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[Display omitted] • The review explores the versatile capabilities of fluorocolorimetric probes developed in Dr S Velmathi's lab at NIT Trichy for analyte detection, imaging, and real-time applications. • They exhibit distinctive visual signals when interacting with specific targets, marking a significant advancement in molecular interaction analysis. • The probes cover a broad spectrum of analytes, including metal ions, anions, biomolecules, ROS, RSS, toxic agents, and nanoparticles. • The review emphasizes the profound impact of fluorocolorimetric probes on developing analytical potential. • These probes contribute to innovative imaging techniques and expand the possibilities of real-time applications. Fluorescent and colorimetric probes are gaining interest in analyte detection, imaging, and real-time applications. However, it faces several challenges in achieving high sensitivity and selectivity, maintaining a strong signal-to-noise ratio, detecting analytes at extremely low concentrations, ensuring stability under different conditions and addressing issues related to cost, accessibility and instrumentation. Additionally, ensuring biological compatibility and enabling real-time monitoring are also challenges that need to overcome. We have investigated the sensing application of probe molecules towards various analytes like metal ions, anions, biomolecules, reactive oxygen species (ROS), reactive sulphur species (RSS), toxic agents and nanoparticles performed by our group together with critically comparing with the existing reports. This featured article provides the impact of fluorescent and colorimetric probes in advancing analytical techniques, imaging and real-time applications that transcend traditional boundaries. [ABSTRACT FROM AUTHOR]

Micro- and nanoplastics (MNPs) are ubiquitous in the environment, resulting in the uptake of MNPs by a variety of organisms, including humans, leading to particle-cell interaction. Human macrophages derived from THP-1 cell lines take up Polystyrene (PS), a widespread plastic. The question therefore arises whether primary human macrophages also take up PS micro- and nanobeads (MNBs) and how they react to this stimulation. Major aim of this study is to visualize this uptake and to validate the isolation of macrophages from peripheral blood mononuclear cells (PBMCs) to assess the impact of MNPs on human macrophages. Uptake of macrophages from THP-1 cell lines and PBMCs was examined by transmission electron microscopy (TEM), scanning electron microscopy and live cell imaging. In addition, the reaction of the macrophages was analyzed in terms of metabolic activity, cytotoxicity, production of reactive oxygen species (ROS) and macrophage polarization. This study is the first to visualize PS MNBs in primary human cells using TEM and live cell imaging. Metabolic activity was size- and concentration-dependent, necrosis and ROS were increased. The methods demonstrated in this study outline an approach to assess the influence of MNP exposure on human macrophages and help investigating the consequences of worldwide plastic pollution. [Display omitted] • Human monocyte-derived macrophages as novel in vitro model to study effects of MNP. • Concentration-dependent cytoplasmic uptake of PS MNBs visualized by TEM and SEM. • Uptake of PS MNBs by primary human macrophages is visible in live cell imaging. • Effects of different sized, non-fluorescent PS-MNBs in physiological concentration. • PS MNBs induce nitric oxide formation and cytotoxic effects in human macrophages. [ABSTRACT FROM AUTHOR]

Emerging studies demonstrates that mitochondrial G-quadruplex (G4) may be a prospective diagnostic and therapeutic target for cancer. Fluorescent probes targeting mitochondrial G4 are crucial for in-depth research on G4 function and mechanism and developing new diagnostic techniques. Here, we report a benzothiazole derivative named ThT-E, which can be prepared with high yield through only one step of reaction. ThT-E exhibits superior detection sensitivity compared to existing probes, and also demonstrates reliable performance for in situ characterization of mitochondrial G4 in living cells. Interestingly, ThT-E has been used to differentiate tumor cells from normal cells by quantifying the formation of mitochondrial G4s, implicating that ThT-E may have wide applications in tumor diagnosis. • A benzothiazole derivative for monitoring mitochondrial G-quadruplexes. • More pronounced fluorescence enhancement in tumor cells than normal cells. • More G-quadruplex formation in tumor mitochondria. • Mitochondrial G-quadruplex as a potential cancer biomarker. • New probe tool for tumor diagnosis. [ABSTRACT FROM AUTHOR]

DNA-functionalized nanomaterials represent the cutting edge of the convergence between nanoscience and life sciences. The gold nanoparticles (AuNPs), with their versatile attributes, serve as multifunctional carriers in complex environments, supporting diverse physiological activities of DNA. Concurrently, DNA enhances the target specificity and stability of nanomaterials, significantly improving or fine-tuning their performance. This review explores key aspects of DNA-functionalized AuNPs, including their synthesis methods, properties, and biosensing applications. It starts by delving deeply into the interactions between DNA and AuNPs, emphasizing their role in material customization and highlighting the advantages and applicability of each technique. Furthermore, the review thoroughly evaluates the latest synthesis methods developed from shielding the charger repulsion and extremely compressing the physical space. It showcases the potential of DNA-functionalized AuNPs in addressing complex challenges for biosensing applications. This synergistic relationship has propelled advancements in nanotechnology, facilitating the integration of life sciences, medicine, and nanotechnology, and expanding the application domains of these novel biomimetic nanocomposite materials. Therefore, there is an urgent need for in-depth research into the synthesis, structure and function of DNA-functionalized nanomaterials to unravel their pivotal mechanisms. Illustration of the application of DNA-functionalized gold nanomaterials in the field of biosensing. [Display omitted] • Mechanisms behind those milestones during the evolution of synthesizing DNA-functionalized AuNPs have been extensively discussed. • Engineering DNA-AuNPs enables biosensing of biomolecules for more intuitive and accurate access to spatiotemporal information. • Continuous innovation of synthesis enhances the efficiency in designing more functionalized nanomaterials across diverse fields. [ABSTRACT FROM AUTHOR]

Determining the activity of lysosomal β-hexosaminidase in cells is of great importance for understanding the roles that these enzymes play in pathophysiological events. Herein, we designed the new fluorescent probe, βGalNAc-Rhod-CM(NEt 2), which consisted of a βGalNAc-linked rhodol unit serving as a β-hexosaminidase reactive fluorogenic moiety and a N,N′ -diethylaminocoumarin (CM(NEt 2)) group acting as a fluorescence marker for determining the degree of cell permeabilization. Treatment of βGalNAc-Rhod-CM(NEt 2) with β-hexosaminidase promoted generation of Rhod-CM(NEt 2), thereby leading to an increase in the intensity of fluorescence of Rhod. However, this probe did not respond to the functionally related glycosidase, O-GlcNAcase. The detection limit of βGalNAc-Rhod-CM(NEt 2) for β-hexosaminidase was determined to be 0.52 nM, indicating that it has high sensitivity for this enzyme. Furthermore, the probe functioned as an excellent fluorogenic substrate for β-hexosaminidase with k cat and K m values of 17 sec−1 and 22 μM, respectively. The results of cell studies using βGalNAc-Rhod-CM(NEt 2) showed that levels of β-hexosaminidase activity in cells can be determined by measuring the intensity of fluorescence arising from Rhod and that the intensity of fluorescence of CM(NEt 2) can be employed to determine the degree of cell permeabilization of the probe. Utilizing the new probe, we assessed β-hexosaminidase activities in several types of cells and evaluated the effect of glucose concentrations in culture media on the activity of this enzyme. [Display omitted] • Development of the sensitive and selective fluorescent probe for β-hexosaminidase. • Determination of levels of β-hexosaminidase activities in several types of cells. • Evaluation of effect of glucose concentrations in media on β-hexosaminidase activity. [ABSTRACT FROM AUTHOR]

Advances in fluorescence microscopy can greatly facilitate research in regenerative health. Specifically, live cell imaging is a powerful tool to understand the underlying mechanisms of tissue regeneration, which is characterised by a dynamic interplay at cellular and molecular level. Recent advances in microscopy have aimed to overcome some of the most challenging limitations, such as slow acquisition speed, the resolution limit of light, low signal to noise ratio in thick samples, as well as photobleaching and phototoxicity. In applications such as lightsheet fluorescence microscopy and intra-vital multi-photon microscopy, improved deep tissue imaging have been achieved, and super-resolution technologies have shown to improve optical resolution far beyond the diffraction limit of light for better visualisation at the cellular and molecular level. By combining certain techniques, researchers can now image live samples at much higher resolution for a prolonged time. Advances in analytical technologies will enable researchers to gain an even better understanding of the processes involved to ultimately translate stem cell research into therapeutic interventions. • Advances in fluorescence microscopy allows for in vivo imaging of live animals. • 3D deep tissue imaging is a leap from the conventional 2D applications. • Super–resolution microscopy continues to improve. • Combining microscopy technologies overcome limitations of live cell imaging. • Increased imaging speed of thick samples at sub-resolution level is now possible. [ABSTRACT FROM AUTHOR]

The sensor MH-4 has a high sensitivity and selectivity to Cu2+ over a wide range of other metal ions in acetonitrile (ACN)/water (v/v 1:1). The fluorescence imaging studies demonstrated the permeability of MH-4 in L929 living cells and the sensing of Cu2+ in the cells. [Display omitted] • A diformyl phenol based optical sensor (MH-4) was developed for the detection of Cu2+. • MH-4 showed high selectivity and sensitivity Cu2+ over other metal ions/anions tested. • MH-4 can detect Cu2+ at nanomolar level. • Mh-4 is suitable for imaging Cu2+ in living cells. Given the critical role of trace copper ion in human health, its sensitive detection in environmental and biological samples is crucial. We, herein, designed and synthesized a diformyl phenol-based turn-off fluorescent sensor ((5-(tert -butyl)-2-hydroxy-1,3-phenylene)bis (methanylylidene)) bis (azanylylidene)) bis(4-hydroxy-2H-chromen-2-one) (MH-4)) for the spectrofluorometric detection of Cu2+ ion, which has sensitive and selective fluorescence quenching over other competitive metal ions. The efficiency and fast response of the sensor were confirmed by UV–Visible, fluorescence spectral changes, which may be due to the formation of coordination bond between MH-4 and Cu2+ ions. The binding stoichiometry of MH-4 with Cu2+ was examined with the help of UV–Vis and fluorescence titrations, and Job's plot, which gives a 1:1 coordination stoichiometry; this is further confirmed through MS studies. The binding constant of the complex was found to be 9.4 x 107 M−1. Then, the absorbance and fluorescence-based detection limits were calculated to define the sensitivity range of the proposed sensor and were found to be 69.30 nM and 37.40 nM, respectively. Finally, a live cell image study of MH-4 in L929 (murine aneuploid fibrosarcoma) cells was also performed to test the cell permeability of MH-4 and its effectiveness for selective detection of Cu2+ in living cells. [ABSTRACT FROM AUTHOR]

• A Light Origami Multi-Beam Interference – Digital Holographic Microscope (LOMBI-DHM) is proposed for live cell imaging that leverages single-shot acquisition and double field-of-view (FoV). • The concept of the proposed study to extend the FoV is based on the optical spatial multiplexing of two distinct regions of the object beam in a single hologram by adding a cube beam splitter in the path of the object beam. • The two distinct imaging regions corresponding to the two recorded FoVs, can be simultaneously retrieved during the reconstruction process. • The experimental findings of the proposed LOMBI-DHM point to a number of potential uses for this system, including optical metrology and biological imaging, with several advantages. In the field of scientific, industrial, and biological research, digital holographic microscopy (DHM) has established itself as a potential optical instrument due to its three-dimensional (3D) imaging capabilities and non-destructive nature. Like any imaging system, the field-of-view (FoV) of DHM is constrained by the image sensor's finite size. To address this challenge, in the present study, we propose the Light Origami Multi-Beam Interference (LOMBI)-DHM technique for live cell imaging that leverages single-shot acquisition and double FoV. The concept of the proposed study to extend the FoV is based on the optical spatial multiplexing of two distinct regions of the object beam by introducing a cube beam splitter in the path of the object beam. The beam splitter is angled so that it produces two object beams with different object information that are propagating in the same direction and collected within the area of the image sensor. The image sensor records a multiplexed digital hologram in a single-shot as a result of the interference of three beams: two object beams with different FoVs and one reference beam. The two distinct imaging regions corresponding to the two recorded FoVs, can be simultaneously retrieved during the reconstruction process. The experimental results of imaging different areas of a standard resolution target, microlens array, and living plant cells, are shown to demonstrate the capability of the proposed single-shot off-axis double FoV LOMBI-DHM. Furthermore, we dynamically monitor the time-lapse live-cell imaging of tobacco plant cells by the proposed system. [ABSTRACT FROM AUTHOR]

[Display omitted] Isatin derivatives have attracted a lot of interest for their potential in the development of new anticancer drugs. A library of 38 isatin derivatives, created through an Ugi four-component reaction, underwent an initial screening in a panel of six human solid tumor cell lines. The four most active derivatives were then selected for further testing. These compounds showed selectivity towards the non-small cell lung cancer (NSCLC) cell line SW1573, whilst NSCLC A549 cells were barely affected. The combination of phenotypic assays, including wound healing, clonogenic and continuous live cell imaging provided a deeper understanding of the compounds' mode of action. In particular, the latter demonstrated that isatin derivatives were able to induce necroptosis in SW1573 cells. The kinetics of cell death showed that necroptosis appeared after 2.5 h of exposure, which could be delayed to 7 h when co-treated with necrostatin-1. Interaction between the isatin derivatives and the KRAS G12C protein variant was discarded after in silico studies. Further studies are warranted to identify the cellular target responsible for the observed selectivity among cell lines. [ABSTRACT FROM AUTHOR]

Multidrug resistance (MDR) remains a significant challenge in cancer therapy, with inherent and acquired resistance distinct. While conventional drug selection processes enable the isolation of cancer cells with acquired multidrug resistance, identifying cancer cells with inherent drug resistance remains challenging. Herein, we proposed a molecular beacon (MB)-based strategy to identify and isolate the inherent MDR cancer cells. A lipid/PLGA core-shell nanoparticulate system (DNCP) was designed to deliver MB for intracellular MDR1 mRNA imaging. DNCP-MB – possess a surface potential of −8 mV and a size of 150 nm – demonstrated effective delivery of MB, remarkable selectivity towards the selected intracellular mRNA targets, and low cytotoxicity. Following DNCP transfection, fluorescence-activated cell sorting (FACS) was employed to differentiate MCF-7 cells into two distinct sub-populations: the Top 10 cells with a high level of MDR gene expression and the Bottom 10 cells with a low level of MDR gene expression, which represent inherent drug-resistant and non-drug-resistant cells, respectively. Intriguingly, we observed a positive correlation between elevated MDR1 mRNA expression and increased migration, enhanced proliferation rate, and tighter spheroid formation. Moreover, we conducted RNA sequencing analysis on the Top 10, Bottom 10, and MCF-7/ADR cells. The findings revealed a notable disparity in the gene ontology enrichment analysis of differentially expressed genes between the Top 10 and Bottom 10 cells when compared to the Bottom 10 and MCF-7/ADR cells. This novel approach provides a promising avenue for isolating inherent drug-resistant cells and holds significant potential in unraveling the mechanisms underlying inherent drug resistance. [ABSTRACT FROM AUTHOR]

The present study highlights the development, identification, and solvent-regulated chemosensing property of a Schiff base chemosensor (E)-2-(2-((5-chloro-2-hydroxybenzylidene)amino)propyl)-3′,6′-bis(diethylamino)spiro[isoindoline-1,9′-xanthen]-3-one (RBAS). The Schiff base RBAS is developed by the simple condensation of 5-chloro salicylaldehyde and rhodamine-1,2 diamino propane derivative (RBAP). Interestingly this probe can optically as well as fluorometrically exhibit pH sensor activity and the spirolactam ring opening and the conversion from enol to keto form in lower and higher pH regions respectively are the main key factors to show pH sensor activity. Several spectroscopic, Dynamic Light Scattering (DLS), and scanning electron microscopy (SEM) studies confirm the AIE active property of the probe in 2:8 DMSO-water and ethanol-water mediums where the probe exhibits strong luminescence properties. Interestingly in ethanol-water and DMSO-water medium, it can detect Hg(II) via turn-on and off fluorescence respectively. Actually, in different solvent mediums, the presence of Hg(II) plays a crucial role in the spirolactam ring opening and closing of the developed probe. Simultaneously in an ethanol-water medium, it can detect picric acid by turning off fluorescence. By using mass and NMR spectrum analysis, the 1:1 stoichiometric addition of the probe and target analytes has been verified and the density functional theory (DFT) study is implemented in optimizing the probe-analyte structure. The live cell imaging study gives the approval of the biosensing ability of the probe. Impressively the probe can be successfully used to detect Hg(II) in real soil samples. [Display omitted] • Development and spectroscopic identification of a novel rhodamine-based chemosensor (RBAS). • RBAS can be effectively used as pH sensor and spirolactam ring closing or opening controls the pH sensors performance. • DLS, SEM, UV and fluorescence probes the AIE effect of RBAS in two different mediums. [ABSTRACT FROM AUTHOR]

[Display omitted] The ability to execute live-cell, localized glyco-editing is of pivotal importance for revolutionizing the field of glycobiology but no effective tool has been developed. We report herein the design of protein-specific glyco-editing probes, operating through localized enzyme decaging (LED), for live-cell editing of carbohydrate units on the protein of interest. The integration of aptamer recognition and molecular cloaking/uncloaking-based glyco-enzyme caging/decaging mechanism ensures the protein-specific editing. Proof-of-concept demonstration has been achieved for mucin 1 (MUC1)-specific terminal galactose/ N -acetylgalactosamine (Gal/GalNAc) editing and sialic acid (Sia) trimming on live cells. A unique advantage of our LED strategy is the modular adaptability for performing cascade localized glyco-editing (CALOGE) manipulation through sequential aptamer docking-erasing-docking processes. Moreover, in vivo MUC1-specific Gal/GalNAc editing has also been realized, which enables the fluorescence visualization of Gal/GalNAc on MUC1 in murine bladder. In particular, specific trimming of MUC1-bound Sia on live cells has demonstrated, for the first time, the ability to directly reveal the significant effects of protein-specific glycoform on intracellular signaling and cell migration. These results suggest the probe as a generically applicable investigation tool for the revelation and manipulation of biological functions in association with protein-specific glycoforms. [ABSTRACT FROM AUTHOR]

[Display omitted] • This study was performed using live cell imaging for four hours. • Methadone and fentanyl induced significant changes to mitochondrial morphology. • Morphine had no effect on mitochondrial morphology. The important role of mitochondria in maintaining normal brain cell function has been demonstrated in several neurodegenerative diseases where mitochondrial dysfunction is a prominent feature. Accumulating evidence indicates that opioids may induce neuronal cell death and inhibit neurogenesis, two factors that are dependent on normal mitochondrial function. The aim of the present study was to examine the effects of morphine, methadone, and fentanyl on MitoTracker-stained mitochondria. Cells from the neuroblastoma/glioma hybrid cell-line NG108−15 were seeded on 96-well cell culture plates and treated with MitoTracker for 30 min prior to opioid treatment. Morphine, methadone, and fentanyl were added at various concentrations and images of mitochondria were acquired every 30 min for four hours using a high-content imaging device. The parameters total mitochondrial area, mitochondrial network, as well as the number and mean area of mitochondrial objects were analyzed using automated image analysis. Methadone and fentanyl, but not morphine, decreased the mitochondrial network, the number of mitochondrial objects, and increased the mean area of mitochondrial objects. Both methadone and fentanyl altered mitochondrial morphology with no effects seen from morphine treatment. These data suggest that methadone and fentanyl impact mitochondrial morphology negatively, which may be associated with neuronal cell death. [ABSTRACT FROM AUTHOR]

[Display omitted] • Two novel near-infrared emissive probes were designed and synthesized for mercury ions detection. • CyHg-Cl probe exhibited high photostability, low background fluorescence and biotoxicity. • Mercury ions in living cells and real water samples were successfully monitored. Mercury sensing and imaging in the bio-system is essential for comprehending its toxicity and therapies. Based on the merocyanine scaffold, we designed and synthesized two novel near-infrared (NIR) fluorescent probes for detecting Hg2+. The release of chloro-substituted merocyanine structure on the probe CyHg-Cl enables fluorescence enhancement rapidly by introducing Hg2+. In addition, the probe CyHg-Cl exhibits NIR emission and a low detection limit of 0.59 µM. Finally, the probe CyHg-Cl was used to detect Hg2+ in live cells and real water samples. [ABSTRACT FROM AUTHOR]

[Display omitted] • Highly fluorescent N, B-doped CNDs discerning interactions with Fe(II) and Fe(III). • The "urn-Off" optical probe relies on a concentration-dependent fluorescence decrease. • The N, B-doped CNDs are selective towards Fe(II) ions in the presence of other metals. • The CNDs can monitor Fe(II) presence in living cells by fluorescent imaging. The interactions between metal ions and fluorescent probes are fundamentals for discerning the metal ions. Co-doped carbon nanodots (CNDs) have demonstrated the capacity to optically detect a wide variety of targets, including metal ions. In this work, the quenching of highly fluorescent, nitrogen (N) and boron (B) co-doped CNDs by Fe(II) ions was discovered and employed to differentiate Fe2+ in water samples and live cells. The fluorescence of the N,B-CNDs was quenched in a concentration-dependent manner with a need of very short reaction time (30 s), and the CNDs show good selectivity towards Fe(II) ions relative to other metal ions including Fe(III). The limit of detection (LOD) was calculated as 1.8 nM and the linear response range was 7 nM to 1 μM. The recovery percentage was determined to be 97%, 101%, and 103% for spiked Fe(II) concentrations of 31, 125, and 1000 nM, respectively, in tap water. The N,B-CNDs were able to monitor the presence of Fe(II) in living cells using the optical imaging of Fe(II) ions, and the quenching in cells was completed within one hour. Regarding the quenching mechanism, the photoinduced electron transfer (PET) procedure is proposed with support of cyclic voltammetry study on the CNDs upon addition of Fe(II). Overall, the N,B-CNDs promise as optical probes for the detection of Fe2+ in water and living cells, and demonstrated a satisfactory level of sensitivity and selectivity. [ABSTRACT FROM AUTHOR]

Deregulation of the receptor tyrosine kinase MET/hepatocyte growth factor (HGF) pathway results in several pathological processes involved in tumor progression and metastasis. In a different context, MET can serve as an entry point for the bacterium Listeria monocytogenes , when activated by the internalin B (InlB) protein during infection of non-phagocytic cells. We have previously demonstrated that MET requires CD44v6 for its ligand-induced activation. However, the stoichiometry and the steps required for the formation of this complex, are still unknown. In this work, we studied the dynamics of the ligand-induced interaction of CD44v6 with MET at the plasma membrane. Using Förster resonance energy transfer-based fluorescence lifetime imaging microscopy in T-47D cells, we evidenced a direct interaction between MET and CD44v6 promoted by HGF and InlB in live cells. In the absence of MET, fluorescence correlation spectroscopy experiments further showed the dimerization of CD44v6 and the increase of its diffusion induced by HGF and InlB. In the presence of MET, stimulation of the cells by HGF or InlB significantly decreased the diffusion of CD44v6, in line with the formation of a ternary complex of MET with CD44v6 and HGF/InlB. Finally, similarly to HGF/InlB, disruption of liquid-ordered domains (Lo) by methyl-β-cyclodextrin increased CD44v6 mobility suggesting that these factors induce the exit of CD44v6 from the Lo domains. Our data led us to propose a model for MET activation, where CD44v6 dimerizes and diffuses rapidly out of Lo domains to form an oligomeric MET/ligand/CD44v6 complex that is instrumental for MET activation. [Display omitted] • CD44v6 directly interacts with MET upon HGF or InlB induction in live cells. • HGF and InlB promote the dimerization of CD44v6. • MET activation is independent of liquid-ordered domains. [ABSTRACT FROM AUTHOR]

[Display omitted] • A novel nicotinic hydrazone-based turn-on fluorescence probe 3 has been synthesized for the detection of carbonate ions in an aqueous medium. • The probe showed excellent selectivity, sensitivity, and good reversibility toward CO 3 2– ions. • The deprotonation mechanism of sensing was elucidated by 1H NMR, ESI-MS spectra, and DFT calculations. • An INHIBIT-type molecular logic gate was successfully constructed using probe 3- CO 3 2– and CH 3 COOH. • Probe 3 effectively senses CO 3 2– ions in the real water samples and live cells. A novel turn-on fluorescent probe 3 was synthesized by condensing salicylaldehyde and nicotinic hydrazide for the selective detection of CO 3 2– in aqueous medium. Probe 3 exhibited a turn-on fluorescence response toward CO 3 2– with excellent selectivity, sensitivity (DL = 2.76 μM), and good reversibility. The binding constant (K) of probe 3 with CO 3 2– was calculated to be 5 × 103 M−1 (log K 3.69). The 1:1 stoichiometry of the complex between probe 3 and CO 3 2– ions was confirmed by Job's plot and ESI-MS spectra. Deprotonation and hydrogen-bonding interactions are involved in the recognition of CO 3 2– ion, which was also suggested by 1H NMR, ESI-MS spectra, and Density Functional Theory (DFT) calculations. Moreover, an INHIBIT type molecular logic gate was constructed by using 3:CO 3 2– and CH 3 COOH as inputs and current signal as output. Owing to the practical applications, probe 3 demonstrated its efficiency in quantifying CO 3 2– ion in real water samples through standard addition method, thus showcasing its potential in real environment. Further, the MTT assay indicated very low cytotoxicity (IC 50 = 1 mM) of probe 3 and also the cell imaging experiments demonstrated the effective sensing of CO 3 2– ions with probe 3 in the biological systems. [ABSTRACT FROM AUTHOR]

[Display omitted] Chimeric Antigen Receptor T cell (CAR-T) therapy has emerged as a transformative therapeutic strategy for hematological malignancies. However, its efficacy in treating solid tumors remains limited. An in-depth and comprehensive understanding of CAR-T cell signaling pathways and the ability to track CAR-T cell biodistribution and activation in real-time within the tumor microenvironment will be instrumental in designing the next generation of CAR-T cells for solid tumor therapy. This review summarizes the signaling network and the cellular and molecular imaging tools and platforms that are utilized in CAR-T cell-based immune therapies, covering both in vitro and in vivo studies. Firstly, we provide an overview of the existing understanding of the activation and cytotoxic mechanisms of CAR-T cells, compared to the mechanism of T cell receptor (TCR) signaling pathways. We further describe the commonly employed tools for live cell imaging, coupled with recent research progress, with a focus on genetically encoded fluorescent proteins (FPs) and biosensors. We then discuss the utility of diverse in vivo imaging modalities, including fluorescence and bioluminescence imaging, Magnetic Resonance Imaging (MRI), Positron Emission Tomography (PET), and photoacoustic (PA) imaging, for noninvasive monitoring of CAR-T cell dynamics within tumor tissues, thereby providing critical insights into therapy's strengths and weaknesses. Lastly, we discuss the current challenges and future directions of CAR-T cell therapy from the imaging perspective. We foresee that a comprehensive and integrative approach to CAR-T cell imaging will enable the development of more effective treatments for solid tumors in the future. [ABSTRACT FROM AUTHOR]

• We review enzyme-free nucleic acid amplification for DNA repair enzyme imaging. • Their sensing mechanisms and practical applications are highlighted. • The performances of different approaches are compared. • We discuss the potential challenges and future perspectives in this field. To maintain the stability and integrity of cellular genomic DNA, DNA repair enzymes catalyze the repair of DNA damage caused by exogenous and endogenous factors through various DNA repair pathways (e.g., base excision repair pathway). Abnormal DNA repair enzyme activity hampers the timely repair of DNA damage, inducing cell growth stagnation, cell death, transformation of normal cells into tumor cells and eventually the occurrence of human diseases including cancer. DNA repair enzymes have become the valuable biomarkers and potential therapeutic targets in biomedical research and clinical diagnosis. To accurately monitor DNA repair enzymes with low expression levels in cells, signal amplification strategies have been introduced to achieve reliable signal sensing and signal gain, facilitating diagnosis and treatment of malignant diseases. Conventional strategies for amplified detection of DNA repair enzymes involve multiple enzymes, washing/separation steps, and thermal cycling, and they are unable to provide spatiotemporal information of intracellular DNA repair enzymes. In recent years, some enzyme-free nucleic acid amplification approaches have been introduced for real-time imaging of intracellular DNA repair enzymes, providing new insights into the dynamic changes of DNA repair enzymes at the single-cell level. Herein, we review the recent advances in enzyme-free nucleic acid amplification-based fluorescent biosensors for the imaging of intracellular DNA repair enzymes. We highlight the mechanisms and applications of these enzyme-free nucleic acid amplification approaches, and further compare the performances of different enzyme-free nucleic acid amplification approaches for DNA repair enzyme imaging. Furthermore, we discuss the potential challenges and future perspectives in this field. [ABSTRACT FROM AUTHOR]

[Display omitted] • A Zn2+ selective turn-on optical sensor (DIH) with ESIPT process was reported. • DIH showed high selectivity and sensitivity Zn2+ over other metal ions tested. • The detection mechanism was elucidated by ESI–MS, NMR and DFT calculations. • The sensor is suitable for imaging Zn2+ in living cells. Zinc ions (Zn2+) play a significant role in our daily lives and production, which is related to human health and environment protection. Therefore, it is crucial in this field to develop sensors with improved analyte selectivity and sensitivity. We prepared a fluorescent sensor, DIH, through the reaction of 5-(tert -butyl)-2-hydroxyisophthalaldehyde with isonicotinohydrazide. DIH, in free form, can undergo excited state intramolecular proton transfer (ESIPT) process upon excitation. Among a wide range of metal ions tested, only Zn2+ inhibited this ESIPT process, resulting in an increase in fluorescence intensity (13.3-fold) with chelation enhancement fluorescence process (CHEF). The complex (DIH/Zn2+) stoichiometry was determined to be 1:1 and the binding constant of the complex was estimated as 3.18 × 107 M−1. DIH could also be applied to detect zinc ions down to a low concentration level of 11.2 nanomolar with fluorescence spectroscopy. Moreover, the cytotoxicity evaluation of DIH against human colon adenocarcinoma cells lines demonstrated that the sensor was nontoxic and could be used successfully in cellular imaging. [ABSTRACT FROM AUTHOR]

The misfolding and aggregation of specific proteins induced by endogenous oxidation are closely related to some incurable diseases including cancers, diabetes and neurodegenerative diseases. Reactive oxygen species (ROS), as a common oxidizing substance in vivo would induce the oxidation of endogenous biomacromolecules like proteins. However, it remains a challenge to detect protein aggregation induced by specific ROS. Herein, we demonstrate that the developed probe (P2) with a dual function that can simultaneously detect hydrogen peroxide (H 2 O 2) levels and protein aggregates formed by oxidative stress. In this probe, the boronic acid pinacol ester group was conjugated to fluorescent molecular rotor 4-hydroxybenzylidene-imidazolinone (HBI) and served as the H 2 O 2 detection group. Experimental results show that the fluorescence of P2 can be activated by H 2 O 2 and exhibited a positive correlation with the degree of protein aggregation. Moreover, combined with AggTag method P2 was successfully applied to dual-channel imaging of protein aggregation that is stressed by H 2 O 2 in cellular. In general, this work not only provides a potential method to study protein aggregation that associates with oxidation in live cells but also can be generally applied to study other biological processes. • A dual-functional probe activated by hydrogen peroxide (H 2 O 2) and viscosity was designed and synthesized. • The probe responds to H 2 O 2 with high selectivity. • The probe exhibits a cascade response to H 2 O 2 and protein aggregation. • Revealing the process of protein aggregation under H 2 O 2 stress in live cells. [ABSTRACT FROM AUTHOR]

• 120 nm, 500 ms/frame imaging of live osteoblasts identifies ER-Golgi procollagen carriers. • ER-Golgi procollagen carriers contain ERGIC protein ERGIC53 but not HSP47. • HSP47 is released from procollagen at ER exit sites rather than ERGIC or cis-Golgi. • Procollagen carrier formation requires GDP to GTP exchange at ARF1 GTPase. Efficient quality control and export of procollagen from the cell is crucial for extracellular matrix homeostasis, yet it is still incompletely understood. One of the debated questions is the role of a collagen-specific ER chaperone HSP47 in these processes. Most ER chaperones preferentially bind to unfolded polypeptide chains, enabling selective export of natively folded proteins from the ER after chaperone release. In contrast, HSP47 preferentially binds to the natively folded procollagen and is believed to be released only in the ER-Golgi intermediate compartment (ERGIC) or cis-Golgi. HSP47 colocalization with procollagen in punctate structures observed by immunofluorescence imaging of fixed cells has thus been interpreted as evidence for HSP47 export from the ER together with procollagen in transport vesicles destined for ERGIC or Golgi. To understand the mechanism of this co-trafficking and its physiological significance, we imaged the dynamics of fluorescently tagged type I procollagen and HSP47 punctate structures in live MC3T3 murine osteoblasts with up to 120 nm spatial and 500 ms time resolution. Contrary to the prevailing model, we discovered that most bona fide carriers delivering procollagen from ER exit sites (ERESs) to Golgi contained no HSP47, unless the RDEL signal for ER retention in HSP47 was deleted or mutated. These transport intermediates exhibited characteristic rapid, directional motion along microtubules, while puncta with colocalized HSP47 and procollagen similar to the ones described before had only limited, stochastic motion. Live cell imaging and fluorescence recovery after photobleaching revealed that the latter puncta (including the ones induced by ARF1 inhibition) were dilated regions of ER lumen, ERESs, or autophagic structures surrounded by lysosomal membranes. Procollagen was colocalized with HSP47 and ERGIC53 at ERESs. It was colocalized with ERGIC53 but not HSP47 in Golgi-bound transport intermediates. Our results suggest that procollagen and HSP47 sorting occurs at ERES before procollagen is exported from the ER in Golgi-bound transport intermediates, providing new insights into mechanisms of procollagen trafficking. [ABSTRACT FROM AUTHOR]

• Growth factor signaling causes sustained nuclear ERK1/2 activation. • The SCR and BCR/ABL inhibitor dasatinib blocks ERK1/2 and represses cell invasion. • EGF-stimulated cells may escape dasatinib inhibition of invasion through mesenchymal to amoeboid transition. • Combined inhibition of SRC and Rho-kinase signaling is necessary to completely block EGF-induced invasion. Aberrantly activated kinase signaling pathways drive invasion and dissemination in medulloblastoma (MB). A majority of tumor-promoting kinase signaling pathways feed into the mitogen-activated protein kinase (MAPK) extracellular regulated kinase (ERK1/2) pathway. The activation status of ERK1/2 during invasion of MB cells is not known and its implication in invasion control unclear. We established a synthetic kinase activation relocation sensor (SKARS) for the MAPK ERK1/2 pathway in MB cells for real-time measuring of drug response. We used 3D invasion assays and organotypic cerebellum slice culture to test drug effects in a physiologically relevant tissue environment. We found that hepatocyte growth factor (HGF), epidermal growth factor (EGF), or basic fibroblast growth factor (bFGF) caused rapid nuclear ERK1/2 activation in MB cells, which persisted for several hours. Concomitant treatment with the BCR/ABL kinase inhibitor dasatinib completely repressed nuclear ERK1/2 activity induced by HGF and EGF but not by bFGF. Increased nuclear ERK1/2 activity correlated positively with speed of invasion. Dasatinib blocked ERK-associated invasion in the majority of cells, but we also observed fast-invading cells with low ERK1/2 activity. These ERK1/2-low, fast-moving cells displayed a rounded morphology, while ERK-high fast-moving cells displayed a mesenchymal morphology. Dasatinib effectively blocked EGF-induced proliferation while it only moderately repressed tissue invasion, indicating that a subset of cells may evade invasion repression by dasatinib through non-mesenchymal motility. Thus, growth factor-induced nuclear activation of ERK1/2 is associated with mesenchymal motility and proliferation in MB cells and can be blocked with the BCR/ABL kinase inhibitor dasatinib. [ABSTRACT FROM AUTHOR]

With this research we presented a ratiometric and mitochondria-target fluorescent probe (Mito-HT) for detection of H 2 O 2 both in vitro and in live cells. Mito-HT was constructed by direct conjugation of aryl boronate to fluorophore with three synthetic steps. The borate group is cleaved from Mito-HT in the presence of H 2 O 2 , resulting in the exposure of the hydroxyl group of the electron donating group. Then the ICT mechanism was turned on, and the fluorescence emission of Mito-HT at 493 nm was red-shifted to 562 nm, thereby achieving radiometric detection of H 2 O 2. Mito-HT exhibited a highly selectivity towards H 2 O 2 , and this interaction can be completed within 40 min. Mito-HT could be used for quantitative detection of H 2 O 2 (0–200 μM) through ratiometric fluorescence signal readout. And limit of detection (LOD) is approximately 0.33 μM. The relatively high stability and medium fluorescence quantum yield of Mito-HT (0.39) and Mito-HT-OH (0.43) enable clear mitochondria localization and dual-channel fluorescence imaging of H 2 O 2 in live cells with confocal microscopy. Image 1 • A new ratiometric and mitochondria-target fluorescent probe (Mito-HT) was constructed. • Mito-HT exhibited a selective response towards H 2 O 2 through ratiometric fluorescence signal readout. • Mito-HT could be used for quantitative detection of H 2 O 2 (0–200 μM). • Mito-HT localized in mitochondria and allows a dual-channel imaging of H 2 O 2 in live cells. [ABSTRACT FROM AUTHOR]

Although important breakthroughs in our understanding of the hepatitis B virus (HBV) life cycle have been made since the discovery of its main entry factor, the spatiotemporal dynamics of HBV–host interactions remains understudied. Here, we discuss recent advances and continuing challenges to image the HBV life cycle in live cells. [ABSTRACT FROM AUTHOR]

Strategically modified4-(diethylamino)-2-(pyridin-2-ylmethoxy)benzaldehyde appended BODIPY-based probe N,N-SP-BPY detects aspartic acid (Asp) and glutamic acid (Glu) by inhibition of Photoinduced electron transfer (PET) process in water. The probe shows considerable and rapid fluorescence enhancement upon addition of Asp and Glu. Additionally, a distinct colorimetric change from pink to a colorless solution can be observed to recognize traces of Asp and Glu. The probe detects Asp and Glu as low as 1 μM and 5 μM respectively. Furthermore, the probe can efficiently detect intracellular Asp and Glu in live HeLa, A549, MDA-MB-468 and HEK-293T cells under fluorescence microscope without showing any cytotoxic effect. Two model BODIPY derivatives are also synthesized to establish strategic modification. 1H NMR titration and theoretical calculations strongly supports experimental findings. Image 1 • A highly selective BODIPY probe, N,N-SP-BPY detects aspartic acid (Asp) and glutamic acid (Glu) in water. • 'Turn On' fluorescence can be observed by inhibition of Photoinduced Electron Transfer (PET) process. • A distinct colorimetric change from pink to colourless solution can be observed to recognize Asp and Glu. • N,N-SP-BPY efficiently images intracellular Asp and Glu in live cells under fluorescence microscope. [ABSTRACT FROM AUTHOR]

• A fluorescence strategy was proposed for detection and imaging of multiple microRNAs. • A pH-responsive ZnO nanoprobe was designed. • The strategy showed good sensitivity and specificity for multiple microRNAs. • Different levels of miR-21 expression in three types of cells were differentiated. • Simultaneous imaging of miR-21 and miR-373 in same living cells was achieved. Here, a pH-responsive ZnO nanoprobe mediated DNAzyme signal amplification strategy was proposed for sensitive detection and live cell imaging of multiple microRNAs. The nanoprobe including ZnO nanoparticles (ZnO NPs) core as carrier and cofactor provider and polydopamine shell for adsorption the functional hairpin DNAs was designed. When nanoprobe entered the cell through endocytosis, the acidic environment of the cell could decompose the ZnO NPs core to release the functional hairpin DNAs and Zn2+. The Zn2+ could act as cofactor for the DNAzyme cleavage amplification reaction, avoiding additional cell delivery processes. The recognition hairpin DNAs (H1 and H3) recognized microRNA and exposed the DNAzyme. The DNAzyme cleaved cyclically the reporter hairpin DNAs (H2 and H4), producing enhanced fluorescent signal for miR-21 and miR-373 detection with the detection limit of 54 pM and 38 pM, respectively. Expression levels of miR-21 in Hela, HepG-2 and L02 cells were differentiated. Furthermore, simultaneous imaging of miR-21 and miR-373 in same living cells was achieved. These results indicated this strategy could have a potential application in microRNAs assays for the accurate diagnosis and therapy of cancer. [ABSTRACT FROM AUTHOR]

A simple julolidine based chemosensor (JT) was designed and synthesized by single condensation step. JT displayed excellent selectivity and sensitivity with on-off responses towards Zn2+ and Cu2+ over other biologically relevant metal ions in aqueous media. Upon addition of Zn2+ ions, JT exhibited a significant blue shift in emission followed by turn- on enhancement while with Cu2+, the fluorescence intensity of JT was completely vanished. The 1:1 binding affinity between JT and Zn2+/Cu2+ was proposed by Job's plot analysis. The detection limit for Zn2+ and Cu2+ ions reached at 3.5 × 10−8 M and 1.46 × 10−6 M, respectively. The sensing mechanism of JT with Zn2+/Cu2+ was supported by DFT calculations. Based on photophysical studies and its reversibility environment with EDTA, molecular logic gates were fabricated. Furthermore, JT was successfully established to detect intracellular Zn2+ ions in live cells by turn-on response. A bifunctional chemosensor JT was synthesized to distinguish Zn2+ and Cu2+ ions based on emission on-off response. Unlabelled Image • New julolidine based Schiff base derivative (JT) was designed and synthesized and JT served as a selective emission on-off response towards Zn2+ and Cu2+ ions. • The sensor shows excellent sensitivity for Zn2+ and Cu2+ ions with an LOD at 3.5 × 10 -8 M and 1.46 × 10 -6 M, respectively. • The 1:1 binding model of the sensor JT with Zn2+/Cu2+ was established and reversibility of the chemosensor JT was proved by experiments with EDTA. • JT was successfully established to track intracellular Zn2+ ions in HeLa cells. [ABSTRACT FROM AUTHOR]

Transcription–blocking lesions are specifically targeted by transcription-coupled nucleotide excision repair (TC-NER), which prevents DNA damage-induced cellular toxicity and maintains proper transcriptional processes. TC-NER is initiated by the stalling of RNA polymerase II (RNAPII), which triggers the assembly of TC-NER-specific proteins, namely CSB, CSA and UVSSA, which collectively control and drive TC-NER progression. Previous research has revealed molecular functions for these proteins, however, exact mechanisms governing the initiation and regulation of TC-NER, particularly at low UV doses have remained elusive, partly due to technical constraints. In this study, we employ knock-in cell lines designed to target the endogenous CSB gene locus with mClover, a GFP variant. Through live cell imaging, we uncover the intricate molecular dynamics of CSB in response to physiologically relevant UV doses. We showed that the DNA damage-induced association of CSB with chromatin is tightly regulated by the CSA-containing ubiquitin-ligase CRL complex (CRL4CSA). Combining the CSB-mClover knock-in cell line with SILAC-based GFP-mediated complex isolation and mass-spectrometry-based proteomics, revealed novel putative CSB interactors as well as discernible variations in complex composition during distinct stages of TC-NER progression. Our work not only provides molecular insight into TC-NER, but also illustrates the versatility of endogenously tagging fluorescent and affinity tags. • CSB-fluorescently tagged knock-in cell line is a powerful and sensitive tool to monitor TC-NER kinetics in living cells. • Advantage of using the same tag for imaging and protein-association studies. • Development of fluorescence-based single-cell assay to study the mechanism and regulation of TC-NER. [ABSTRACT FROM AUTHOR]

Lipid droplets (LD) are important regulators of lipid metabolism and are implicated in several diseases. However, the mechanisms underlying the roles of LD in cell pathophysiology remain elusive. Hence, new approaches that enable better characterization of LD are essential. This study establishes that Laurdan, a widely used fluorescent probe, can be used to label, quantify, and characterize changes in cell LD properties. Using lipid mixtures containing artificial LD we show that Laurdan GP depends on LD composition. Accordingly, enrichment in cholesterol esters (CE) shifts Laurdan GP from ∼0.60 to ∼0.70. Moreover, live-cell confocal microscopy shows that cells present multiple LD populations with distinctive biophysical features. The hydrophobicity and fraction of each LD population are cell type dependent and change differently in response to nutrient imbalance, cell density, and upon inhibition of LD biogenesis. The results show that cellular stress caused by increased cell density and nutrient overload increased the number of LD and their hydrophobicity and contributed to the formation of LD with very high GP values, likely enriched in CE. In contrast, nutrient deprivation was accompanied by decreased LD hydrophobicity and alterations in cell plasma membrane properties. In addition, we show that cancer cells present highly hydrophobic LD, compatible with a CE enrichment of these organelles. The distinct biophysical properties of LD contribute to the diversity of these organelles, suggesting that the specific alterations in their properties might be one of the mechanisms triggering LD pathophysiological actions and/or be related to the different mechanisms underlying LD metabolism. [Display omitted] • Lipid droplets (LD) are organelles involved in cell homeostasis and in pathology. • Cells have multiple LD populations with distinct biophysical properties. • Laurdan senses changes in LD properties caused by compositional remodeling. • LD hydrophobicity change in response to several stress and pathological conditions. • LD heterogeneity might underly the distinct cellular actions of LD. [ABSTRACT FROM AUTHOR]

The widespread use of cyanide (CN−) in industry results in contamination of various effluents such as drain, lake, and tap water, an imminent danger to the environment and human health. We prepared naphthoquinolinedione (cyclized; 1 – 5) and anthracenedione (un-cyclized) probes (6 – 7) for selective detection of CN−. The addition of CN− to the probe solutions (1 – 5) resulted in a color change from pale green to orange under 365 nm illumination. The nucleophilic addition of CN− to C2 of the imidazolium ring of the probes is responsible for selective CN− detection. Among all probes, 1 gave the lowest fluorescence-based LOD of 0.13 pM. In contrast, the un-cyclized probes (6 and 7) were substantially inferior to the cyclized counterparts (1 and 2 , respectively) for detecting a trace amount of CN−. The notably low LOD displayed by probe 1 was maintained in the detection of CN− in real food samples, human fluids, and human brain cells. This is the first report studying imidazolium-bearing naphthoquinolinedione-based probes for CN− sensing in 100% water. Water-soluble probe 1 displayed a selective CN− sensing in water, along with a very low LOD of 0.13 pM and the probe was successfully applied for CN− sensing in human bio-fluids and food items. [Display omitted] • Several probes 1 – 7 were prepared in a single step. • Probe 1 showed a selective color change upon addition of CN−. • Formation of [probe 1 -CN−] adduct is responsible for fluorescence response. • Probe 1 displayed an effective detection of CN− in food samples and human bio-fluids. • A concentration of CN− as low as 1 μM was detected in human brain cells using probe 1. [ABSTRACT FROM AUTHOR]

Abstract A new simple pyrene based schiff base chemosensor 1 (nicotinic acid pyren-1-ylmethylene-hydrazide) has been constructed and is prepared from 1-pyrenecarboxaldehyde and nicotinic hydrazide. Notably, the chemosensor 1 exhibited remarkable colour changes while in the presence of trivalent metal ions like Bi3+ & Al3+ ion in DMSO-H 2 O, (1:1 v/v, HEPES = 50 mM, pH = 7.4). The UV–Vis spectral investigation of chemosensor 1 showed that the maximum absorption peak appeared at 378 nm. In emission studies, chemosensor 1 develops weak fluorescence, while upon the addition of Bi3+ and Al3+ ions, it exhibits an enhancement of fluorescence intensity. Nevertheless, rest of metal ions have no changes in the emission spectra. The association constant of chemosensor 1 for binding to Bi3+ & Al3+ system had a value of 1.27 × 104 M−1 and 1.53 × 104 M−1. The detection limits were 0.12 µM for Bi3+ and 0.17 µM for Al3+ respectively. The overall results reveal that chemosensor 1 can act as a dual-channel, highly selective, and sensitive probe for Bi3+ and Al3+ ions. Moreover, the fluorescence imaging of chemosensor 1 was applied in RAW 264.7 cell line and cytotoxicity assay prove that this chemosensor 1 is non-toxic as well as highly biocompatible. Graphical abstract A new pyrene based schiff base derivative (chemosensor 1) was designed and synthesized for the detection of dual metal ions Bi3+ and Al3+ ions with live cell imaging. fx1 Highlights • A new simple pyrene based Schiff's base chemosensor 1 was designed and synthesized. • The chemosensor 1 has enhanced fluorescence intensity and dual response to Bi3+ and Al3+ ions. • The binding interaction was confirmed by Job's plot. • Chemosensor 1 utilized for fluorescence imaging of Bi3+ and Al3+ in RAW 264.7 cell line. [ABSTRACT FROM AUTHOR]

A new, bis -thiophene based Schiff base receptor A2 has been synthesized and rationally designed as rapid and highly selective fluorescence sensor for Cu2+ ion in a semi-aqueous medium. Structural characterization of A2 was confirmed by NMR, FT-IR and HRMS spectral techniques. The receptor A2 shows the colorimetric response from yellow to colorless and fluorescence "turn-on" with Cu2+ ions under visible and UV light. Both the UV–Vis and fluorescence studies were confirmed the formation of 1:1 stoichiometry complex with an estimated association constant of 1.07 × 10−5 M−1. The sensor could work in the pH range of 4.0–8.0 without interference in the presence of other interfering ions. The lowest detection limits by colorimetric and fluorescence techniques were found to be 1.18 × 10−7 M and 8.8 × 10−8 M respectively. Further, the probe was applied to image the intracellular Cu2+ ions in the HeLa cell line. The sensing mechanism was studied by ESI-mass analysis and DFT calculations. Unlabelled Image • Synthesized Schiff base receptor A2 exhibited superb selectivity for Cu2+ ion. • The lowest detection limit of Cu2+ (UV–Vis =1.18 × 10−7 M and FL = 8.8 × 10−8 M) for receptor A2 reported. • DFT calculations supported the experimental data. • The receptor A2 was used to observe Cu2+ ion in live cell imaging. • A "Turn-on" Cu2+ sensor with 19-fold fluorescence enhancement was observed. [ABSTRACT FROM AUTHOR]

Abstract A new coumarin-derived hydrazone (1) has been developed as a "turn-on" fluorescent probe for the detection of Cu2+ in two different sensing mechanisms. In anhydrous acetonitrile, the strong fluorescence response of 1 to Cu2+ was mainly attributed to the chelation-controlled C=N isomerization. Besides, S-donor is indispensable in protecting from the Cu2+-induced fluorescence quenching. In aqueous acetonitrile, 1 as a chemodosimeter can highly selectively sense of Cu2+, which was ascribed to the Cu2+-promoted cyclization reaction affording the strong fluorescent cyclization product (2). The proposed cyclization reaction was confirmed by the single-crystal structure of 2. Furthermore, 1 was utilized for imaging intracellular Cu2+ with good performance. Graphical abstract Image 1 Highlights • A fluorescent chemodosimeter for Cu2+ based on Cu2+-promoted cyclization reaction was developed. • The fluorescent product of the Cu2+-promoted reaction was characterized by single-crystal X-ray analysis. • The aza-michael addition based mechanism of the Cu2+-promoted cyclization reaction was proposed. • The chemodosimeter was utilized for imaging intracellular Cu2+ with good performance. • The fluorescent probe can sense Cu2+ in two different sensing mechanisms. [ABSTRACT FROM AUTHOR]

Graphical abstract Highlights • We developed a deep-red fluorogenic probe (BT-NH), with high sensitivity and good selectivity to detect NO. • The probe was successfully used to visualize exogenous/endogenous NO level in living cells. • The probe was further applied to detect NO in PD models. Abstract Nitric oxide (NO), as one of important gaseous signaling molecule in human body, has been associated with a variety of physiologic processes. However, excessive production of NO has been profoundly implicated in the pathogenesis of neurodegenerative disorders in particular, Parkinson's disease (PD). Therefore, accurate and facile detection of NO is of great significance for investigating its functions in PD, and the subsequent diagnosis and/or treatment. Herein, we developed a deep-red fluorogenic probe (BT-NH), with high sensitivity and good selectivity to detect NO, which was successfully used to visualize exogenous/endogenous NO level in living cells, and further applied to in vitro and in vivo PD models. Therefore, BT-NH could be a potential biological fluorogenic tool to explore the roles of NO in PD. [ABSTRACT FROM AUTHOR]

Graphical abstract A new fluorescent rhenium(I) carbonyl complex 1 , bearing {Re(CO) 3 }+ core with ONS donor thioether containing redox noninnocent azo-phenol ligand has been synthesized and characterized. The distorted octahedral geometry of the complex is confirmed by X-ray structure. The complex exhibits low energy emission band at 525 nm with high emission quantum yield (ϕ = 0.115). Cytotoxicity of the complex is studied by MTT method with human breast cancer cell lines (MCF-7) and IC 50 value is found to be 23.6 μM. In presence of the complex (10 μM) a bright green fluorescence image of MCF-7 cell lines is observed under fluorescence microscope. Abstract Herein, we have synthesized a new fluorescent rhenium(I) carbonyl complex 1 , bearing {Re(CO) 3 }+ core with ONS donor thioether containing azo-phenol redox noninnocent ligand. The distorted octahedral geometry of the complex is confirmed by single crystal X-ray diffraction method. Cyclic voltammogram in acetonitrile exhibits irreversible oxidation peak (E pa = 1.36 V) along with quasi-reversible reduction peak at E 1/2 = −0.92 V (Δ E = 210 mV). The complex exhibits low energy emission band at 525 nm with high emission quantum yield (ϕ = 0.115). Cytotoxicity of the complex is studied by MTT method with human breast cancer cell lines (MCF-7) and IC 50 value is found to be 23.6 μM. In presence of the complex (10 μM) a bright green fluorescence image of MCF-7 cell lines is observed under fluorescence microscope. [ABSTRACT FROM AUTHOR]

Graphical abstract Highlights • A new probe RDNAP-PY is developed for selective detection of Cr3+ ion. • RDNAP-PY shows ratiometric fluorescence and colorimetric change in solution. • The limit of detection of RDNAP-PY for Cr3+ ion is 1.81×10−6 M. • The probe efficiently images intracellular Cr3+ in live Hep3B, MCF-7, HeLa, SiHa and HEK 293T cells. Abstract A 2-(1,3-dioxo-1H-benzo[de]isoquinolin-2(3H)-yl)acetaldehydeanchored rhodamine B based probe, RDNAP detects Cr3+ ion by fluorescence resonance energy transfer (FRET) process in aqueous buffered acetonitrile media (7:3, v/v). Conversely, conjugation of 2-(1,3-dioxo-6-(piperidin-1-yl)-1H-benzo[de]isoquinolin-2(3H)-yl)acetaldehyde with rhodamine B provides another probe, RDNAP-PY that undergoes Cr3+assisted ratiometric fluorescence and colorimetric change in the same media. RDNAP-PY provides higher FRET efficiency and detects as low as 1.81×10−6 M Cr3+with an association constant, 15.9 × 104 M−1. Other common ions do not interfere. RDNAP-PY efficiently images intracellular Cr3+ in live Hep3B, MCF-7, HeLa, SiHa and HEK 293T cells under fluorescence microscope in a ratiometric and time dependent manner. 1H NMR titration and DFT studies strongly support experimental findings. [ABSTRACT FROM AUTHOR]

Graphical abstract Highlights • The probe exhibits aggregation induced emission enhancement. • Emits in the broad range covering blue to red in its dry state. • Reversible emission could be achieved simply by changing the excitation wavelength. • The high biocompatibility makes the probe an ideal candidate for fluorescence imaging of live cells. • The probe demonstrates rarely known detection of picric acid in live cells. Abstract In an attempt to achieve photo switching of multicolour photoluminescence on a unimolecular platform, herein we report a benzothiazole based probe as an excitation wavelength sensitive multicolour emitter in its aggregated state. Though in solution the blue emission overwhelms others, but in the dry state the emission in blue, green and red with intermediate yellow could be achieved. Further, the probe in its aggregated state detects picric acid in solution, solid and live cells, where blue emission is quenched in all the three working media while the red emission is activated in solid state and live cells. TD-DFT (time dependent density function theory) calculations were performed to understand the electronic properties and sensing behaviour of the probe. This probe constitutes a first example of detection of picric acid in human cancer cells to the best of our knowledge. The tuneable multicolour emission of the probe suggests its possible implementation in applications related to OLEDs. [ABSTRACT FROM AUTHOR]

Graphical abstract Highlights • A novel fluorescent probe (DFI) was designed and synthesized. • DFI showed good selectivity towards H 2 S with 'turn on' fluorescence emission. • The detection limit of DFI probe towards H 2 S was found to be 0.13 nM. • DFI probe was able to detect H 2 S in live cells. Abstract A simple turn-on fluorescence sensor (2,2′-(2-(2,4-dinitrophenoxy)-5-methyl-1,3-phenylene)bis(1,4,5-triphenyl-1H-imidazole) (DFI) was synthesized, characterized and its sensing properties were investigated through fluorescence spectral studies. The new fluorescence sensor DFI shows a selective sensing of hydrogen sulfide (H 2 S) via enhancement of fluorescence intensity and the detection limit was found to be 0.13 nM. Subsequently, the probe DFI has been applied to be an effective bio-tracker for monitoring hydrogen sulfide in real samples as well as living cells. [ABSTRACT FROM AUTHOR]

Abstract Hydrazino-carbothioamide, 1 , serves as a turn-on fluorescent chemosensor to Zn2+ and the mixture shows green emission (λ em 492) in presence of large number of ions with impressive limit of detection (LOD), 0.59 nM. Intense fluorescence of Zn-complex is selectively turn-off upon adding H 2 PO 4 − only to the limit of detection, 26 μM, while other anions remain silent. The sensing mechanism of 1 is established by suppressing the ESIPT of 1 and complexation with Zn2+ (CHEF) is supported by Job's plot, 1H NMR, and HR-MS data. Turn-on-off sequential detection of Zn2+ and H 2 PO 4 − also have successfully been employed for the engineering of INHIBIT molecular logic gate. The sensor is non-toxic and has been used in identifying Zn2+ in the intracellular region of African green monkey kidney cells (Vero cells). Graphical Abstract Very low detection limit (0.59 nM) is shown by green emission (492 nm) of hydrazino-carbothioamide probe on binding selectively with Zn2+ at pH, 7.2 which is specifically quenched by H 2 PO 4 − with LOD, 26 μM. This probe uses detection of Zn2+ in Vero cells and INHIBIT logic gate is constituted. Unlabelled Image Highlights • Hydrazino-carbothioamide sensor shows green emission with Zn2+. • Highly selective turn-on Zn2+ sensor shows LOD 0.59 nM. • Emission of the complex is quenched specifically upon addition of H 2 PO 4 − and LOD is 26 μM. • The sensor is non-toxic and identifies Zn2+ in the intracellular region of Vero cells. • Emission change at 492 nm with Zn2+ and H 2 PO 4 − can be described to INHIBIT logic gate. [ABSTRACT FROM AUTHOR]

Abstract The male germ-line cysts that occur in annelids appear to be a very convenient model for spermatogenesis studies. Germ-line cysts in the studied earthworm are composed of two compartments: (1) germ cells, where each cell is connected via one intercellular bridge to (2) an anuclear central cytoplasmic mass, the cytophore. In the present paper, confocal and transmission electron microscopy were used to follow the changes in the mitochondrial activity and ultrastructure within the cysts during spermatogenesis. JC-1 was used to visualize the populations of mitochondria with a high and low membrane potential. We used the spot detection Imaris software module to obtain the quantitative data. We counted and compared the 'mitochondrial spots' – the smallest detectable signals from mitochondria. It was found that in all of the stages of cyst development, the majority of mitochondria spots showed a green fluorescence, thus indicating a low mitochondrial membrane potential (MMP). Moreover, the number of active mitochondria spots that were visualized by red JC-1 fluorescence (high MMP) drastically decreased as spermatogenesis progressed. As much as 26% of the total number of mitochondrial spots in the spermatogonial cysts showed a high MMP – 19% in the spermatocytes, 24% in the isodiametric spermatids and 3% and 6%, respectively, in the cysts that were holding early and late elongate spermatids. The mitochondria were usually thread-like and had an electron-dense matrix and lamellar cristae. Then, during spermiogenesis, the mitochondria within both the spermatids and the cytophore had a tendency to form aggregates in which the mitochondria were cemented by an electron-dense material. Highlights • Mitochondria activity changes during all stages of spermatogenesis in germ-line cysts cells were exanimated. • The level of active mitochondria in germ-line cysts visualized by JC-1 never exceeded 26%. • During spermatogenesis progression, the percentage of active mitochondria decreased. • Image analyses suggest that mitochondria form network(s) that entwine the entire cyst and showed tendency to form aggregates. • The formation of aggregates coincided with a drop in mitochondria activity level. [ABSTRACT FROM AUTHOR]

Abstract The synthesis and characterization of meso-piperidine linked Bodipy starting from 4-(2-(piperidine-1-yl)ethoxy)benzaldehyde was described. Meso-piperidine linked Bodipy was subsequently used for the construction of distyryl-piperidine-Bodipy and distyryl-morpholine-Bodipy via Knoevenagel type reaction. The photophysical properties including molar extinction coefficient, fluorescence lifetime and fluorescence quantum yield of Bodipys were investigated in ethanol solution. The targeted compounds were also assessed in live cell imaging and cytotoxicity studies by using breast cancer cell line, MCF-7 in vitro. Graphical abstract fx1 [ABSTRACT FROM AUTHOR]

Abstract Oxidative stress, specifically lipid peroxidation, is a major driving force in neurodegenerative processes. However, the exact role of lipid peroxidation remains elusive as reliable real-time detection and quantification of lipid peroxyl radicals proves to be challenging in vitro and in vivo. Motivated by this methodological limitation, we have optimized conditions for real-time imaging and quantification of lipid peroxyl radical generation in primary neuron cultures using the lipophilic fluorogenic antioxidant H 4 BPMHC (8-((6-hydroxy-2,5,7,8-tetramethylchroman-2-yl)-methyl)-1,5-di(3-chloropropyl)-pyrromethene fluoroborate), an α-tocopherol analog probe. By subjecting neurons to different antioxidant conditions in the presence and absence of lipid peroxidation inducing stressors (Haber-Weiss reagents), we maximized H 4 BPMHC sensitivity and confirmed its potential to temporally resolve subtle and marked differences in lipid peroxidation levels in real-time. Herein we report imaging and quantification of homeostatic and induced lipid peroxidation in primary neuron cultures, supporting the use of this probe for investigating healthy and diseased states. Overall these results provide the necessary foundation and impetus towards using H 4 BPMHC for elucidating and mapping lipid peroxyl radical contributions to ROS-associated pathological processes in neurons. Graphical abstract fx1 Highlights • Lipid peroxyl radicals were imaged and quantified real-time in primary neurons. • Antioxidant load impacted the rate of lipid peroxyl radical formation. • H 4 BPMHC has potential to detect subtle changes in lipid peroxyl radical levels. [ABSTRACT FROM AUTHOR]

Abstract A novel multifunctional ratiometric fluorescent probe has been designed and synthesized for the selective recognition of Cd2+/F− ions. The probe (3)-((2)-(1H-benzoimidazole-2-yl)-phenylimino) methyl-4-chloro-methyl-2H-chromen-2-ene ( BIMC ) displays excellent ratiometric responses towards Cd2+/F− ions over the tested cations/anions. The lowest detection limits observed for Cd2+ and F− are 1.5 × 10−10 mol/l and 1.2 × 10−10 mol/l respectively. Job's plot and Electro spray Ionization mass spectral (ESI-MS) studies confirms 1:1 binding stoichiometry of BIMC with Cd2+/F− ions, which is further evidenced by 1H NMR titration studies. The reversibility studies of BIMC with Cd2+ have been investigated using ethylenediaminetetraacetic acid (EDTA). Upon binding to Cd2+/F− ions, the probe features strong ratiometric response in both UV–Visible and fluorescence spectra due to the inhibition of intramolecular charge transfer (ICT). Furthermore, the mechanism of ICT has been rationalized via solvatochromism and DFT calculations. Graphical Abstract Unlabelled Image Highlights • The multifunctional fluorogenic probe BIMC was synthesized with good yield. • The detection limit of both Cd2+/F− ions is 1.5 × 10−10 mol/L and 1.2 × 10−10 mol/L respectively. • The probe BIMC displays good reversibility towards Cd2+ with high stability. • The probe BIMC is successfully applicable for the intracellular detection of Cd2+ ions in living cells. [ABSTRACT FROM AUTHOR]

Abstract Long-term live cell tracking is desirable and necessary to understand the dynamics and complexity of biological interactions in stem cells and cancer cells. Conventional live cells fluorescence trackers are generally non-degradable and are showing increased toxicity concerns during the long-term application. Previously we developed biodegradable fluorescent poly(citrate)-based hybrid elastomers for bone regeneration applications. Here, we fabricated the photoluminescent poly(citrate-siloxane) nanoparticles (PCSNPs) through an oil/water emulsion method and demonstrated their long-term live stem cells/cancer cells imaging applications. PCSNPs showed a uniform size distribution (mean diameter 120 nm) and highly stable dispersability (above 30 days) in various physiological medium, as well as excellent fluorescent properties and photostability. PCSNPs possess excellent cellular biocompatibility, which could be efficiently internalized by cells and selectively image the cell lysosome with a high photostability. Compared with commercial Cell Tracker™ Green and Cell Tracker™ Red, the adipose-derived mesenchymal stem cells or human hepatoma cells were stably labeled by PCSNPs for over 14 days as they grew and developed (7 passages). Additionally, PCSNPs efficiently tracked cells up to 7 days in vivo through a non-invasively way compared with 1 day of commercial tracker. This study demonstrates an important strategy to design biodegradable multifunctional delivery platforms for biomedical applications such as long-term bioimaging. Graphical abstract Unlabelled Image Highlights • Biodegradable PCS nanoparticles were fabricated through a facile emulsion technology. • PCSNPs showed a monodispersed size (120 nm) and highly stable dispersability. • PCSNPs exhibited excellent fluorescent properties and photostability. • PCSNPs possess excellent cellular biocompatibility and long term cell tracking. [ABSTRACT FROM AUTHOR]

A new easily available “off-on-off” colorimetric and fluorescent probe ( L ) was synthesized and it displayed the selective detection towards Hg 2+ in aqueous solution over other competing metal ions. In presence of Hg 2+ , L showed the incredible color and fluorescent response. More interestingly, the color and fluorescence could be recovered upon the addition of EDTA into the L-Hg 2+ solution. Moreover, this compound can be efficiently applied to molecular logic functions of OR, AND, NOR and NOT gates through the procured spectral results. Based on the reversible and reproducible switching process, we designed a molecular-scale sequential memory unit exhibits the “Writing-Reading-Erasing-Reading” and “Multi-write” activities in the form of binary logic. Density Functional Theory (DFT) calculations were theoretically supported the photo physical changes. Furthermore, the probe efficacy was studied by monitoring changes in intracellular Hg 2+ ions in HeLa cells. [ABSTRACT FROM AUTHOR]

In this communication, we have developed an optical chemosensor 2-amino N-(6-bromo-1,3-dioxo-1H-benzo[ de ]isoquinolin-2(3H)-yl)benzamide ( NAPH ) for selective detection of Al 3+ by reacting 4-bromo-1,8-naphthalic anhydride with 2-aminobenzohydrazide. In (DMSO:H 2 O, 1:1, v/v) medium, the selective and specific nature of NAPH towards Al 3+ was observed by the quenching along with a blue-shift in the absorption of NAPH at 465 nm that resulted in the distinct colour change from light brown to colourless. The selective complexation that occurred between NAPH and Al 3+ was investigated by 1 H NMR and DFT methods. Under similar conditions, the weakly fluorescent receptor NAPH showed a distinct fluorescence enhancement at 555 nm in the presence of Al 3+ among the other tested metal ions and anions. The NAPH ·Al 3+ complex formation is reversible upon addition of strong chelating agent EDTA. The receptor NAPH can be applied to detect Al 3+ down to 2.9 μM without any interference from other tested metal ions. In addition, the receptor NAPH was successfully applied to detect Al 3+ in live HeLa cells. [ABSTRACT FROM AUTHOR]

Abstract Neutrophils can release their chromatin to form neutrophil extracellular traps (NETs), a process known as NETosis. Although NET formation can be induced by various stimuli, recent evidence suggests that these stimuli do so via different mechanisms. Here, we have analysed NET formation induced by lipopolysaccharide (LPS), phorbol 12‑myristate 13‑acetate (PMA) and the calcium (Ca2+) ionophore A23187. Our results show distinct peroxidase and neutrophil elastase activities in both culture supernatant and NETs. Especially stimulation with A23187 led to pronounced peroxidase and elastase release and yielded high peroxidase activity on the resulting NETs. In contrast to LPS and PMA, A23187 did not induce morphological changes of the nuclei. Histone H3 citrullination was more extensively observed upon induction by A23187 and particularly in LPS- and PMA-induced NETs the detection of citrullinated H3 was dependent on the inhibition of neutrophil proteases, which suggests that NET-associated citrullinated histones are readily cleaved by these proteases. With live cell imaging techniques, differences in the rate of plasma membrane permeabilization were observed, not only for the different inducers, but also among individual neutrophils. LPS and PMA, but not A23187, induced early calcium oscillations and the cytosolic calcium concentrations gradually increased upon LPS and PMA stimulation until the plasma membrane ruptured. The levels of reactive oxygen species rose rapidly after PMA stimulation and much later in neutrophils exposed to LPS and A23187. Taken together, the observed molecular and dynamic differences indicate that NET formation induced by LPS, PMA and A23187 proceeds via different pathways. Graphical abstract Unlabelled Image Highlights • NET formation induced by LPS, PMA and A23187 proceeds via different pathways. • LPS, PMA and A23187 induce distinct cell morphological changes. • Activated neutrophils show differences in peroxidase, elastase and deiminase activities. • Variable calcium dynamics upon induction of NETosis by LPS, PMA and A23187 • Relatively fast and high ROS production by neutrophils is induced by PMA. [ABSTRACT FROM AUTHOR]

Abstract Förster resonance energy transfer (FRET) probes are unique tools in biology, as they allow for a non-destructive monitoring of a certain state of a biomolecule or of an artificial substrate within living cells in real time. FRET substrates indicate their relative cleavage rate and thus the in situ activity of a given enzyme. In contrast to quenched probes or turn-on probes, one of the two separate signals of the FRET probes can be used as internal reference, which makes ratio-imaging and quantitation of cleavage events independent of cellular delivery possible. In this review, we describe the first examples of sphingolipid FRET probes in comparison to different alternative probes. Finally, we give an outlook on future probes and their potential application. [ABSTRACT FROM AUTHOR]

Graphical abstract Highlights • Naphthalene dianhydride based novel probe R1 useful for the detection of Fe+3. • The performance of probe R1 over the other di/trivalent ions. • The PET mechanism on fluorescent turn-on cell permeable probe. • The efficacy of R1 towards live-cells. Abstract A green emissive PET operating fluorescent turn-on cell permeable novel probe R1 has been successfully developed and utilized for the detection of Fe+3 in the pure aqueous system at sub-nanomolar level. Moreover, probe R1 demonstrate highly sensitive and selective towards Fe+3 over the other divalent and trivalent metal ions and was established by using fluorescence spectroscopy. The efficiency and aid of R1 was demonstrated by the fluorescence imaging of captured Fe+3 within Pollen grains by using fluorescence microscopy. These results indicate that, this is the first fluorescent turn-on PET probe to detect sub-nanomolar Fe+3 in the pure aqueous system and in cellular level. [ABSTRACT FROM AUTHOR]