Dye Cy5 Research Articles

Quantitative Analysis of Hepatitis D Virus Using gRNA-Sensitive Semiconducting Polymer Dots.

Hepatitis D virus (HDV) significantly influences the progression of liver diseases. Through clinical observations and database analyses, it has been established that patients coinfected with HDV and hepatitis B virus (HBV) experience accelerated progression toward cirrhosis, hepatocellular carcinoma (HCC), and liver failure compared to those infected solely with HBV. A higher viral load correlates with increased replicative activity, enhanced infectivity, and more severe disease manifestations. In this study, we use HDV gRNA-sensitive semiconducting polymer dots (Pdots) as the nanoprobes for the quantitative analysis of HDV copy number variations. The surface of the Pdots is engineered with a clamp design that includes a pair of reporter sequences, protection sequences, and capture sequences tailored to the conserved sequence length of the HDV genome. The capture sequence, comprising leading and trailing chains, specifically binds to the gRNA of the target virus. The protection sequence shields the Pdots from external interference, while the reporter sequence detects the presence of target gRNA through the degradation of fluorescent dye Cy5.5dt. We demonstrate the effectiveness of this assay in a stably transfected cell line derived from HepG2-HDV cells and its translational application in clinical samples from patients. Additionally, this nanobiosensor can accurately detect gRNA at femtomolar (fM) levels, a sensitivity unachievable by previously reported methods. This novel virus quantification system offers significant potential for clinical and virological applications, enhancing screening, early diagnosis, and personalized treatment strategies.

An oxygen-scavenging system without impact on DNA mechanical properties in single-molecule fluorescence experiments.

Oxygen scavenging systems (OSSs) are critical for dye stability in single-molecule fluorescence (SMF) experiments. However, the commonly used protocatechuic acid (PCA)/protocatechuate-3,4-dioxygenase (PCD) OSS alters DNA mechanical properties, limiting its applicability. To address this limitation, we examine the bilirubin oxidase (BOD) OSS, which had not been previously used in single-molecule experiments, alongside the pyranose oxidase and catalase (POC) OSS. Our results revealed that POC OSS affected DNA mechanics in a buffer-dependent manner, while BOD OSS had no discernible effect across all tested buffer conditions. Furthermore, BOD OSS significantly extended the photobleaching lifetimes of Cy3 and Cy5 dyes and caused minimal pH changes compared to PCD OSS. Collectively, these findings highlight the superior performance of BOD OSS, suggesting its potential for widespread application, particularly in experiments combining SMF with single-molecule force spectroscopy (SMFS) measurements.

Super-Resolved Fluorescence Lifetime Imaging of Single Cy3 Molecules and Quantum Dots Using Time-Correlated Single Photon Counting with a Four-Pixel Fiber Optic Array Camera.

Time-resolved single molecule localization microscopy (TR-SMLM) with a 2 × 2 pixel fiber optic array camera was combined with time-correlated single photon counting (TCSPC) to obtain super-resolved fluorescence lifetime images of individual Cy3 dye molecules and individual colloidal CdSe/CdS/ZnS core/shell/shell semiconductor quantum dots (QDs). The characteristic blinking and bleaching behavior of the Cy3 and the blinking behavior of the QD emitters were used as distinguishing optical characteristics to isolate them and determine their centroid locations with spatial resolution below the optical diffraction limit. TCSPC was used to characterize the fluorescence lifetime and intensity corresponding to each emitter location. The mean centroid locations of the QDs could be determined with a precision of ∼1-4 nm, and the mean centroid locations of the Cy3 molecules could be determined with a precision of ∼2-9 nm, depending on the number of photons collected during the observation time. In a super-resolved fluorescence lifetime image with a single Cy3 dye molecule and a single QD separated by ∼34 nm, the two emitters were distinguished based on the average photon arrival times with respect to the excitation laser pulse observed during time intervals when only one emitter was in the on state, ∼6 ns for Cy3 and ∼17 ns for the QD. The mean distance between the two emitters was determined with a precision of ∼8 nm. The feasibility of using this super-resolved fluorescence lifetime imaging technique to investigate QD-dye complexes that use Förster resonance energy transfer (FRET) and/or electron transfer to form optical biosensors is discussed.

Near-infrared fluorescent molecular probes with cetuximab in the in vivo fluorescence imaging for epithelial ovarian cancer

BackgroundNear-infrared fluorescence (NIRF) imaging is an excellent choice for image-guided surgery due to its simple operation and non-invasiveness. Developing tumor-specific fluorescent molecular probes is key to fluorescence imaging-guided surgery. EGFR (epidermal growth factor receptor) is closely related to the proliferation and growth of tumor cells and is highly expressed in epithelial ovarian cancer (EOC). The study aims to construct a NIR fluorescent molecular probe using cetuximab (an EGFR monoclonal antibody) and investigate its feasibility for targeting EOC in vivo through fluorescence imaging.MethodsWe determined the expression of EGFR in EOC. NIR fluorescent molecular probe with cetuximab (cetuximab-Cy7) was chemically engineered and identified. The subcutaneous xenografted tumor model of EOC was induced using SKOV3-Luc cell line with positive expression of EGFR. Cetuximab-Cy7 was used for in vivo fluorescence imaging, and phosphate-buffered saline, free Cy7 dye and mouse isotype immunoglobulin G-Cy7 were used as controls. NIRF imaging system was performed to study the distribution and targeting of the probes. Tumors were imaged in situ and ex vivo, and fluorescent intensity was quantified. Resected specimens were analyzed to confirm diagnosis, and immunohistochemical (IHC) staining was used to identify EGFR expression.ResultsEGFR expression was increased in EOC tissues than fallopian tube tissues. The high expression of EGFR was significantly correlated with well-differentiation, residual lesions ≤ 1 cm, no recurrence and increased survival. NIRF imaging showed that the cetuximab-Cy7 enabled detection of tumor lesions in EOC-bearing mice with the optimal dose of 30 µg. The suitable imaging time window may be 24–96 h post-injection. Ex vivo fluorescence imaging indicated that fluorescent signal was mainly detected in the tumor and the lung. IHC results confirmed that xenografts were EGFR positive.ConclusionCetuximab-Cy7 can specifically target the tumors of EOC xenografted nude mice. This research lays the foundation for future studies on EOC surgery navigation.

Atom‐Efficient Synthesis of Trimethine Cyanines Using Formaldehyde as a Single‐Carbon Source

AbstractHerein, we present an innovative and atom‐efficient synthesis of trimethine cyanines (Cy3) using formaldehyde (FA) as a single‐carbon reagent. The widespread application of Cy3 dyes in bioimaging and genomics/proteomics is often limited by synthetic routes plagued by low atom economy and substantial side‐product formation. Through systematic investigation, we have developed a practical and efficient synthetic pathway for both symmetrical and unsymmetrical Cy3 derivatives, significantly minimizing resource utilization. Notably, this approach yields water as the by‐product, in alignment with sustainable chemistry principles. Moreover, the efficient one‐pot synthesis facilitates the detection of intracellular FA levels, utilizing the fluorescence signal of Cy3 in live cells. It is also possible to detect endogenous FA in the intestinal tissues. We observed a significant decrease in FA in the small intestine of inflammatory bowel disease (IBD) mice as compared to healthy mice. This methodological advancement not only enhances the scope of fluorescent dye synthesis but also contributes to sustainable practices within chemical manufacturing, offering a significant leap forward in the development of environmentally friendly synthetic strategies.

Atom-Efficient Synthesis of Trimethine Cyanines Using Formaldehyde as a Single-Carbon Source.

Herein, we present an innovative and atom-efficient synthesis of trimethine cyanines (Cy3) using formaldehyde (FA) as a single-carbon reagent. The widespread application of Cy3 dyes in bioimaging and genomics/proteomics is often limited by synthetic routes plagued by low atom economy and substantial side-product formation. Through systematic investigation, we have developed a practical and efficient synthetic pathway for both symmetrical and unsymmetrical Cy3 derivatives, significantly minimizing resource utilization. Notably, this approach yields water as the by-product, in alignment with sustainable chemistry principles. Moreover, the efficient one-pot synthesis facilitates the detection of intracellular FA levels, utilizing the fluorescence signal of Cy3 in live cells. It is also possible to detect endogenous FA in the intestinal tissues. We observed a significant decrease in FA in the small intestine of inflammatory bowel disease (IBD) mice as compared to healthy mice. This methodological advancement not only enhances the scope of fluorescent dye synthesis but also contributes to sustainable practices within chemical manufacturing, offering a significant leap forward in the development of environmentally friendly synthetic strategies.

Tumor agnostic ultrasmall nanoprobes for fluorescence-guided surgical resection in peritoneal metastasis.

Surgical excision of metastases is the only curative treatment strategy in peritoneal carcinomatosis management, and the completeness of tumor resection determines the success of the surgery. Tumor-specific fluorescence-guided probes can improve the outcomes of cytoreductive surgery and thereby prognosis. This study aimed to develop and evaluate the feasibility of fluorescently labeled ultrasmall porous silica nanoparticles (UPSN) for image-guided resection of peritoneally disseminated tumors of different origins. Ultrasmall fluorescent nanoprobes were synthesized and characterized for their physicochemical properties and stability. Tumor-specific uptake and biodistribution profiles were evaluated in syngeneic CT26 colorectal and KPC-689 pancreatic cancer murine models. The practicability of real-time optical UPSN-guided resection was examined in the CT26 colorectal cancer model using a surgical stereomicroscope. Quantitative measurements of tumor sensitivity and specificity were performed. Histopathological examination validated in vivo findings about tumor-specific accumulation and safety of ultrasmall fluorescent probes. As-synthesized UPSNs were successfully surface modified with Cy5 or Cy3 dyes maintaining sub-15nm size and near neutral charge which is beneficial for optimized in vivo pharmacokinetics. UPSN-Cy5 demonstrated high tumor-specific uptake and favorable biodistribution profiles in peritoneal metastasis models of CT26 and KPC tumors. Dye-conjugated UPSN enabled resection of microscopic lesions and achieved a higher tumor-to-background ratios in comparison to FDA-approved indocyanine green (ICG) dye in both models. Microscopic evaluation showed tumor localization and off-target safety profile of the UPSN-Cy5. Ultrasmall fluorescent probes were effective in surgical resection of peritoneal metastases with high sensitivity and specificity, thus emerging as promising tumor agnostic agents for image-guided cancer surgery.

A PD-L1-Targeted Probe Cy5.5-A11 for In Vivo Imaging of Multiple Tumors.

PD-L1 is an immune checkpoint molecule mediating cancer immune escape, and its expression level in the tumor has been used as a biomarker to predict response to immune checkpoint inhibitor (ICI) therapy. Our previous study reveals that an 11 amino acid-long ANXA1-derived peptide (named A11) binds and degrades the PD-L1 protein in multiple cancers and is a potential peptide for cancer diagnosis and treatment. Near-infrared fluorescence (NIF) optical imaging of tumors offers a noninvasive method for detecting cancer and monitoring therapeutic responses. In this study, an NIF dye Cy5.5 was conjugated with A11 peptide to develop a novel PD-L1-targeted probe for molecular imaging of tumors and monitor the dynamic changes in PD-L1 expression in tumors. In vitro imaging studies showed that intense fluorescence was observed in triple-negative breast cancer MDA-MB-231, nonsmall cell lung cancer H460, and melanoma A375 cells incubated with Cy5.5-A11, and the cellular uptake of Cy5.5-A11 was efficiently inhibited by coincubation with unlabeled A11 or knockdown of cellular PD-L1 by shRNA. In vivo imaging studies showed accumulation of Cy5.5-A11 in the MDA-MB-231, H460, and A375 xenografts with good contrast from 0.5 to 24 h after intravenous injection, indicating that Cy5.5-A11 possesses the strong ability for in vivo tumor imaging. Moreover, the fluorescent signal of A11-Cy5.5 in the xenografts was successfully blocked by coinjection of unlabeled A11 peptide or knockdown of cellular PD-L1 by shRNA, indicating the specificity of Cy5.5-A11 targeting PD-L1 in tumor imaging. Our data demonstrate that Cy5.5-A11 is a novel tool for tumor imaging of PD-L1, which has the potential for detecting cancer and predicting ICI therapeutic responses.

Red Fluorescent Aminoferrocene (Pro)Drugs for in Cellulo and in Vivo Imaging.

Red fluorescent dyes are usually charged, lipophilic molecules with relatively high molecular weight, which tend to localize in specific intracellular locations, e. g., a cyanine dye Cy5 is biased towards mitochondria. They are often used as markers of biomolecules including nucleic acids and proteins. Since the molecular weight of the dyes is much smaller than that of the biomolecules, the labelling has a negligible effect on the properties of the biomolecules. In contrast, conjugation of the dyes to low molecular weight (pro)drugs can dramatically alter their properties. For example, conjugates of Cy5 with lysosome-targeting aminoferrocenes accumulate in mitochondria and exhibit no intracellular effects characteristic for the parent (pro)drugs. Herein we tested several neutral and negatively charged dyes for labelling lysosome-targeting aminoferrocenes 7 and 8 as well as a non-targeted control 3. We found that a BODIPY derivative BDP-TR exhibits the desired unbiased properties: the conjugation does not disturb the intracellular localization of the (pro)drugs, their mode of action, and cancer cell specificity. We used the conjugates to clarify the mechanism of action of the aminoferrocenes. In particular, we identified new intermediates, explained why lysosome-targeting aminoferrocenes are more potent than their non-targeted counterparts, and evaluated their distribution in vivo.

Molecular Dynamical and Quantum Mechanical Exploration of the Site-Specific Dynamics of Cy3 Dimers Internally Linked to dsDNA.

Performing spectroscopic measurements on biomolecules labeled with fluorescent probes is a powerful approach to locating the molecular behavior and dynamics of large systems at specific sites within their local environments. The indocarbocyanine dye Cy3 has emerged as one of the most commonly used chromophores. The incorporation of Cy3 dimers into DNA enhances experimental resolution owing to the spectral characteristics influenced by the geometric orientation of excitonically coupled monomeric units. Various theoretical models and simulations have been utilized to aid in the interpretation of the experimental spectra. In this study, we employ all-atom molecular dynamics simulations to study the structural dynamics of Cy3 dimers internally linked to the dsDNA backbone. We used quantum mechanical calculations to derive insights from both the linear absorption spectra and the circular dichroism data. Furthermore, we explore potential limitations within a commonly used force field for cyanine dyes. The molecular dynamics simulations suggest the presence of four possible Cy3 dimeric populations. The spectral simulations on the four populations show one of them to agree better with the experimental signatures, suggesting it to be the dominant population. The relative orientation of Cy3 in this population compares very well with previous predictions from the Holstein-Frenkel Hamiltonian model.

DNA-mediated assembly of Au bipyramids into anisotropic light emitting kagome superlattices.

Colloidal crystal engineering with DNA allows one to design diverse superlattices with tunable lattice symmetry, composition, and spacing. Most of these structures follow the complementary contact model, maximizing DNA hybridization on building blocks and producing relatively close-packed lattices. Here, low-symmetry kagome superlattices are assembled from DNA-modified gold bipyramids that can engage only in partial DNA surface matching. The bipyramid dimensions and DNA length can be engineered for two different superlattices with rhombohedral unit cells, including one composed of a periodic stacking of kagome lattices. Enabled by the partial facet alignment, the kagome lattices exhibit lattice distortion, bipyramid twisting, and planar chirality. When conjugated with Cy-5 dyes, the kagome lattices serve as cavities with high-density optical states and large Purcell factors along lateral directions, leading to strong dipole radiation along the z axis and facet-dependent light emission. Such complex optical properties make these materials attractive for lasers, displays, and quantum sensing constructs.

Mechanistic Insight into the Thermal "Blueing" of Cyanine Dyes.

In recent work to develop cyanine dyes with especially large Stokes shifts, we encountered a "blueing" reaction, in which the heptamethine cyanine dye Cy7 (IUPAC: 1,3,3-trimethyl-2-((1E,3E,5E)-7-((E)-1,3,3-trimethylindolin-2-ylidene)hepta-1,3,5-trien-1-yl)-3H-indol-1-ium) undergoes shortening in two-carbon steps to form the pentamethine (Cy5) and trimethine (Cy3) analogs. Each step blue-shifts the resulting absorbance wavelength by ca. 100 nm. Though photochemical and oxidative chain-shortening reactions had been noted previously, it is simple heating alone or with amine bases that effects this unexpected net C2H2 excision. Explicit acetylene loss would be too endothermic to merit consideration. Our mechanistic studies using 2H labeling, mass spectrometric and NMR spectroscopic analyses, and quantum chemical modeling point instead to electrocyclic closure and aromatization of the heptamethine chain in Cy7 forming Fischer's base FB (1,3,3-trimethyl-2-methyleneindoline), a reactive carbon nucleophile that initiates chain shortening of the cyanine dyes by attack on their polymethine backbones. The byproduct is the cationic indolium species TMP (IUPAC: 1,3,3 trimethyl-2-phenyl indolium).

Screening of Methyl-β-cyclodextrins as an Antifading Agent for Cyanine Dye-Labeled Streptavidin to Improve the Performance of Genotyping Chips.

As a photostabilizing agent for cyanine dye, methyl-β-cyclodextrin (MβCD) was investigated as a possible antifading agent for cyanine dye-labeled proteins. Cyanine-3 (Cy3)-labeled streptavidin (SA-Cy3) solutions containing MβCD exhibited improved resistance against photobleaching. Further research revealed that MβCD can be used as a coating material on the surface of gene chips. Chips loaded with cyanine dye (Cy3 and Cyanine-5 (Cy5))-conjugated model microbeads exhibited resistance against photobleaching with MβCD coatings. MβCD coatings improved the imaging quality of model chips and resulted in higher discrimination ratios (DR) of single base recognition by a set of control beads (NP68). In a whole genome genotyping assay for human samples, the MβCD-coated samples were found to have a better clustering performance than the noncoated ones for a group of randomly picked single nucleotide polymorphisms (SNPs).

An antioxidative-enhanced endoplasmic reticulum-targeted cyanine dye for efficient tumor immunotherapy

Excessive generation of reactive oxygen species (ROS) triggers intense endoplasmic reticulum (ER) stress, emerging as a key mechanism for arousing autoimmune therapy. Therefore, the development of an ER-targeted ROS generator is crucial for advancing self-immunotherapy. However, despite a clear demand for ER-targeting ROS generators, the high-efficiency agents are still limited. In this study, the pentamethine cyanine dye (Cy5) is used as a parent structure to explore the efficient ER-targeted immune activator. Three variants (PCy5-H, PCy5-Cl, and PCy5-2Cl) were synthesized by introducing benzene, chlorobenzene, and dichlorobenzene structures at the meso-position of the Cy5 scaffold. The electron-withdrawing groups (EWGs) effectively reduce the electron density on the conjugate chain, facilitating the intersystem crossing (ISC) process and enhancing the antioxidant capability. Consequently, the antioxidant properties and ROS generation capacity of PCy5-2Cl were enhanced. Furthermore, encapsulating PCy5-2Cl into liposomes significantly improves its retention at the tumor site, inducing robust ER stress. Upon accumulation at the ER sites in tumor cells, PCy5-2Cl nanoparticles induced strong ER stress under near-infrared (NIR) light irradiation. Exposure to necrotic fragments reverses the immunosuppressive microenvironment within the tumor, ultimately promoting dendritic cell maturation and infiltration of cytotoxic T lymphocytes (CTLs). As a result, significant inhibition is observed in both primary and distant tumors, underscoring the efficacy of this EWGs modification strategy for enhancing tumor immunotherapy.

Energy Transfer Between i-Motif DNA Encapsulated Silver Nanoclusters and Fluorescein Amidite Efficiently Visualizes the Redox State of Live Cells.

The redox regulation, maintaining a balance between oxidation and reduction in living cells, is vital for cellular homeostasis, intricate signaling networks, and appropriate responses to physiological and environmental cues. Here, a novel redox sensor, based on DNA-encapsulated silver nanoclusters (DNA/AgNCs) and well-defined chemical fluorophores, effectively illustrating cellular redox states in live cells is introduced. Among various i-motif DNAs, the photophysical property of poly-cytosines (C20)-encapsulated AgNCs that sense reactive oxygen species (ROS) is adopted. However, the sensitivity of C20/AgNCs is insufficient for evaluating ROS levels in live cells. To overcome this drawback, the ROS sensing mechanism of C20/AgNCs through gel electrophoresis, mass spectrometry, and small-angle X-ray scattering is primarily defined. Then, by tethering fluorescein amidite (FAM)and Cyanine 5 (Cy5)dyes to each end of the C20/AgNCs sensor, an Energy Transfer (ET) between AgNCs and FAM is achieved, resulting in intensified green fluorescence upon ROS detection. Taken together, the FAM-C20/AgNCs-Cy5 redox sensor enables dynamic visualization of intracellular redox states, yielding insights into oxidative stress-related processes in live cells.

A non-spherical nanoparticles system for the delivery of peptides using polymer grafted nanocellulose

AbstractPeptide drugs are increasingly used to treat a variety of diseases ranging from cancer, and infections to cardiovascular diseases. However, peptides can suffer from low stability in the bloodstream. Entrapment of peptides into nano-sized carriers of various types has widely been explored, but all of them have spherical shapes. Nanocellulose can in contrast serve as a non-spherical nanoparticle with a high aspect ratio. After the isolation of nanocellulose by TEMPO-mediated oxidation, the material needs to be modified with polymers to generate nanoparticles with high water-solubility that can also favourably interact with peptide drugs. We have here chosen insulin as the model drug, which can strongly interact with cationic polymers. As it is known that cationic polymer may retain charged drugs too tightly, we have selected poly(2-(dimethylamino)ethyl acrylate) PDMAEA as a degradable polymer that undergoes self-hydrolysis to poly(acrylic acid) in water. This polymer was compared to poly(N-(3-(dimethylamino)propyl) acrylamide) PDMAPAA, which is a stable cationic polymer. The cationic polymer was co-grafted with poly(2-hydroxyethyl acrylate) PHEA as a water-soluble neutral polymer using the three-component Passerini reaction. A combination of fluorescence and UV-Vis techniques were used to quantify the amount of polymer that was conjugated to the surface. The polymer-coated nanocellulose was labelled with the fluorescent cyanine dye Cy5 while insulin was labelled with Cy3 creating a FRET system that allows monitoring of the interaction between insulin and polymer in cell growth media. We observed that despite the self-hydrolysis of PDMAEA into a negatively charged polymer, the negatively charged insulin was not released in buffer solution according to the FRET studies. Only the addition of serum-supplemented cell growth media led to insulin release. The limited release was explained with the fact that insulin, as well as other peptides, have a mixture of negative and positive charges, with the pH value and the isoelectric point determining the balance between both. Negative-charged polymers can therefore still interact favourably with negatively charged peptides by interacting with cationic amino acids. Graphical Abstract

PH-responsive albumin-mimetic synthetic nanoprobes for magnetic resonance/fluorescence imaging of thyroid cancer.

The combination of magnetic resonance and fluorescence imaging in dual-modality imaging not only resolves the limitations of conventional single molecular imaging techniques in terms of specificity, sensitivity, and resolution but also expands the possibilities of molecular imaging techniques in diagnostics and therapeutic monitoring. Herein, a novel pH-responsive magnetic resonance/near-infrared fluorescence (MR/NIRF) nanoprobe (MnO2@BSA-Cy5.5) was successfully prepared by biomineralizing manganese dioxide (MnO2) with bovine serum albumin (BSA) while coupling fluorescent dye Cy5.5 for precise tumor detection and visualization. The synthesized MnO2@BSA-Cy5.5 nanoprobes were spherical particles of approximately 22.62 ± 3.31 nm in size, and their relaxation rates and T1 imaging signals were activated-enhanced in an acidic environment. Cytotoxicity assay and hematoxylin and eosin staining demonstrated that MnO2@BSA-Cy5.5 had low cytotoxicity and good biocompatibility. More importantly, active targeting via solid tumor albumin-binding protein receptor and enhanced permeability and retention effect, the probe can be specifically aggregated to the tumor site of the 8305C tumor model and exhibit excellent MR/NIRF imaging properties. Our results show that MnO2@BSA-Cy5.5 has high resolution and sensitivity in tumor imaging and is expected to be applied as an MR/NIRF contrast agent for accurate diagnosis of thyroid cancer.

Squaraine Dyes Exhibit Spontaneous Fluorescence Blinking That Enables Live-Cell Nanoscopy.

Hampered by their susceptibility to nucleophilic attack and chemical bleaching, electron-deficient squaraine dyes have long been considered unsuitable for biological imaging. This study unveils a surprising twist: in aqueous environments, bleaching is not irreversible but rather a reversible spontaneous quenching process. Leveraging this new discovery, we introduce a novel deep-red squaraine probe tailored for live-cell super-resolution imaging. This probe enables single-molecule localization microscopy (SMLM) under physiological conditions without harmful additives or intense lasers and exhibits spontaneous blinking orchestrated by biological nucleophiles, such as glutathione or hydroxide anion. With a low duty cycle (∼0.1%) and high-emission rate (∼6 × 104 photons/s under 400 W/cm2), the squaraine probe surpasses the benchmark Cy5 dye by 4-fold and Si-rhodamine by a factor of 1.7 times. Live-cell SMLM with the probe reveals intricate structural details of cell membranes, which demonstrates the high potential of squaraine dyes for next-generation super-resolution imaging.

Magnetic-optical dual-modality imaging monitoring chemotherapy efficacy of pancreatic ductal adenocarcinoma with a low-dose fibronectin-targeting Gd-based contrast agent.

Pancreatic ductal adenocarcinoma (PDAC) is a lethal hypovascular tumor surrounded by dense fibrosis. Albumin-bound paclitaxel and gemcitabine (AG) chemotherapy is the mainstay of PDAC treatment through depleting peritumoral fibrosis and killing tumor cells; however, it remains challenging due to the lack of a noninvasive imaging method evaluating fibrotic changes during AG chemotherapy. In this study, we developed a dual-modality imaging platform that enables noninvasive, dynamic, and quantitative assessment of chemotherapy-induced fibrotic changes through near-infrared fluorescence molecular imaging (FMI) and magnetic resonance imaging (MRI) using an extradomain B fibronectin (EDB-FN)-targeted imaging probe (ZD2-Gd-DOTA-Cy7). The ZD2-Gd-DOTA-Cy7 probe was constructed by conjugating a peptide (Cys-TVRTSAD) to Gd-DOTA and the near-infrared dye Cy7. PDAC murine xenograft models were intravenously injected with ZD2-Gd-DOTA-Cy7 at a Gd concentration of 0.05 mmol/kg or free Cy7 and Gd-DOTA as control. The normalized tumor background ratio (TBR) on FMI and the T1 reduction ratio on MRI were quantitatively analyzed. For models receiving AG chemotherapy or saline, MRI/FMI was performed before and after treatment. Histological analyses were performed for validation. The ZD2-Gd-DOTA-Cy7 concentration showed a linear correlation with the fluorescence intensity and T1 relaxation time in vitro. The optimal imaging time was 30min after injection of the ZD2-Gd-DOTA-Cy7 (0.05 mmol/kg), only half of the clinic dosage of gadolinium. Additionally, ZD2-Gd-DOTA-Cy7 generated a 1.44-fold and 1.90-fold robust contrast enhancement compared with Cy7 (P < 0.05) and Gd-DOTA (P < 0.05), respectively. For AG chemotherapy monitoring, the T1 reduction ratio and normalized TBR in the fibrotic tumor areas were significantly increased by 1.99-fold (P < 0.05) and 1.78-fold (P < 0.05), respectively, in the control group compared with those in the AG group. MRI/FMI with a low dose of ZD2-Gd-DOTA-Cy7 enables sensitive imaging of PDAC and the quantitative assessment of fibrotic changes during AG chemotherapy, which shows potential clinical applications for precise diagnosis, post-treatment monitoring, and disease management.

Cy5 Dye Cassettes Exhibit Through-Bond Energy Transfer and Enable Ratiometric Fluorescence Sensing.

The chemosensor literature contains many reports of fluorescence sensing using polyaromatic hydrocarbon fluorophores such as pyrene, tetraphenylethylene, or polyaryl(ethynylene), where the fluorophore is excited with ultraviolet light (<400 nm) and emits in the visible region of 400-500 nm. There is a need for general methods that convert these "turn-on" hydrocarbon fluorescent sensors into ratiometric sensing paradigms. One simple strategy is to mix the responsive hydrocarbon sensor with a second non-responsive dye that is excited by ultraviolet light but emits at a distinctly longer wavelength and thus acts as a reference signal. Five new cyanine dye cassettes were created by covalently attaching a pyrene, tetraphenylethylene, or biphenyl(ethynylene) component as the ultraviolet-absorbing energy donor directly to the pentamethine chain of a deep-red cyanine (Cy5) energy acceptor. Fluorescence emission studies showed that these Cy5-cassettes exhibited large pseudo-Stokes shifts and high through-bond energy transfer efficiencies upon excitation with ultraviolet light. Practical potential was demonstrated with two examples of ratiometric fluorescence sensing using a single ultraviolet excitation wavelength. One example mixed a Cy5-cassette with a pyrene-based fluorescent indicator that responded to changes in Cu2+ concentration, and the other example mixed a Cy5-cassette with the fluorescent pH sensing dye, pyranine.