Search Results (16)

Water quality control employs techniques mostly targeting individual analytes; group detection is also practiced, but the choice of group methods is limited, which supports interest in developing such methods. We have examined the interaction of hypochlorite with a chlorine-containing heptamethine carbocyanine dye in the presence of 30 organic and inorganic model analytes that were found to induce diverse color changes in the system. The main supposed mechanisms are retardation of the dye oxidation with hypochlorite (presumably by scavenging chlorine radicals) and substitution of chlorine atom in the dye by the most nucleophilic analytes (amines, amino acids, proteins, DNA, phenol). The grass-green substitution product is more contrastingly visible against the dark-purple hypochlorite oxidation product of the dye than against the original emerald-green dye. The indicator reaction is monitored photographically for 10–40 min and the images are processed using principal component analysis (PCA) or linear discriminant analysis (LDA), allowing for data convolution for the complex color transitions. Nitrogen compounds are discriminated from the others, and more reactive analytes (tryptophan, cysteine, bovine serum albumin, and DNA) are detected in the presence of less reactive ones in natural water. The system is promising for the development of group assays for dissolved organic matter and the discrimination of water samples. Full article

Super-resolution single-molecule localization microscopy (SMLM) of presynaptic active zones (AZs) and postsynaptic densities contributed to the observation of protein nanoclusters that are involved in defining functional characteristics and in plasticity of synaptic connections. Among SMLM techniques, direct stochastic optical reconstruction microscopy (dSTORM) depends on organic fluorophores that exert high brightness and reliable photoswitching. While multicolor imaging is highly desirable, the requirements necessary for high-quality dSTORM make it challenging to identify combinations of equally performing, spectrally separated dyes. Red-excited carbocyanine dyes, e.g., Alexa Fluor 647 (AF647) or Cy5, are currently regarded as “gold standard” fluorophores for dSTORM imaging. However, a recent study introduced a set of chemically modified rhodamine dyes, including CF583R, that promise to display similar performance in dSTORM. In this study, we defined CF583R’s performance compared to AF647 and CF568 based on a nanoscopic analysis of Bruchpilot (Brp), a nanotopologically well-characterized scaffold protein at Drosophila melanogaster AZs. We demonstrate equal suitability of AF647, CF568 and CF583R for basal AZ morphometry, while in Brp subcluster analysis CF583R outperforms CF568 and is on par with AF647. Thus, the AF647/CF583R combination will be useful in future dSTORM-based analyses of AZs and other subcellularly located marker molecules and their role in physiological and pathophysiological contexts. Full article

In the present study, we conducted a scoping review to provide an overview of the existing literature on the carbocyanine dye DiI, in human neuroanatomical tract tracing. The PubMed, Scopus, and Web of Science databases were systematically searched. We identified 61 studies published during the last three decades. While studies incorporated specimens across human life from the embryonic stage onwards, the majority of studies focused on adult human tissue. Studies that utilized peripheral nervous system (PNS) tissue were a minority, with the majority of studies focusing on the central nervous system (CNS). The most common topic of interest in previous tract tracing investigations was the connectivity of the visual pathway. DiI crystals were more commonly applied. Nevertheless, several studies utilized DiI in a paste or dissolved form. The maximum tracing distance and tracing speed achieved was, respectively, 70 mm and 1 mm/h. We identified studies that focused on optimizing tracing efficacy by varying parameters such as fixation, incubation temperature, dye re-application, or the application of electric fields. Additional studies aimed at broadening the scope of DiI use by assessing the utility of archival tissue and compatibility of tissue clearing in DiI applications. A combination of DiI tracing and immunohistochemistry in double-labeling studies have been shown to provide the means for assessing connectivity of phenotypically defined human CNS and PNS neuronal populations. Full article

Mitochondria are the energy factories of a cell, and depending on the metabolic requirements, the mitochondrial morphology, quantity, and membrane potential in a cell change. These changes are frequently assessed using commercially available probes. In this study, we tested the suitability of three commercially available probes—namely 5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolo-carbocyanine iodide (JC-1), MitoTracker Red CMX Rox (CMXRos), and tetramethylrhodamine methyl ester (TMRM)—for assessing the mitochondrial quantity, morphology, and membrane potential in living human mesoangioblasts in 3D with confocal laser scanning microscope (CLSM) and scanning disk confocal microscope (SDCM). Using CLSM, JC-1, and CMXRos—but not TMRM—uncovered considerable background and variation. Using SDCM, the background signal only remained apparent for the JC-1 monomer. Repetitive imaging of CMXRos and JC-1—but not TMRM—demonstrated a 1.5–2-fold variation in signal intensity between cells using CLSM. The use of SDCM drastically reduced this variation. The slope of the relative signal intensity upon repetitive imaging using CLSM was lowest for TMRM (−0.03) and highest for CMXRos (0.16). Upon repetitive imaging using SDCM, the slope varied from 0 (CMXRos) to a maximum of −0.27 (JC-1 C1). Conclusively, our data show that TMRM staining outperformed JC-1 and CMXRos dyes in a (repetitive) 3D analysis of the entire mitochondrial quantity, morphology, and membrane potential in living cells. Full article

Drug delivery systems play a pivotal role in targeted pharmaceutical transport and controlled release at specific sites. Liposomes, commonly used as drug carriers, constitute a fundamental part of these systems. Moreover, the drug–liposome model serves as a robust platform for investigating interaction processes at both cellular and molecular levels. To advance our understanding of drug carrier uptake mechanisms, we employed fluorescence lifetime imaging microscopy (FLIM) and fluorescence correlation spectroscopy (FCS), leveraging the unique benefits of two-photon (2P) excitation. Our approach utilized giant unilamellar vesicles (GUVs) as a simplified model system for cell membranes, labelled with the amphiphilic fluorescent dye 3,3′-dioctadecyloxa-carbocyanine (DiOC₁₈(3)). Additionally, large unilamellar vesicles (LUVs) functioned as a drug carrier system, incorporating the spectrally distinct fluorescent sulforhodamine 101 (SRh101) as a surrogate drug. The investigation emphasized the diverse interactions between GUVs and LUVs based on the charged lipids employed. We examined the exchange kinetics and structural alterations of liposome carriers during the uptake process. Our study underscores the significance of employing 2P excitation in conjunction with FLIM and FCS. This powerful combination offers a valuable methodological approach for studying liposome interactions, positioning them as an exceptionally versatile model system with a distinct technical advantage. Full article

The fluorine atom possesses many intrinsic properties that can be beneficial when incorporated into small molecules. These properties include the atom’s size, electronegativity, and ability to block metabolic oxidation sites. Substituents that feature fluorine and fluorine-containing groups are currently prevalent in drugs that lower cholesterol, relieve asthma, and treat anxiety disorders, as well as improve the chemical properties of various medications and imaging agents. The dye scaffolds (fluorescein/rhodamine, coumarin, BODIPY, carbocyanine, and squaraine dyes) reported will address the incorporation of the fluorine atom in the scaffold and the contribution it provides to its application as an imaging agent. It is also important to recognize radiolabeled fluorine atoms used for PET imaging in the early detection of diseases. This review will discuss the many benefits of incorporating fluorine atoms into small molecules and give examples of fluorinated molecules used in the pharmaceutical industry and imaging techniques. Full article

Optical “fingerprints” are widely used for chemometrics-assisted recognition of samples of different types. An emerging trend in this area is the transition from obtaining “static” spectral data to reactions analyzed over time. Indicator reactions are usually carried out in aqueous solutions; in this study, we developed reactions that proceed in an organic solvent, thereby making it possible to recognize fat-soluble samples. In this capacity, we used 5W40, 10W40, and 5W30 motor oils from four manufacturers, with six samples in total. The procedure involved mixing a dye, sample, and reagents (HNO₃, HCl, or tert-butyl hydroperoxide) in an ethanolic solution in a 96-well plate and measuring absorbance or near-infrared fluorescence intensity every several minutes for 20–55 min. The obtained photographic images were processed by linear discriminant analysis (LDA) and the k-nearest neighbors algorithm (kNN). Discrimination accuracy was evaluated by a validation procedure. A reaction of oxidation of a dye by nitric acid allowed us to recognize all six samples with 100% accuracy for LDA. Merging of data from the four reactions that did not provide complete discrimination ensured an accuracy of 93% for kNN. The newly developed indicator systems have good prospects for the discrimination of other fat-soluble samples. Overall, the results confirm the viability of the kinetics-based discrimination strategy. Full article

Optical sensor arrays are widely used in obtaining fingerprints of samples, allowing for solutions of recognition and identification problems. An approach to extending the functionality of the sensor arrays is using a kinetic factor by conducting indicator reactions that proceed at measurable rates. In this study, we propose a method for the discrimination of proteins based on their oxidation by sodium hypochlorite with the formation of the products, which, in turn, feature oxidation properties. As reducing agents to visualize these products, carbocyanine dyes IR-783 and Cy5.5-COOH are added to the reaction mixture at pH 5.3, and different spectral characteristics are registered every several minutes (absorbance in the visible region and fluorescence under excitation by UV (254 and 365 nm) and red light). The intensities of the photographic images of the 96-well plate are processed by principal component analysis (PCA) and linear discriminant analysis (LDA). Six model proteins (bovine and human serum albumins, γ-globulin, lysozyme, pepsin, and proteinase K) and 10 rennet samples (mixtures of chymosin and pepsin from different manufacturers) are recognized by the proposed method. The method is rapid and simple and uses only commercially available reagents. Full article

Array-based optical sensing is an efficient technique for the determination and discrimination of small organic molecules. This study is aimed at the development of a simple and rapid strategy for obtaining an optical response from a wide range of low-molecular-weight organic compounds. We have suggested a colorimetric and fluorimetric sensing platform based on the combination of two response mechanisms using carbocyanine dyes: aggregation and oxidation. In the first one, the analyte forms ternary aggregates with an oppositely charged surfactant wherein the dye is solubilized in the hydrophobic domains of the surfactant accompanied with fluorescent enhancement. The second mechanism is based on the effect of the analyte on the catalytic reaction rate of dye oxidation with H₂O₂ in the presence of a metal ion (Cu²⁺, Pd²⁺), which entails fluorescence waning and color change. The reaction mixture in a 96-well plate is photographed in visible light (colorimetry) and the near-IR region under red light excitation (fluorimetry). In this proof-of-concept study, we demonstrated the feasibility of discrimination of nine medicinal compounds using principal component analysis: four cephalosporins (ceftriaxone, cefazolin, ceftazidime, cefotaxime), three phenothiazines (promethazine, promazine, chlorpromazine), and two penicillins (benzylpenicillin, ampicillin) in an aqueous solution and in the presence of turkey meat extract. The suggested platform allows simple and rapid recognition of analytes of various nature without using spectral equipment, except for a photo camera. Full article

Imaging-guided delivery is developed for hydrophobic drugs, and to a much lesser extent, hydrophilic ones. In this work we have designed a novel strategy for real-time monitoring of hydrophilic drug delivery. Traditionally, the drug and the dye are covalently attached to a nanocarrier or are electrostatically adsorbed. Recently, we found an efficient way to bind the drug by ion-paring with an appropriate counter-ion to form the aggregate that embeds a hydrophobic dye with a considerable fluorescence enhancement. We synthesized a series of carbocyanine dyes of hydrophobicity sufficient for solubilization in hydrophobic ion pairs, which restores their emission in the near-infrared (NIR) region upon the formation of the ternary aggregates. To avoid using toxic surfactants, we applied an amphiphilic polymer-oligomer poly(hexamethylene guanidine) (PHMG) as a counter-ion. Сeftriaxone was used as a model hydrophilic drug ensuring the highest fluorescent signal. The so-formed drug–counter-ion–dye aggregates were encapsulated into a cross-linked maleated chitosan carrier. Confocal laser scanning microscopy (CLSM) studies have demonstrated internalization of the encapsulated model drug by breast adenocarcinoma cells at 40 min after treatment. These results suggest the potential application of hydrophobic ion pairs containing an NIR dye in imaging-guided delivery of hydrophilic compounds. Full article

G-quadruplexes (G4) are the most actively studied non-canonical secondary structures formed by contiguous repeats of guanines in DNA or RNA strands. Small molecule mediated targeting of G-quadruplexes has emerged as an attractive tool for visualization and stabilization of these structures inside the cell. Limited number of DNA and RNA G4-selective assays have been reported for primary ligand screening. A combination of fluorescence spectroscopy, AFM, CD, PAGE, and confocal microscopy have been used to assess a dimeric carbocyanine dye B6,5 for screening G4-binding ligands in vitro and in cellulo. The dye B6,5 interacts with physiologically relevant DNA and RNA G4 structures, resulting in fluorescence enhancement of the molecule as an in vitro readout for G4 selectivity. Interaction of the dye with G4 is accompanied by quadruplex stabilization that extends its use in primary screening of G4 specific ligands. The molecule is cell permeable and enables visualization of quadruplex dominated cellular regions of nucleoli using confocal microscopy. The dye is displaced by quarfloxin in live cells. The dye B6,5 shows remarkable duplex to quadruplex selectivity in vitro along with ligand-like stabilization of DNA G4 structures. Cell permeability and response to RNA G4 structures project the dye with interesting theranostic potential. Our results validate that B6,5 can serve the dual purpose of visualization of DNA and RNA G4 structures and screening of G4 specific ligands, and adds to the limited number of probes with such potential. Full article

Visualizing biological events and states to resolve biological questions is challenging. Tissue clearing permits three-dimensional multicolor imaging. Here, we describe a pH-adjustable tissue clearing solution, Seebest (SEE Biological Events and States in Tissues), which preserves lipid ultrastructures at an electron microscopy level. Adoption of polyethylenimine was required for a wide pH range adjustment of the tissue clearing solution. The combination of polyethylenimine and urea had a good tissue clearing ability for multiple tissues within several hours. Blood vessels stained with lipophilic carbocyanine dyes were deeply visible using the solution. Adjusting the pH of the solution was important to maximize the fluorescent intensity and suppress dye leakage during tissue clearing. The spatial distribution of doxorubicin and oxidative stress were observable using the solution. Moreover, spatial distribution of liposomes in the liver was visualized. Hence, the Seebest solution provides pH-adjustable, rapid, sufficient tissue clearing, while preserving lipid ultrastructures, which is suitable for drug delivery system evaluations. Full article

Here, we report the syntheses of two pentamethine cyanine dyes containing quinolinium rings and substituted with either hydrogen (3) or bromine (4) at the meso carbon. The electron withdrawing bromine atom stabilizes dye 4 in aqueous buffer, allowing complex formation to occur between the dye and double-helical DNA. UV–visible, CD, and fluorescence spectra recorded at low DNA concentrations suggest that dye 4 initially binds to the DNA as a high-order aggregate. As the ratio of DNA to dye is increased, the aggregate is converted to monomeric and other low-order dye forms that interact with DNA in a non-intercalative fashion. The brominated dye 4 is relatively unreactive in the dark, but, under 707–759 nm illumination, generates hydroxyl radicals that cleave DNA in high yield (pH 7.0, 22 °C). Dye 4 is also taken up by ES2 ovarian carcinoma cells, where it is non-toxic under dark conditions. Upon irradiation of the ES2 cells at 694 nm, the brominated cyanine reduces cell viability from 100 ± 10% to 14 ± 1%. Our results suggest that 2-quinolinium-based carbocyanine dyes equipped with stabilizing electron withdrawing groups may have the potential to serve as sensitizing agents in long-wavelength phototherapeutic applications. Full article

Alternative routes of administration are one approach that could be used to bypass the blood–brain barrier (BBB) for effective drug delivery to the central nervous system (CNS). Here, we focused on intranasal delivery of polymer nanoparticles. We hypothesized that surface modification of poly(lactic-co-glycolic acid) (PLGA) nanoparticles with rabies virus glycoprotein (RVG29) would increase residence time and exposure of encapsulated payload to the CNS compared to non-targeted nanoparticles. Delivery kinetics and biodistribution were analyzed by administering nanoparticles loaded with the carbocyanine dye 1,1′-Dioctadecyl-3,3,3′,3′-Tetramethylindotricarbocyanine Iodide (DiR) to healthy mice. Intranasal administration yielded minimal exposure of nanoparticle payload to most peripheral organs and rapid, effective delivery to whole brain. Regional analysis of payload delivery within the CNS revealed higher delivery to tissues closest to the trigeminal nerve, including the olfactory bulb, striatum, midbrain, brainstem, and cervical spinal cord. RVG29 surface modifications presented modest targeting benefits to the striatum, midbrain, and brainstem 2 h after administration, although targeting was not observed 30 min or 6 h after administration. Payload delivery to the trigeminal nerve was 3.5× higher for targeted nanoparticles compared to control nanoparticles 2 h after nanoparticle administration. These data support a nose-to-brain mechanism of drug delivery that closely implicates the trigeminal nerve for payload delivery from nanoparticles via transport of intact nanoparticles and eventual diffusion of payload. Olfactory and CSF routes are also observed to play a role. These data advance the utility of targeted nanoparticles for nose-to-brain drug delivery of lipophilic payloads and provide mechanistic insight to engineer effective delivery vectors to treat disease in the CNS. Full article

One of the important features of polymethine (cyanine) dyes is isomerization about one of C–C bonds of the polymethine chain. In this review, spectral properties of the isomers, photoisomer-ization and thermal back isomerization of carbocyanine dyes, mostly meso-substituted carbocy-anine dyes, are considered. meso-Alkyl-substituted thiacarbocyanine dyes are present in polar solvents mainly as cis isomers and, hence, exhibit no photoisomerization, whereas in nonpolar solvents, in which the dyes are in the trans form, photoisomerization takes place. In contrast, the meso-substituted dyes 3,3′-dimethyl-9-phenylthiacarbocyanine and 3,3′-diethyl-9-(2-hydroxy-4-methoxyphenyl)thiacarbocyanine occur as trans isomers and exhibit photoisomerization in both polar and nonpolar solvents. The behavior of these dyes may be ex-plained by the fact that the phenyl ring of the substituent in their molecules can be twisted at some angle, removing the substituent from the plane of the molecule and reducing its steric ef-fect on the conformation of the trans isomer. In some cases, photoisomerization of cis isomers of meso-substituted carbocyanine dyes is also observed (for some meso-alkyl-substituted dyes com-plexed with DNA and chondroitin-4-sulfate; for 3,3′-diethyl-9-methoxythiacarbocyanine in moderate polarity solvents). The cycle photoisomerization–thermal back isomerization of cya-nine dyes can be used in various systems of information storage and deserves further investiga-tion using modern research methods. Full article