Fluorescent Probe For Imaging Research Articles

Tetrazine-derived chromones as conditionally activated solvatochromic fluorescent probes for dual imaging of lipid droplets and mitochondria

Tetrazine-derived chromones were developed as conditionally activated solvatochromic fluorescent probes and applied for LD and mitochondrial imaging.

Bioorthogonally activated probes for precise fluorescence imaging.

Over the past two decades, bioorthogonal chemistry has undergone a remarkable development, challenging traditional assumptions in biology and medicine. Recent advancements in the design of probes tailored for bioorthogonal applications have met the increasing demand for precise imaging, facilitating the exploration of complex biological systems. These state-of-the-art probes enable highly sensitive, low background, in situ imaging of biological species and events within live organisms, achieving resolutions comparable to the size of the biomolecule under investigation. This review provides a comprehensive examination of various categories of bioorthogonally activated in situ fluorescent labels. It highlights the intricate design and benefits of bioorthogonal chemistry for precise in situ imaging, while also discussing future prospects in this rapidly evolving field.

D-A-D organic fluorescent probes for NIR-II fluorescence imaging and efficient photothermal therapy of breast cancer.

Near-infrared second region (NIR-II) fluorescent probes are used in the diagnosis of early cancer due to their high tissue penetration. However, there are still few reports on organic small molecule fluorescent probes with NIR-II fluorescence imaging (NIR-II FI) combined with efficient photothermal therapy (PTT). In this study, planar cyclopentadithiophene (CPDT) was incorporated into the twisted structural skeleton (D-A-D), and the strong acceptor TTQ molecule (A) and the donor triphenylamine (D) were introduced to synthesize an organic small molecule (TCT) with enhanced NIR-II fluorescence emission performance. To improve the hydrophilicity of TCT molecules, we used the nanoprecipitation method to coat DSPE-mPEG2000 on the TCT molecules and obtained nanoparticles (TCT-NPs) with a strong absorption band, good water dispersibility, and NIR-II FI ability, which realized NIR-II FI-guided PTT for breast cancer tumors. Due to their effective near-infrared absorption, TCT-NPs exhibit high photothermal conversion efficiency (η = 40.1%) under 660 nm laser irradiation, making them a photothermal therapeutic agent with good performance. Therefore, TCT-NPs have the potential to diagnose, eliminate, and monitor the diffusion of cancer.

Recent advances in fluorescent probes for fatty liver imaging by detecting lipid droplets.

Fatty liver, a major health problem worldwide, is closely associated with aberrant accumulation and alteration of energy storage organelles, lipid droplets (LDs), in the disease process. Fluorescent probes with excellent optical performance, high sensitivity/selectivity and real-time monitoring have emerged as an attractive tool for the detection of LDs used in the diagnosis of fatty liver. In this review, we summarize various probes based on different response mechanisms to image LDs in the fatty liver process using different excitation imaging modes and emission wavelengths, including the visible to the near-infrared, two/three-photon, and the second near-infrared region. The perspectives and barriers associated with the reported lipid droplet (LD) probes for future development are also discussed.

A lipid droplet-targeted fluorescent probe for fluorescence imaging of cell and zebrafish viscosity

Lipid droplets (LDs) represent highly dynamic organelles found in almost all organisms and play a pivotal role in regulating the balance of intracellular lipid homeostasis.

Construction of cationic meso-thiazolium-BODIPY AIE fluorescent probes for viscosity imaging in dual organelles.

Two new cationic meso-thiazolium-BODIPY-based water-soluble and red-shifted fluorescent probes were constructed for the first time. They can monitor cellular viscosity in dual organelles and show aggregation-induced emission (AIE), which is ascribed to the efficient restricted rotation of meso-thiazolium in viscous or hindered systems. Probe 3 with an N-benzyl group shows better AIE as compared to probe 2 with an N-methyl group.

A hydrogen peroxide activated near-infrared ratiometric fluorescent probe for ratio imaging in vivo.

A sensitive near-infrared ratiometric fluorescence sensing platform was designed and structured. The platform consisted of carbon dots and a small organic molecule probe with pinacol phenylborate as the recognition group, and has been applied for ratio imaging of hydrogen peroxide in vivo.

A novel high signal-to-noise ratio fluorescent probe for real-time mitochondrial viscosity detection and imaging in vitro and in vivo.

Mitochondrial viscosity serves as a critical indicator for assessing mitochondrial functionality and offers valuable insights into cellular homeostasis. Continuous, real-time monitoring of mitochondrial viscosity is indispensable for understanding and diagnosing diseases associated with these dynamic changes. In this study, we introduce a novel mitochondrial viscosity-responsive probe named "JL-JC" which is designed by using a molecular strategy, with a classic "D-π-A" molecular structure. Leveraging the distinctive twisted intramolecular charge transfer (TICT) properties of the probe, JL-JC exhibits exceptional sensitivity and a high signal-to-noise ratio, enabling precise detection of viscosity variations within its microenvironment while remaining unaffected by other factors. Upon rapid cellular uptake, JL-JC can efficiently evaluate the mitochondrial viscosity changes under diverse physiological and pathological conditions. Notably, this probe also enables viscosity imaging in zebrafish, offering insights into mitochondrial states in vivo. Our findings present JL-JC as a promising tool and potential diagnostic platform for mitochondria-related diseases.

Synthesis and Application of a Near-Infrared Light-Emitting Fluorescent Probe for Specific Imaging of Cancer Cells with High Sensitivity and Selectivity.

The preclinical diagnosis of tumors is of great significance to cancer treatment. Near-infrared fluorescence imaging technology is promising for the in-situ detection of tumors with high sensitivity. Here, a fluorescent probe was synthesized on the basis of Au nanoclusters with near-infrared light emission and applied to fluorescent cancer cell labeling. Near-infrared methionine-N-Hydroxy succinimide Au nanoclusters (Met-NHs-AuNCs) were prepared successfully by one-pot synthesis using Au nanoclusters, methionine, and N-Hydroxy succinimide as frameworks, reductants, and stabilizers, respectively. The specific fluorescence imaging of tumor cells or tissues by fluorescent probe was studied on the basis of SYBYL Surflex-DOCK simulation model of LAT1 active site of overexpressed receptor on cancer cell surface. The results showed that Met-NHs-AuNCs interacted with the surface of LAT1, and C_Score scored the conformation of the probe and LAT1 as five. Characterization and in vitro experiments were conducted to explore the Met-NHs-AuNCs targeted uptake of cancer cells. The prepared near-infrared fluorescent probe (Met-NHs-AuNCs) can specifically recognize the overexpression of L-type amino acid transporter 1 (LAT1) in cancer cells so that it can show red fluorescence in cancer cells. Meanwhile, normal cells (H9c2) have no fluorescence. The fluorescent probe demonstrates the power of targeting and imaging cancer cells.

Mitochondrial membrane potential-independent near-infrared fluorescent probes for viscosity-exclusive imaging.

Elucidating the intrinsic relationship between disease and mitochondrial viscosity is crucial for early diagnosis. However, current mitochondrial viscosity fluorescent probes are highly dependent on mitochondrial membrane potential (MMP) and are sensitive to other mitochondrial microenvironment parameters. To address these issues, a mitochondria-targeting MMP-independent and viscosity exclusive near-infrared (NIR) fluorescent probe, ACR-DMA, was developed. ACR-DMA consists of thiophene acetonitrile as the skeleton and viscosity-sensitive unit, a pyridinium cation for the mitochondria-targeting group, and a benzyl bromide subunit for mitochondrial immobilization. It is very sensitive to viscosity and shows significant "turn-on" fluorescence behavior at 710 nm with a more than 150-fold fluorescence intensity increase. Furthermore, ACR-DMA can be firmly immobilized in mitochondria and can monitor viscosity changes induced by nystain, monensin, and lipopolysaccharide. Additionally, it was successfully used to visualize mitochondrial viscosity changes resulting from tumors, inflammation, and drug-induced acute kidney injury, revealing the relationship between viscosity and disease both in vitro and in vivo. ACR-DMA is expected to be a promising candidate for diagnosing mitochondrial viscosity-related diseases.

Xanthene-based near-infrared chromophores for high-contrast fluorescence and photoacoustic imaging of dipeptidyl peptidase 4

A class of new xanthene-based NIR chromophores which exhibited analyte-tunable spirocyclic “closed” and zwitterionic “open” forms allowed the development of high-contrast fluorescence and PA imaging probes for imaging dipeptidyl peptidase 4.

Miniature fluorescence sensor for quantitative detection of brain tumour.

Fluorescence-guided surgery has emerged as a vital tool for tumour resection procedures. As well as intraoperative tumour visualisation, 5-ALA-induced PpIX provides an avenue for quantitative tumour identification based on ratiometric fluorescence measurement. To this end, fluorescence imaging and fibre-based probes have enabled more precise demarcation between the cancerous and healthy tissues. These sensing approaches, which rely on collecting the fluorescence light from the tumour resection site and its "remote" spectral sensing, introduce challenges associated with optical losses. In this work, we demonstrate the viability of tumour detection at the resection site using a miniature fluorescence measurement system. Unlike the current bulky systems, which necessitate remote measurement, we have adopted a millimetre-sized spectral sensor chip for quantitative fluorescence measurements. A reliable measurement at the resection site requires a stable optical window between the tissue and the optoelectronic system. This is achieved using an antifouling diamond window, which provides stable optical transparency. The system achieved a sensitivity of 92.3% and specificity of 98.3% in detecting a surrogate tumour at a resolution of 1 × 1 mm2. As well as addressing losses associated with collecting and coupling fluorescence light in the current 'remote' sensing approaches, the small size of the system introduced in this work paves the way for its direct integration with the tumour resection tools with the aim of more accurate interoperative tumour identification.

A ratiometric fluorescent probe for imaging the fluctuation of HOBr during endoplasmic reticulum stress.

Endoplasmic reticulum (ER) stress is closely associated with cell apoptosis, autophagy, DNA damage, metabolism, and migration. When ER stress occurs, a large number of reactive oxygen species, including hypobromous acid (HOBr), are generated. The degree of ER stress can be understood by accurately detecting the HOBr concentration in the ER. Unfortunately, no ER-targetable probes for detecting HOBr have been reported to date. To solve this problem, we developed a naphthalimide-based fluorescent probe (ER-NABr) for imaging HOBr in the ER. Upon reaction with HOBr, a red shift in the fluorescence spectrum occurs due to the difference in the molecular conjugation between the original ER-NABr and the reaction product. ER-NABr showed a fast response (within 30 s) and high selectivity towards HOBr, with a ratiometric quantitative response (5-40 μM) and high sensitivity (138 nM). With its excellent biocompatibility and remarkable ER-targetable ability, ER-NABr was successfully utilized to ratiometrically image intracellular HOBr, particularly during ER stress, which is beneficial for revealing the role of HOBr in ER-associated diseases.

Glutathione and viscosity double-locked response fluorescent probe for imaging and surgical navigation of hepatocellular carcinoma.

Early diagnosis and precise treatment of hepatocellular carcinoma (HCC) are crucial for human health. Therefore, addressing the potential markers of HCC, glutathione (GSH) and viscosity, we constructed a fluorescent probe (PG-V) activated cascadically by GSH/viscosity. PG-V possessed excellent photophysical properties and biocompatibility, and could specifically illuminate tumor tissue, achieving fluorescence imaging of HCC, and imaging-guided tumor resection.

CB[8]- and triarylboron-based supramolecular organic framework for microRNA detection, tumor-targeted drug delivery, and photodynamic therapy.

Supramolecular organic frameworks (SOFs) are widely used for biological detection and drug delivery. In this study, a SOF system was fabricated through the self-assembly of photosensitive triarylboron (TAB), TAB-6-methyl, and CB[8]. The maximum fluorescence emission of TAB-6-methyl was greatly enhanced and red-shifted from 560 nm to 610 nm after SOF formation. The SOF can specifically respond to a microRNA by dissembling and then combining with microRNA, which is accompanied by a fluorescence shift from 610 nm to 560 nm, thus providing a ratiometric readout for microRNA detection. The photosensitivity of TAB-6-methyl can be further improved by forming a SOF, which can be used in photodynamic therapy. By constructing another guest molecule, TAB-5-1-cRGD, we successfully embedded cRGD in the SOF system to improve its tumor-targeting ability. Moreover, we used this SOF system as a fluorescence imaging probe for targeted tumor imaging and as a drug carrier system for loading DOX to achieve combined photodynamic and chemotherapy treatment of tumors.

Construction of a super large Stokes shift near-infrared fluorescent probe for detection and imaging of superoxide anion in living cells, zebrafish and mice

As one of the major reactive oxygen species (ROS), superoxide anion (O2•−) is engaged in maintaining redox homeostasis in the cell microenvironment. To identify the pathological roles in related disorders caused by abnormal expression of O2•−, it is of great significance to monitor and track the fluctuation of O2•− concentration in vivo. However, the low concentration of O2•− and the interference caused by tissue autofluorescence make the development of an ideal detection methodology full of challenges. Herein, a “Turn-On” chemical response near-infrared (NIR) fluorescence probe Dcm-Cu-OTf for O2•− detection in inflamed models, was constructed by conjugating the NIR fluorophore (dicyanisophorone derivative) with an O2•− sensing moiety (trifluoromethanesulfonate). Dcm-Cu-OTf exerted about 140-fold fluorescence enhancement after reacting 200 μM O2•− with an excellent limited of detection (LOD) as low as 149 nM. Additionally, Dcm-Cu-OTf exhibited a super large Stokes shift (260 nm) and high selectivity over other bio-analytes in stimulated conditions. Importantly, Dcm-Cu-OTf showed low toxicity and enabled imaging of the generation of O2•− in the Lipopolysaccharide (LPS)-stimulated HeLa cells, zebrafish, and LPS-induced inflamed mice. The present study provided a potential and reliable detection tool to inspect the physiological and pathological progress of O2•− in living biosystems.

Synthesis of a fluorescent chemical probe for imaging of L-type voltage-gated calcium channels

Herein, we report the synthesis of a chemical probe for real-time visualization of L-type voltage-gated calcium channels (LTCCs) using fluorescence microscopy. The probe, FluoBar2, is based on a barbiturate ligand selective for LTCCs. FluoBar2 contains the organic fluorophore 5-carboxyfluorescein and was synthesized in seven total steps with a 22% overall yield.

Aptamer Conjugate-Based Ratiometric Fluorescent Probe for Precise Imaging of Therapy-Induced Cancer Senescence.

Therapy-induced cellular senescence has been increasingly recognized as a key mechanism to promote various aspects of carcinogenesis in a nonautonomous manner. Thus, real-time imaging monitoring of cellular senescence during cancer therapy is imperative not only to further elucidate its roles in cancer progression but also to provide guidance for medical management of cancer. However, it has long been a challenging task due to the lack of effective imaging molecule tools with high specificity and accuracy toward cancer senescence. Herein, we report the rational design, synthesis, and evaluation of an aptamer conjugate-based ratiometric fluorescent probe for precise imaging of therapy-induced cancer senescence. Unlike traditional senescence imaging systems, our probe targets two senescence-associated markers at both cellular and subcellular dimensions, namely, aptamer-mediated membrane marker recognition for active cell targeting and lysosomal marker-triggered ratiometric fluorescence changes of two cyanine dyes for site-specific, high-contrast imaging. Moreover, such a two-channel fluorescence response is activated after a one-step reaction and at the same location, avoiding the diffusion-caused signal decay previously encountered in dual-marker activated probes, contributing to spatiotemporally specific imaging of therapy-induced cancer senescence in living cells and three-dimensional multicellular tumor spheroids. This work may offer a valuable tool for a basic understanding of cellular senescence in cancer biology and interventions.

Multifunctional Probe Based on "Chemical Antibody-Aptamer" for Noninvasive Detection of PD-L1 Expression in Cancer.

Immune checkpoint inhibitors (ICIs) therapy based on programmed cell death ligand 1 (PD-L1) has shown significant development in treating several carcinomas, but not all patients respond to this therapy due to the heterogeneity of PD-L1 expression. The sensitive and accurate quantitative analysis of in vivo PD-L1 expression is critical for treatment decisions and monitoring therapy. In the present study, an aptamer-based dual-modality positron emission tomography/near-infrared fluorescence (PET/NIRF) imaging probe was developed, and its specificity and sensitivity to PD-L1 were assessed in vitro and in vivo. The probe precursor NOTA-Cy5-R1 was prepared by using automated solid-phase oligonucleotide synthesis. PET/NIRF dual-modality probe [68Ga]Ga-NOTA-Cy5-R1 was successfully synthesized and radiolabeled. The binding specificity of [68Ga]Ga-NOTA-Cy5-R1 to PD-L1 was evaluated by flow cytometry, fluorescence imaging, and cellular uptake in A375-hPD-L1 and A375 cells, and it showed good fluorescence properties and stability in vitro. In vivo PET/NIRF imaging studies illustrated that [68Ga]Ga-NOTA-Cy5-R1 can sensitively and specifically bind to PD-L1 positive tumors. Meanwhile, the rapid clearance of probes from nontarget tissues achieved a high signal-to-noise ratio. In addition, changes of PD-L1 expression in NCI-H1299 xenografts treated with cisplatin (CDDP) were sensitivity monitored by [68Ga]Ga-NOTA-Cy5-R1 PET imaging, and ex vivo autoradiography and western blot analyses correlated well with the change of PD-L1 expression in vivo. Overall, [68Ga]Ga-NOTA-Cy5-R1 showed notable potency as a dual-modality PET/NIRF imaging probe for visualizing tumors and monitoring the dynamic changes of PD-L1 expression, which can help to direct and promote the clinical practice of ICIs therapy.

Bio-orthogonally Activatable Fluorescent Probe for Specific Imaging of Myeloid Cell Leukemia 1 Protein.

The antiapoptotic protein myeloid cell leukemia 1 (Mcl-1) has been increasingly identified as a promising potential therapeutic target attributed to its critical regulation effect in diverse cellar physiopathological events. Current fluorescence imaging strategies tend to be susceptible to the cellular microenvironment, and straightforward mapping of Mcl-1's level variation remains challenging. In this paper, an activatable "off-on" fluorescence strategy for Mcl-1 specific labeling was presented based on bio-orthogonal chemistry by introducing tetrazine-functionalized borondipyrromethene (TB) as a fluorescent reporter and trans-cyclooctyne-derived indole-2-carboxylic acid (TI) as an Mcl-1 targeting moiety. With the click pair of TB and TI, the Mcl-1 expression level in vitro and in vivo was successfully mapped straightforward. Also, the level changes of Mcl-1 upon drug challenge were demonstrated. This work provides a robust fluorescence strategy for Mcl-1 in situ imaging, and the results would further facilitate the comprehensive revelation of the Mcl-1 biological effect.