live-cell imaging.

Fluorescence imaging is a powerful technique for visualizing biological events in living samples with high temporal and spatial resolution. Fluorescent probes emitting far-red to near infrared (NIR) fluorescence are particularly advantageous for in vivo imaging due to their high tissue permeability and low autofluorescence, as well as their suitability for multicolor imaging. Among the far-red to NIR fluorophores, Si-rhodamine is one of the most practical fluorophores for the development of tailor-made NIR fluorescent probes because of the relative ease of synthesis of various derivatives, the unique intramolecular spirocyclization behavior, and the relatively high water solubility and high photostability of the probes. This review summarizes these features of Si-rhodamines and presents recent advances in the synthesis and applications of far-red to NIR fluorescent probes based on Si-rhodamines, focusing on live-cell imaging applications such as fluorogenic probes, super-resolution imaging and dye-protein hybrid-based indicators.