Cellular Imaging Applications Research Articles

Two-Photon Cellular and Intravital Imaging of Hypochlorous Acid by Fluorescent Probes That Exhibit a Synergistic Excited-State Intramolecular Proton Transfer-Intramolecular Charge Transfer Mechanism Enabling Near-Infrared Emission with a Large Stokes Shift.

To develop highly effective molecular tools for intravital imaging of hypochlorous acid (HOCl), in this study, we initially designed two-photon hybrid fluorophores, SDP and P-SDP, by conjugating the classical dye 2-(2'-hydroxyphenyl)benzothiazole with the two-photon hydroxylphenyl-butadienylpyridinium fluorophore. The designed fluorophores exhibit a synergistic interaction between excited-state intramolecular proton transfer and intramolecular charge transfer mechanisms, enabling near-infrared (NIR) emission and significant Stokes shifts. Subsequently, using these fluorophores, we developed two HOCl fluorescent probes, SDP-SN and P-SDP-SN, by further incorporating N,N-dimethylthiocarbamate as a specific recognition group for HOCl. Toward HOCl, both SDP-SN and P-SDP-SN demonstrate an ultrafast response (less than 3 s), NIR emission at wavelengths of 714 and 682 nm, and remarkable Stokes shifts of 303 and 271 nm, respectively. Leveraging these advantages in conjunction with their ability to cross the blood-brain barrier, the probes find successful application in two-photon cellular and intravital imaging of HOCl. This includes visualizing endogenous generation of HOCl in cellular models related to inflammation, hyperglycemia, and ferroptosis, as well as mapping in vivo generation of HOCl within the brain and abdominal cavity using a murine model of systemic inflammation.

Colorimetric and fluorescent probes for cysteine detection: Applications in food safety and cellular imaging

In this study, three BODIPY-based fluorescent probes were designed and synthesized. The ultraviolet-visible spectra, fluorescence spectra, smartphone color recognition application and bioimaging were utilized to evaluate the capacity of the probes. By comparing key parameters, BDP-SIN had optimal performances including fastest response (10 min), highest signal-to-noise ratio (815 times) and lowest limit of detection (LOD = 49 nM). The recovery rate ranged from 92.04 % to 103.25 %. Meanwhile, BDP-SIN was triumphantly employed for determination of Cys in different daily food samples. Moreover, the test strips and microporous filter membrane loaded with BDP-SIN were developed for the portable real-time visualization and quantitative detection of Cys in food samples, which the contents ranged from 0.27 μM to 0.49 μM. Besides, BDP-SIN could image Cys in the living cells and mice. The novelty of this work was that developed an effective tool for researching the roles of Cys in food industry and living organisms.

Real-Time Monitoring of mtDNA Aggregation and Mitophagy Induced by a Fluorescent Platinum Complex in Living Cells.

Mitochondrial DNA (mtDNA) is pivotal for mitochondrial morphology and function. Upon mtDNA damage, mitochondria undergo quality control mechanisms, including fusion, fission, and mitophagy. Real-time monitoring of mtDNA enables a deeper understanding of its effect on mitochondrial function and morphology. Controllable induction and real-time tracking of mtDNA dynamics and behavior are of paramount significance for studying mitochondrial function and morphology, facilitating a deeper understanding of mitochondria-related diseases. In this work, a fluorescent platinum complex was designed and developed that not only induces mitochondrial DNA (mtDNA) aggregation but also triggers mitochondrial autophagy (mitophagy) through the MDV pathway for damaged mtDNA clearance in living cells. Additionally, this complex allows for the real-time monitoring of these processes. This complex may serve as a valuable tool for studying mitochondrial microautophagy and holds promise for broader applications in cellular imaging and disease research.

Unraveling the ability of electronically diverse 9H-carbazoles as potential fluorophores in cellular imaging applications with BSA binding affinities

In the present study, fluorophores based on 9H-carbazole have been synthesized from readily accessible 2-nitrochalcones in a single-step reductive cyclization reaction with methyl acetoacetate. The molecular geometry was confirmed based on IR, NMR and mass analysis. The ground state geometry for all the synthesized compounds was optimized using DFT-based methods to explore the localization of frontier molecular orbitals in electronically diverse carbazoles. The photophysical studies revealed a higher Stokes shift for PPM 17 due to the presence of the naphthyl group at C-1 of carbazole. The high stability of PPM14, as evidenced by high chemical hardness, prompted us to explore its BSA binding affinity using fluorescence titration, synchronous,3-D fluorescence and energy transfer analysis. The probe (PPM 14) displayed a “turn-off” response in the presence of Fe3+ ions. The less cytotoxic behavior of PPM 14, its “turn-off” fluorescence response with Fe3+, and its utility in cell imaging application establish it as a potential small organic framework-based bio-sensor.

Selective and Sensitive Detection of Cu+ in Living Cells Based on Chelator Modified Carbon Dots

AbstractCuprous ions (Cu+) are essential for various biological activities. However, it is still a great challenge to detect Cu+ efficiently in cells in situ. As emerging 0‐D carbon photoluminescence nanomaterials, carbon dots (CDs) exhibit non‐toxicity and ultra stability, which shows great advantages in biosensing and bioimaging. In this work, CDs are prepared by a one‐step microwave‐assisted hydrothermal method. To enhance the selectivity and efficiency of the CDs‐based sensor, the NS4‐CDs are synthesized by surface modification of CDs using Cu+ chelating molecules. The NS4‐CDs, that carry specific detection molecules, exhibit remarkable selectivity and high sensitivity with an extraordinarily low detection limit (26 nmol L−1). It is noteworthy that the detection response is extremely fast, which only takes 20 s. The PL response mechanism of NS4‐CDs is also thoroughly investigated. In addition, the NS4‐CDs possess great biocompatibility and non‐cytotoxicity, which is vital for advanced cellular imaging applications. Finally, the NS4‐CDs are successfully applied to Cu+ detection in cells in situ. This work paves a new path for the design and efficient construction of CDs‐based biosensors for ion detection in cells, which expands the application area of carbon nanomaterials.

Biomass derived carbon dots for enhanced wastewater remediation: ANN-optimized synthesis and cellular imaging applications

Water pollution is a significant problem in today’s modern world, with dye molecules being the most challenging pollutants to eliminate. Carbon dots (CDs) have demonstrated high efficacy in water and wastewater treatment. This research aimed to develop CDs using banana peel as a carbon precursor through a facile hydrothermal method. These CDs were utilized to eliminate methylene blue (MB) dye. The CDs exhibited blue fluorescence with an improved quantum yield (QY) of up to 19 %. They showed an adsorption efficiency of 71.942 mg g−1, with maximum adsorption occurring within a pH range from 2 to 10. The adsorption kinetic data were well described by the pseudo-second-order and Freundlich isotherm models. Thermodynamic studies revealed that the adsorption process was spontaneous and exothermic. Moreover, the CDs demonstrated excellent re-usability potential over five successive cycles of adsorption. Additionally, a prediction study was performed using an Artificial Neural Network (ANN) to forecast the adsorption of dye by CDs. The CDs were further investigated for cytotoxic properties and cellular internalization in breast cancer cells (MCF-7). The findings indicated that cellular viability significantly decreased with increasing dosage, reaching 50 % (IC50) at a concentration of 128 μg mL−1. Successful internalization of CDs by cancer cells with blue emission suggests their potential in biomedical applications. Thus, the synthesized nanomaterial is highly promising for both bio-sensing applications and wastewater treatment.

Enhancing Emission Efficiencies Through Side‐Chain Engineering of Luminogens to Control Molecular Motion

AbstractControlling the efficient emission of organic luminogens in both monomeric and various aggregated states remains a challenging task. In this work, the motion of an organic luminophores is synergistically regulated by introducing side alkyl chains with appropriate length and the aromatic rotor triphenylamine. This study finds that the incorporation of alkyl chains not only increased the rotational barriers in the molecule, but also influenced the packing modes. The as‐prepared organic luminogen, FIMPA‐C6, exhibited bright emissions in solution, polymorph, and amorphous states. Furthermore, the emission of FIMPA‐C6 can be widely tuned from yellow to deep red in different aggregated states. The exceptional luminescent properties of FIMPA‐C6 make it a promising functional dye for cellular imaging applications.

BODIPY-fused uracil: synthesis, photophysical properties, and applications.

Fluorescent nucleobase and nucleic acid analogs are important tools in chemical and molecular biology as fluorescent labelling of nucleobases has applications in cellular imaging and anti-tumor activity. Boron-dipyrromethene (BODIPY) dyes exhibiting high brightness and good photostability are extensively used as fluorescent labelling agents and as type II photosensitizers for photodynamic therapy. Thus, the combination of nucleobases and BODIPY to obtain new compounds with both anti-tumor activity and fluorescent imaging functions is the focus of our research. We synthesized two new nucleobase analogs 1 and 2 by fusing the BODIPY core directly with uracil which resulted in favorable photophysical properties and high emission quantum efficiencies particularly in organic solvents. Further, we explored the newly synthesized derivatives, which possessed good singlet oxygen generation efficiencies and bio-compatibility, as potential PDT agents and our results show that they exhibit in vitro anti-tumor activities.

A rapid access to activity-based and mitochondria targeting rhodol-like chromenoxanthene dyes for selective bioimaging

Chromenoxanthenes are colorful dyes which do not suffer from any fluorescence turn-off induced by spirocyclic forms as usually observed for conventional rhodamine and fluorescein xanthene analogues. In that way, wide variety of applications in particular in bioimaging can be foreseen for those underexplored fluorophores. Herein, a one-pot synthesis of rhodol-like chromenoxanthene dyes is reported, which are further modified at the periphery of the core structure to tune the optical properties. Resulting neutral derivatives are bright fluorophores in the orange-red spectral window, while cationic counterparts are almost non-emissive. These optical properties are fully characterized and are also rationalized via first-principles calculations. In particular, the occurrence of twisted intramolecular charge transfers is shown to account for the dark-state quenching occurring in the charged compounds. This feature is harvested in the construction of an activity-based fluorescent probe for mitochondria imaging. Excellent organelle localization is achieved, while the emission signal is proportional to the hydrogen peroxide concentration in the mitochondria. Furthermore, the absence of cytotoxicity bestows rhodol-like chromenoxanthene dyes a bright future in cellular imaging applications.

Chronological development of functional fluorophores for bio-imaging.

Functional fluorophores represent an emerging research field, distinguished by their diverse applications, especially in sensing and cellular imaging. After the discovery of quinine sulfate and subsequent elucidation of the fluorescence mechanism by Sir George Stokes, research in the field of fluorescence gained momentum. Over the past few decades, advancements in sophisticated instruments, including super-resolution microscopy, have further promoted cellular imaging using traditional fluorophores. These advancements include deciphering sensing mechanisms via photochemical reactions and scrutinizing the applications of fluorescent probes that specifically target organelles. This approach elucidates molecular interactions with biomolecules. Despite the abundance of literature illustrating different classes of probe development, a concise summary of newly developed fluorophores remains inadequate. In this review, we systematically summarize the chronological discovery of traditional fluorophores along with new fluorophores. We briefly discuss traditional fluorophores ranging from visible to near-infrared (NIR) in the context of cellular imaging and in vivo imaging. Furthermore, we explore ten new core fluorophores developed between 2007 and 2022, which exhibit advanced optical properties, providing new insights into bioimaging. We illustrate the utilization of new fluorophores in cellular imaging of biomolecules, such as reactive oxygen species (ROS), reactive nitrogen species (RNS), and proteins and microenvironments, especially pH and viscosity. Few of the fluorescent probes provided new insights into disease progression. Furthermore, we speculate on the potential prospects and significant challenges of existing fluorophores and their potential biomedical research applications. By addressing these aspects, we intend to illuminate the compelling advancements in fluorescent probe development and their potential influence across various fields.

Novel mono substituted pyridoimidazoisoquinoliniums via a silver-catalyzed intramolecular cyclization and their applications in cellular imaging.

Cationic heterocycles, an important class of organic compounds soluble in polar solvents, have been gaining attention in the construction of fluorescent probes. This paper reports the quick synthesis of novel pyrido[1',2';2,3]imidazo[5,1-a]isoquinoliniums starting from 2-(2-ethynylphenyl)imidazo[1,2-a]pyridines at room temperature via intramolecular cyclization by employing a catalytic amount of silver trifluoromethanesulfonate in addition to lithium trifluoromethanesulfonate and silica gel as the counter anion source and additive, respectively. The designed pyridoimidazoisoquinoliniums consisted of an imidazo[1,2-a]pyridine fused isoquinolinium. The X-ray diffraction results revealed that pyrido[1',2';2,3]imidazo[5,1-a]isoquinolinium trifluoromethanesulfonate contained considerable planar parent skeletons and interacted by π-π stacking with neighbouring molecules. Furthermore, in a methanol solution the designed 6-phenyl derivative exhibited strong fluorescence in the 420-450 nm region in addition to strong mitochondrial specificity in a cell staining assay.

DNA nanotechnology-based nucleic acid delivery systems for bioimaging and disease treatment.

Nucleic acids, including DNA and RNA, have been considered as powerful and functional biomaterials owing to their programmable structure, good biocompatibility, and ease of synthesis. However, traditional nucleic acid-based probes have always suffered from inherent limitations, including restricted cell internalization efficiency and structural instability. In recent years, DNA nanotechnology has shown great promise for the applications of bioimaging and drug delivery. The attractive superiorities of DNA nanostructures, such as precise geometries, spatial addressability, and improved biostability, have enabled them to be a novel category of nucleic acid delivery systems for biomedical applications. In this review, we introduce the development of DNA nanotechnology, and highlight recent advances of DNA nanostructure-based delivery systems for cellular imaging and therapeutic applications. Finally, we propose the challenges as well as opportunities for the future development of DNA nanotechnology in biomedical research.

Green approach for the fabrication of dual-functional S/N doped graphene tagged ZnO nanograins for in vitro bioimaging and water pollutant remediation

Dual-functional S/N (sulfur and nitrogen) doped graphene-tagged zinc oxide nanograins were synthesized for bioimaging applications and light-dependent photocatalytic activity. Applying the green synthesis approach, graphene was synthesized from kimchi cabbage through a hydrothermal process followed by tagging it with synthesized zinc oxide nanoparticles (ZnO-NPs). The 2D/0D heterostructure prepared by combining both exhibited exceptional advantages. Comprehensive characterizations such as TEM, SEM, XRD, FTIR, XPS, and UV–Vis spectra have been performed to confirm the structures and explore the properties of the synthesized nanocomposite. The graphene/ZnO-NP composite produced exhibited more intense fluorescence, greater chemical stability and biocompatibility, lower cytotoxicity, and better durability than ZnO NPs conferring them with potential applications in cellular imaging. While tagging the ZnO NPs with carbon derived from a natural source containing hydroxyl, sulfur, and nitrogen-containing functional group, the S/N doped graphene/ZnO heterostructure evidences the high photocatalytic activity under UV and visible irradiation which is 3.2 and 3.8 times higher than the as-prepared ZnO-NPs. It also demonstrated significant antibacterial activity which confers its application in removing pathogenic contaminant bacteria in water bodies. In addition, the composite had better optical properties and biocompatibility, and lower toxicity than ZnO NPs. Our findings indicate that the synthesized nanocomposite will be suitable for various biomedical and pollutant remediation due to its bright light-emitting properties and stable fluorescence.

A near-infrared AIE fluorescent probe for accurate detection of sulfur dioxide derivatives and visualization of fingerprints

In most biophysiological processes, sulfur dioxide (SO2) is an important intracellular signaling molecule that plays an important role. The change of SO2 in cells are closely related to various diseases such as neurological disorders and lung cancer, so it is necessary to develop fluorescent probes with the ability to accurately detect SO2 during physiological processes. In this work, we designed and synthesized a multifunctional fluorescent probe TIS. TIS has excellent properties such as near-infrared emission, large stokes shift, excellent SO2 detection capabilities, low detection limit, high specificity and visualization of color change before and after reaction. Simultaneously, TIS has low cytotoxicity, good biocompatibility, clear cell imaging capability and mitochondrial targeting ability. In addition, the ability of TIS to be applied to different material surfaces for latent fingerprint fluorescence imaging was also explored. TIS provides an excellent method for the accurate detection of SO2 derivatives and shows great potential applications in near-infrared cellular imaging and latent fingerprint fluorescence imaging.

Eu-doped carbon quantum dot as a selective probe for visualizing and monitoring sulfite in biological systems.

The detection of SO3 2- in complex environments and its visualization at the cellular level are critical for understanding its role in biological processes. In this study, we developed an Eu-doped long-wavelength fluorescent carbon quantum dot (CD2) and investigated the detection mechanism, interference effects and cellular imaging applications of the fluorescent probe CD2. The results show that the addition of SO3 2- induces an electronic rearrangement that restores CD2 to its original structure, leading to a rapid increase in fluorescence intensity. Selectivity experiments showed that CD2 has excellent selectivity to SO3 2-, with minimal interference from common anions. In addition, CD2 shows good biocompatibility for cellular imaging applications, as evidenced by the high cell viability observed in HeLa cells. Using confocal microscopy, we detected a significant enhancement of red fluorescence in HeLa cells after addition of exogenous SO3 2-, demonstrating the potential of CD2 as a probe for monitoring cellular SO3 2- levels. These findings highlight the promise of CD2 as a selective SO3 2- detection probe in complex environments and its utility in cellular imaging studies. Further studies are necessary to fully exploit the potential of CD2 in various biological and biomedical applications.

Design, Synthesis, and Cellular Imaging Application of a Fluorescent Probe Based on Fluoride Ion-Induced Cyclization of Phenothiazine Derivatives.

Fluoride is both necessary and potentially harmful in excessive amounts, making its detection crucial. Fluorescent probes provide a sensitive and selective means for this purpose. In this study, we developed and synthesized a fluorescent probe for LDT using phenothiazine derivatives and aryl vinyl nitrile. Initially non-fluorescent, the probe undergoes a Si-O bond breakage in the presence of fluoride ions, resulting in the formation of a larger conjugated system and subsequent fluorescence emission. The probe exhibits superior selectivity and sensitivity towards fluoride ions, with a detection limit of 0.35 µM. Moreover, cellular imaging experiments demonstrated the probe's effectiveness in recognizing fluoride ions within HepG2 cells.

Amino-SBBF (Single Benzene-based Fluorophore) Library: Its Synthesis, Photophysical Property, and Cellular Imaging Application

Single benzene-based fluorophore (SBBF) is a representative small-size fluorescent dye which has an electron push-pull structure within a benzene backbone. In this work, we disclosed a new amino-SBBF (ASBBF) library that has various alkyl-amines and aromatic-amines on the electron-donating site. The synthesis, photophysical properties, quantum chemical calculation, two-photon responsiveness, and bioimaging applicability of ASBBF in cells were successfully identified. We believe that our present work provides a guide to the development of new fluorescent materials and molecular probes in various research fields.

Establishing Riboglow-FLIM to visualize noncoding RNAs inside live zebrafish embryos

The central role of RNAs in health and disease calls for robust tools to visualize RNAs in living systems through fluorescence microscopy. Live zebrafish embryos are a popular system to investigate multicellular complexity as disease models. However, RNA visualization approaches in whole organisms are notably underdeveloped. Here, we establish our RNA tagging and imaging platform Riboglow-FLIM for complex cellular imaging applications by systematically evaluating FLIM capabilities. We use adherent mammalian cells as models for RNA visualization. Additional complexity of analyzing RNAs in whole mammalian animals is achieved by injecting these cells into a zebrafish embryo system for cell-by-cell analysis in this model of multicellularity. We first evaluate all variable elements of Riboglow-FLIM quantitatively before assessing optimal use in whole animals. In this way, we demonstrate that a model noncoding RNA can be detected robustly and quantitatively inside live zebrafish embryos using a far-red Cy5-based variant of the Riboglow platform. We can clearly resolve cell-to-cell heterogeneity of different RNA populations by this methodology, promising applicability in diverse fields.

Mulberry Juice-Derived Carbon Quantum Dots as a Cu2+ Ion Sensor: Investigating the Influence of Fruit Ripeness on the Optical Properties

This study synthesized carbon quantum dots (CQDs) with green photoluminescence through a hydrothermal method that utilized mulberry juice as the carbon source. The influence of fruit ripeness on the physical and chemical properties, focusing on the fluorescence spectra, has been explored. Fourier-transform infrared spectroscopy (FT-IR) and energy dispersive X-ray analysis (EDX) showed that there were oxygen-containing groups, and X-ray diffraction (XRD) showed that the carbon quantum dots (CQDs) were graphitic. The results revealed that the CQDs had an average size of around 7.4 nm and 9.7 nm for unripe and ripe mulberry juice, respectively. These CQDs emitted green light at 500 nm and 510 nm in unripe and ripe mulberry juice, respectively, when excited at a wavelength of 400 nm. The prepared CQDs exhibited excitation-dependent photoluminescence (PL) emission behavior, demonstrating their dependence on the excitation light. The impact of fruit ripeness on optical properties was explored by examining fluorescent spectra from different fruits (including tomato and blackberry), demonstrating comparable behaviors observed in mulberry fruit. In addition, the prepared CQDs were utilized as a fluorescent sensor with high specificity to detect Cu2+ ions. The detection limit (DL) for this sensor was determined to be 0.2687 µM, and the limit of qualification (LOQ) is 0.814 µM. The linear range for detection lies between 0.1 and 1 µM. The selectivity of the CQDs towards Cu2+ ions was confirmed by recording the PL response for Cu2+ ions compared to the weak response of other metal ions. According to these results, the CQDs can be applied in various cellular imaging and biology applications, bio-sensing, optoelectronics, and sensors.

Sustainable preparation of luminescent carbon dots from syringe waste and hyaluronic acid for cellular imaging and antimicrobial applications

Addressing the global challenge of persistent waste through an eco-conscious strategy to transform it into valuable and versatile materials holds great significance in today's swiftly evolving world. By adopting a sustainable approach, we can repurpose waste syringes composed of polytetrafluoroethylene (PTFE) into fluorescent carbon dots (CDs) using a simple hydrothermal process. This research harnessed hyaluronic acid to carbonize and modify discarded plastic syringes, resulting in the creation of luminescent syringe carbon dots (SCDs). Rigorous analysis employing diverse techniques delved into their optical attributes, size distribution, and surface characteristics. Extensive biocompatibility assessments using established assay methods confirmed the safety of the derived SCDs, unveiling their potential antibacterial and antifungal traits. Additionally, a confocal microscope was employed to evaluate the cellular imaging capabilities of SCDs on HeLa cells. Notably, at bactericidal concentrations, SCDs exhibited mild cytotoxicity towards mammalian cells, showcasing cell viability surpassing 91.07% at 1 mg/mL. This pioneering exploration paves the way for potential applications of SCD-based nano-bactericides across various biomedical domains. The initial outcomes established herein mark a significant stride towards the creation of cost-effective and ecologically sound fluorescent probes for biomedical imaging, aimed at combating microbial infections. By ingeniously reutilizing polyethylene terephthalate (PET), this investigation offers a sustainable remedy to address the ecological predicaments linked with plastic waste. In doing so, it charts a course towards contributing to the development of affordable, eco-friendly solutions, heralding a promising prospect for a cleaner, healthier environment.