Fluorescent Probe For Imaging Research Articles

A Highly Selective and Sensitive Carbazole-Based Fluorescent Probe for Peroxynitrite Detection and Cellular Imaging

A Highly Selective and Sensitive Carbazole-Based Fluorescent Probe for Peroxynitrite Detection and Cellular Imaging

Clog P-Guided Development of Multi-Colored Buffering Fluorescent Probes for Super-Resolution Imaging of Lipid Droplet Dynamics.

Super-resolution fluorescence imaging of live cells increasingly demands fluorescent probes capable of multi-color and long-term dynamic imaging. Understanding the mechanisms of probe-target recognition is essential for the engineered development of such probes. In this study, it is discovered that the molecular lipid solubility parameter, Clog P, determines the staining performance of fluorescent dyes on lipid droplets (LDs). Fluorescent dyes with Clog P values between 2.5 and 4 can form buffering pools outside LDs, replacing photobleached dyes within LDs to maintain constant fluorescence intensity in LDs, thereby enabling dynamic super-resolution imaging of LDs. Guided by Clog P, four different colored buffering LD probes spanning the visible light spectrum have been developed. Using Structured Illumination Microscopy (SIM), the role of LD dynamics have been tracked during cellular ferroptosis with the secretion, storage, and degradation of overexpressed ACSL3 proteins. It is found that LDs serve as storage sites for these proteins through membrane fusion, and further degrade overexpressed proteins via interactions with organelles like lysosomes or through lipophagy, thereby maintaining cellular homeostasis.

Sensing and Imaging Agents for Cyclooxygenase Enzyme.

In this concept, we present a comprehensive study on the development and application of COX-2-specific fluorescent probes for cancer imaging and diagnosis. Targetting cancer cells and measuring cancer-related activities in specific organelles quickly and accurately are crucial factors for early diagnosis and research on cancer pathology and treatment. This concept explores a variety of probes which include BDIMC1-2, TPI-IMC, YZP1, IQ-1, ANQ-IMC-6, Niblue-C6-IMC, AZB-IMC2, CMP, CF3-Fluorocoxib, IBPC1, CoxFluor, etc. and each one demonstrates unique mechanisms and high selectivity towards COX-2 enzymes. These probes were designed to enhance fluorescence upon binding to COX-2 which enable precise visualization of tumor and inflamed tissues. The research emphasizes the importance of COX-2 as a biomarker in cancer diagnostics, particularly in identifying cancer stem cells and inflamed tissues. This concept highlights the potentiality of these probes in non-invasive imaging techniques which offering significant advancements in cancer diagnosis and monitoring. The in vivo and in vitro experiments, including applications in mouse models and human tissue samples, confirm the efficacy of these probes in providing detailed imaging for clinical and research applications.

Multivalent supramolecular fluorescent probes for accurate disease imaging.

Optical imaging is a powerful tool for early disease detection and effective treatment planning, but its accuracy is often compromised by the uptake of imaging materials by the mononuclear phagocyte system (MPS). Herein, we leverage multivalent host-guest interactions between cyanine dyes and β-cyclodextrin polymers to develop supramolecular probes with enhanced stability, optical, and transport profiles for accurate in vivo imaging. These multivalent interactions not only ensure the stability of the probes but also enhance fluorescence efficiency by minimizing nonradiative decay. Our self-assembly approach effectively modulates probe size and surface properties, enabling evasion of MPS clearance and promoting prolonged bloodstream circulation, thereby improving the signal-to-background ratio for imaging. The effectiveness of our design is demonstrated by substantial advancements in the early diagnosis of acute kidney injury and by providing high-contrast imaging and precise surgical navigation across various tumor models. Our strategy not only advances optical imaging materials toward clinical translation but also establishes a versatile platform applicable to multiple imaging modalities.

Selective Recognition of the Dimeric NG16 Parallel G-Quadruplex Structure Using Synthetic Turn-On Red Fluorescent Protein Chromophore.

Red fluorescent protein (RFP)-based fluorescent probes that can selectively interact with specific nucleic acids are of great importance for therapeutic and bioimaging applications. Herein, we have reported the synthesis of RFP chromophores for selective recognition of G-quadruplex nucleic acids in vitro and ex vivo. We identified DFHBI-DM as a fluorescent turn-on probe that binds to the dimeric NG16 parallel quadruplex with superior selectivity and sensitivity over various parallel, antiparallel, and hybrid topologies. The binding of DFHBI-DM to NG16 exhibited excellent photophysical properties, including high binding affinity, large Stokes shift, high photostability, and quantum yield. The MD simulation study supports the 1:1 binding stoichiometry. It confirms the planar conformation of DFHBI-DM, which makes strong binding interactions with a flat quartet of NG16 compared to other antiparallel and hybrid topologies. The cell imaging and MTT assays revealed that DFHBI-DM is a biocompatible and efficient fluorescent probe for intracellular imaging of NG16. Overall, these results demonstrated that DFHBI-DM could be an effective fluorescent G4-stabilizing agent for the dimeric NG16 parallel quadruplex, and it could be a promising candidate for further exploration of bioimaging and therapeutic applications.

Improved ex vivo fluorescence imaging of human head and neck cancer using the peptide tracer TPP-IRDye800 targeting membrane-bound Hsp70 on tumor cells.

The primary goal of surgery in HNSCC is the complete resection of tumor cells with maximum preservation of normal tissue. The membrane Hsp70-targeting fluorescence labelled peptide TPP-IRDye800 represents a promising tool for real-time intraoperative tumor visualization, enabling the detection of true tumor margins, critical isles of high-grade dysplasia and LN metastases. Membrane Hsp70 (mHsp70) expression on HNSCC cell lines and primary HNSCC was determined by flow cytometry and fluorescence microscopy using FITC-conjugated mAb cmHsp70.1 and TPP. TPP-IRDye800 was sprayed on freshly resected tumor material of immunohistochemically confirmed HNSCC and LN metastases for tumor imaging. TBRs were compared using TPP-IRDye800 and Cetuximab-IRDye680, recognizing EGFR. mHsp70 expressing HNSCC cells specifically bind and internalize TPP in vitro. The TBR (2.56 ± 0.39) and AUC [0.98 CI, 0.95-1.00 vs. 0.91 CI, 0.85-0.97] of TPP-IRDye800 on primary HNSCC was significantly higher than Cetuximab-IRDye680 (1.61 ± 0.39) (p = 0.0068) and TPP-IRDye800 provided a superior tumor delineation. Fluorescence imaging showed higher AUC values than a visual inspection by surgeons [0.97 CI, 0.94-1.00 vs. 0.92 CI, 0.88-0.97] (p = 0.048). LN metastases could be visualized using TPP-IRDye800. Real-time tissue delineation was confirmed using the clinically applied KARL-STORZ imaging system. TPP-IRDye800 is a promising fluorescence imaging probe for HNSCC.

Novel anti-CEA affibody for rapid tumor-targeting and molecular imaging diagnosis in mice bearing gastrointestinal cancer cell lines.

Gastrointestinal cancer is a common malignant tumor with a high incidence worldwide. Despite continuous improvements in diagnosis and treatment strategies, the overall prognosis of gastrointestinal tumors remains poor. Carcinoembryonic antigen (CEA) is highly expressed in various types of cancers, especially in gastrointestinal cancers, making it a potential target for therapeutic intervention. Therefore, the expression of CEA can be used as an indication of the existence of tumors, chosen as a target for molecular imaging diagnosis, and effectively utilized in the targeted therapy of gastrointestinal cancers. In this study, we report the selection and characterization of affibody molecules (ZCEA539, ZCEA546, and ZCEA919) specific to the CEA protein. Their ability to bind to recombinant and native CEA protein has been confirmed by surface plasmon resonance (SPR), immunofluorescence, and immunohistochemistry assays. Furthermore, Dylight755-labeled ZCEA affibody showed accumulation within the tumor site 1 h post injection and was continuously enhanced for 4 h. The Dylight755-labeled ZCEA affibody exhibited high tumor-targeting specificity in CEA+ xenograft-bearing mice and possesses promising characteristics for tumor-targeting imaging. Overall, our results suggest the potential use of ZCEA affibodies as fluorescent molecular imaging probes for detecting CEA expression in gastrointestinal cancer.

Excitation energy transfer based naphthalimide-boron-dipyrromethene dyads as two-photon fluorescent probes for palladium(0) detection and live cell imaging

Increasing industrial demand has accelerated the consumption and contamination of palladium (Pd) worldwide. The implementation of fluorescent probes, especially with two-photon excitation, holds great promise for Pd detection in environmental and biological analysis. In this work, two naphthalimide (NPI)-boron-dipyrromethene (BDP) dyads (named NBH and NBN) were developed as two-photon fluorescent probes for Pd(0) detection based on an excitation energy transfer (EET) strategy, employing NPI as the energy donor and BDP as the acceptor. Spectroscopic studies revealed that both probes exhibited a fast response to Pd(0) in a pronounced fluorescence "ON-OFF" manner. The Pd-catalyzed Tsuji-Trost reaction activated an intramolecular photoinduced electron transfer (PET) process between the aniline and BDP moieties, resulting in the fluorescence quenching of the BDP fluorophore. The reaction-based probes exhibited excellent sensitivity and selectivity for Pd(0) species with a detection limit as low as 2.4 nM. These probes were successfully applied to the determination of Pd residues in drug, soil, and water samples. A simple smartphone-based platform was also constructed to determine Pd(0) via an RGB reader. With negligible cytotoxicity, confocal imaging study validated their feasibility of monitoring Pd(0) in living cells through multiple excitation channels.

The Influence of the Functional Group on the Physicochemical and Biological Properties of New Phenanthro[9,10-d]-Imidazole Derivatives.

The search for safe, cheap, and repeatable diagnostic methods is a fundamental research goal. Currently, great hope is placed on fluorescence imaging. However, the development of this method mainly depends on efficient fluorescent probes. Designing and obtaining new probes with potential applications in fluorescence imaging is very difficult because compounds of this type must meet several requirements related to their properties. Therefore, this article attempted to obtain and study new phenanthro[9,10-d]-imidazole derivatives (PK1-PK3) with potential application as fluorescent probes for fluorescence imaging. The main goal of the work was to assess the effect of two functional groups (such as the formyl group (PK2) and rhodanine-3-acetic acid (PK3)) on selected physicochemical properties and possibilities of practical application of the considered compounds. The conducted studies proved that the influence of the functional group is significant, as it causes a bathochromic shift in both absorption and emission results (by the order PK1 < PK2 < PK3). Moreover, all compounds could stain live cells cultured in vitro. The staining efficiency was not affected by the cell line, thanks to which we obtained the correct staining of both mouse and human cell lines. PK3 was the most attractive of the tested compounds due to its staining potential of live cells and retention after fixation. Our results also showed some antibacterial and antifungal activity of the newly synthesized compounds (PK1-PK3). Among them, PK3 showed the highest antimicrobial effect, especially against Gram-positive bacteria.

Water-soluble sulfur quantum dots as a potential sensitive fluorescent probe for quercetin detection and cell imaging

A simple fluorescent quenching probe based on polyethylene glycol-400 capped sulfur quantum dots (PEG-SQDs) was fabricated to determine quercetin (QT) quantitatively. As anticipated, the PEG-SQDs exhibited favourable luminescent properties, stability and low cytotoxicity. QT effectively quenched the fluorescence of the PEG-SQDs through static quenching and the inner filter effect. Moreover, the PEG-SQDs showed rapid QT detection within a linear range of 0.100–45.0 μM, with a limit of detection of 0.014 μM (3σ/k). This fluorescent probe successfully detected QT in human serum, quercetin supplement capsules and red wine, achieving a standard recovery of 92.6 %–105 %.

An intramolecular charge transfer based fluorescent probe for imaging of OCl–

The discovery and utilization of new fluorescent chromophore is indispensable to exploit high performance probes for biological research. Stokes shift is one of the most important properties of chromophore accounting for super-resolution fluorescence imaging. Intramolecular charge transfer (ICT) is one of the fundamental mechanisms for fluorescence that accompanied by large Stokes shifts. Based on the conformational changes between ground and excited states, ICT models can be divided into two types: conformation-steady ICT, whose conformation remains unchanged, and conformation-changeable ICT, which is characterized by the rotation of the chromophore around an axis upon excitation. Herein, we report a new chromophore whose donor and acceptor parts took a butterfly geometry with a dihedral angle of 21° in ground state and a planar conformation upon photo excitation. The bent conformation might be ascribed to the extra conjugated double bond, which made the coplanarity of the chromophore in ground state get worse. The chromophore shows a remarkable Stokes shift over 150 nm and a high fluorescence quantum yieldof 0.62. The limit of detection is 41 nM, which enabled the imaging of basal as well as induced OCl– in different cells. Moreover, the pronounced spectroscopic properties ensure the in vivo monitoring of OCl– in arthritic mice. This finding would shed light on the exploitation of small molecule probes based on new fluorescence chromophore for precise biological imaging.

Renal-Clearable Organic Probes From D-A-D Type Aza-BODIPY Fluorophores for Multiphoton Deep-Brain Imaging.

Bright near-infrared (NIR) fluorescent probes play an important role in in vivo optical imaging. Here, renal-clearable nanodots prepared from Aza-BODIPY are reported fluorophores for multiphoton brain imaging. The design of donor-acceptor-donor (D-A-D) type conjugated structures endowed the fluorophores with large three-photon absorption cross-section for both 1620 and 2200nm excitation. The side chain modification and lipid encapsulation yield ultrasmall nanodots (≈4nm) and a high fluorescence quantum yield (≈0.35) at 720nm emission in the aqueous phase. The measured three-photon action cross-section of a single Aza-BODIPY fluorophore in the nanodots is ≈30 times higher than the commonly used Sulforhodamine 101 dye. Three-photon deep brain imaging of subcortical structures is demonstrated, reaching a depth of 1900µm below the brain surface in a live mouse study. The nanodots enabled blood flow measurement at a depth of 1617µm using line scanning three-photon microscopy (3PM). This work provides superior fluorescent probes for multiphoton deep-brain imaging.

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.

A dual-responsive fluorescent probe for cellular hypoxia and tumor imaging based on nitroreductase and viscosity synergy

Nitroreductase (NTR), an enzyme that operates within the cytoplasm, relies on flavin mononucleotide or flavin adenine dinucleotide for its activity. In tumor cells, hypoxic conditions often lead to an increase in NTR levels. Consequently, the development of fluorescent probes capable of detecting NTR activity is crucial for the monitoring of hypoxia in biological systems. This study presents the one-step synthesis of a near-infrared fluorescent probe, DSM-NO2. The probe was highly sensitive to NTR and caused a fluorescence enhancement by 50-fold, with a detection limit as low as 1 ng/mL. Under hypoxic conditions in tumor cells, not only does NTR increase, but the cellular viscosity also rises. In high-viscosity environments, the fluorescence enhancement could reach up to 220-fold, and the detection limit could be further reduced to 55 pg/mL. The reaction mechanism was thoroughly investigated using mass spectrometry, molecular docking, and theoretical calculations. Moreover, the probe was successfully applied in the real-time monitoring and visualization analysis of NTR during the growth of Escherichia coli and in the hypoxic state of cancer cells. Additionally, through nebulized inhalation, the probe was successfully employed for in situ fluorescent imaging studies in a murine model of lung cancer.

Molecular engineering of BODIPY-bridged fluorescent probes for lysosome imaging - a computational study.

Lysosome imaging plays an important role in diagnosing many diseases and understanding various intracellular processes. Recently, B0 was reported as a fluorescent probe capable of detecting lysosomal viscosity changes. BODIPY is fused into the molecule as a bridge between the acceptor and donor components of B0, yielding nine new B molecules. Computational design and analysis of their optoelectronic properties were conducted to evaluate their effectiveness as fluorescent probes for lysosome imaging, with a specific target of HSA inside lysosomes. Optimized geometries reveal excellent π electron delocalization, resulting in nearly planar molecular structures. Frontier molecular orbital analysis suggests intramolecular charge transfer, along with π-π* transitions, from donor to bridge. TD-DFT calculations were performed to study absorption properties in the solvent phase, with B3PW91 showing good agreement with experiments. Molecular docking studies indicate that B derivatives can bind with HSA, and molecular dynamics simulations confirm their HSA targeting ability. This investigation highlights the introduction of BODIPY as a bridge for developing new probes capable of producing NIR fluorescence for bio-imaging, aiding in disease diagnosis.

Dual-responsive two-photon probe for specific lipid droplets near-infrared fluorescence imaging in the brain of epileptic mice

Abnormal lipid metabolism in glial cells is a key pathological feature of epilepsy. The identification of lipid droplets (LDs) is essential for investigating lipid metabolism, disease progression, and potential therapeutic interventions. Two-photon imaging technology enables real-time visualization of the spatial distribution and temporal dynamics of LDs in epilepsy models. In this study, we developed a novel two-photon excited dual-responsive near-infrared fluorescent probe, CabA, based on viscosity and polarity, to monitor dynamic changes in LDs. The fluorescence of CabA at 670 nm exhibits a significant increase in response to low polarity and high viscosity due to the twisted intramolecular charge transfer and intramolecular charge transfer mechanisms. The LDs-targeting capability of CabA at the cellular level and the process of LDs generation between neurons and astrocytes during the pathological advancement of epilepsy have been validated. In situ synchronous imaging experiments in epileptic and normal mice using CabA revealed abnormal LDs accumulation in the brain during seizures. Two-photon fluorescence imaging further demonstrated LDs accumulation in the brains of epileptic mice at a penetration depth of 100 μm. This study offers a valuable tool for enhancing the understanding of LDs in physiological and pathological processes, potentially aiding in the early diagnosis of epilepsy.

Catalyst Free Synthesis of 4‐Aryl/Alkylamino‐1,2‐Naphthoquinones: Applications in Fluorescence Microscopy

AbstractAn efficient one‐step reagent‐free synthesis of 4‐Aryl/alkylamino‐1,2‐naphthoquinones in water and iPrOH (1 : 1) mixture was carried out at room temperature (RT) using 4‐amino‐3‐hydroxynaphthalene‐1‐sulfonic acid and various primary and secondary aryl and alkyl amines. The reaction did not proceed in the absence of air or oxygen. In all cases, good yields (isolated yield: ≥65 %) were observed. The compounds exhibited fluorescence emission bands (400–470 nm) in the visible region upon excitation at their respective λmax values (330–350 nm). We evaluated in vitro cytotoxicity of some of the derivatives against two human breast cancer cell lines (MCF‐7, and MDA‐MB‐231) using the WST‐1 assay. The naphthoquinone derivatives did not show any cytotoxic effects at concentration levels relevant to cellular fluorescence imaging. The imaging experiments revealed that a few of the derivatives hold the potential to be used as cellular fluorescent imaging probes.

Thiol-chromene click reaction-triggered mitochondria-targeted ratiometric fluorescent probe for intracellular biothiol imaging.

Chromene as the efficient biothiol recognition site was widely used to develop fluorescent probes based on thiol-chromene click reaction. However, chromene-based fluorescent probes with the both properties of ratiometric measurement and mitochondria-targeted function have not been reported and remain challenging. In this paper, we skillfully designed and synthesized the first mitochondria-targeted ratiometric fluorescent probe (Probe 1) for biothiols based on chromene. Upon addition of biothiols (Cys, Hcy, and GSH), the absorption and fluorescence spectra of Probe 1 changed from 490 to 426nm and from 567 to 498nm respectively, accompanied by color changes from orange to pale yellow under natural light and from orange to blue under a 365-nm UV lamp, which can be attributed to the click reaction of biothiols with α,β-unsaturated ketone of chromene moiety, subsequent pyran ring-opening, and phenol formation as well as 1,6-elimination of p-hydroxybenzyl moiety. Probe 1 not only exhibited high sensitivity (LODs of 149nM, 133nM, and 116nM for Cys, GSH, and Hcy respectively), rapid response, and excellent selectivity for biothiols (Cys, Hcy, and GSH), but also could target in mitochondria and ratiometrically image the fluctuation of intracellular biothiols. Moreover, the novel design strategy of modifying chromene to the N atom of pyridine was proposed for the first time.

Activatable near-infrared fluorescence probe for real-time imaging of PD-L1 expression in tumors.

In clinical practice, determining programmed death-ligand 1 (PD-L1) expression is crucial for selecting patients and monitoring immune checkpoint blockade therapies. Currently, PD-L1 expression is quantified using immunohistochemistry (IHC). However, IHC-based methods do not capture the heterogeneous and dynamic nature of PD-L1 expression. Thus, there is a pressing need for a rapid and efficient method for monitoring PD-L1 expression both in vitro and in vivo, which would considerably aid in prognosis and treatment selection. In this study, we present for the first time an activatable near-infrared (NIR) fluorescence imaging probe (Q-Atezol) for the real-time monitoring of PD-L1 expression in vitro and in vivo. The ability of Q-Atezol to detect PD-L1 expression quickly and in real-time was evaluated in both tumor spheroid and lung cancer xenograft models. An always-on optical probe (ON-Atezol) was synthesized and tested for comparison. In vivo NIR fluorescence imaging studies were conducted on A549 and H1975 tumor-bearing mice, and their tumor-to-background ratios (TBRs) were analyzed. The quenched NIR fluorescence of Q-Atezol is activated upon binding to PD-L1 proteins on the surface of cancer cells, thereby enabling PD-L1 detection in the three-dimensional (3D) tumor spheroids without a washing step. Notably, PD-L1-positive H1975 tumors were clearly visualized with a high TBR 6 hours after Q-Atezol injection, whereas ON-Atezol treatment could not detect H1975 tumors even 24 hours post-injection. The activatable fluorescence probe Q-Atezol demonstrated great potential as an exceptional sensor for assessing PD-L1 expression in 3D cell structures and for in vivo applications.

BOPHY Derivatives as Phototherapeutic Agents for the Photodynamic Inactivation of Microorganisms

AbstractThe improvement of photodynamic inactivation (PDI) significantly depends on the development of new families of photosensitizers (PSs). In this sense, three BOPHY derivatives (BP, BP‐Br and BP‐I) were synthetized, studied, and compared to assess their antimicrobial photodynamic properties. BP is an interesting fluorescent probe for cell imaging, while the halogenated analogs (BP‐Br and BP‐I) are excellent oxygen photosensitizing agents. BP compound presented a fluorescence quantum yield close unity and showed no reactive oxygen species (ROS) production. In contrast, BP‐I did not show emission properties but exhibited a high production of ROS through both photodynamic mechanisms, generating singlet oxygen (type II) and superoxide radical anion (type I) under aerobic light irradiation. BP‐Br presented an adequate balance between ROS production and emission properties. The photokilling action and the binding to bacterial cells of these macrocycles were evaluated in vitro against methicillin‐resistant Staphylococcus aureus (MRSA) and Escherichia coli bacteria. Our results demonstrated that the halogenated BOPHY derivatives were effective PSs in inactivating MRSA using shorter irradiation periods. In addition, the antimicrobial action sensitized by these BOPHYs was potentiated by adding KI. The combination of halogenated BOPHY and KI led to a complete elimination of both Gram‐positive and Gram‐negative bacteria. Hence, BP‐Br and BP‐I prove to be potent broad‐spectrum antimicrobial PSs. To the best of our knowledge, this is the first time that BOPHY derivatives have been applied to photokill pathogenic microorganisms.