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The famous "light-switch" ruthenium complex [Ru(bpy)2(dppz)](PF6)2 (1) has been long known for its DNA binding properties in vitro. However, the biological utility of this compound has been hampered by its poor cellular uptake in living cells. Here we report a bioimaging application of 1 as cell viability probe in both 2D cells monolayer and 3D multi-cellular tumor spheroids of various human cancer cell lines (U87, HepG2, A549). When compared to propidium iodide, a routinely used cell viability probe, 1 was found to enhance the staining of dead cells in particular in tumor spheroids. 1 has high photostability, longer Stokes shift, and displays lower cytotoxicity compared to propidium iodide, which is a known carcinogenic. Finally, 1 was also found to displace the classical DNA binding dye Hoechst in dead cells, which makes it a promising dye for time-dependent imaging of dead cells in cell cultures, including multi-cellular tumor spheroids. [ABSTRACT FROM AUTHOR]

To fill the need for environmentally sensitive fluorescent unnatural amino acids able to operate in the red region of the spectrum, we have designed and synthesized Alared, a red solvatochromic and fluorogenic amino acid derived from the Nile Red chromophore. The new unnatural amino acid can be easily integrated into bioactive peptides using classical solid‐phase peptide synthesis. The fluorescence quantum yield and the emission maximum of Alared‐labeled peptides vary in a broad range depending on the peptide's environment, making Alared a powerful reporter of biomolecular interactions. Due to its red‐shifted absorption and emission spectra, Alared‐labeled peptides could be followed in living cells with minimal interference from cellular autofluorescence. Using ratiometric fluorescence microscopy, we were able to track the fate of the Alared‐labeled peptide agonists of the apelin G protein‐coupled receptor upon receptor activation and internalization. Due to its color‐shifting environmentally sensitive emission, Alared allowed for distinguishing the fractions of peptides that are specifically bound to the receptor or unspecifically bound to different cellular membranes. [ABSTRACT FROM AUTHOR]

We present a facile strategy to achieve color-tunability room-temperature phosphorescence (RTP) nanoprobes by doping mineral acids (i.e., boric acid and phosphoric acid) in an organic silicon scaffold through a cross-linking process. Such RTP nanoprobes exhibit inherent tunable phosphorescence (from 420–650 nm) with long lifetime (emission lasting for ∼5–15 s, RTP lifetime: ∼0.53–2.11 s) and high quantum yields (∼13.1–43.0%). Therefore, the as-prepared nanoprobes enable multiple imaging in live cells with a high signal-to-background ratio value of ∼52. [ABSTRACT FROM AUTHOR]

In filamentous fungi, microtubules are important for polar growth and morphological maintenance and serve as rails for intracellular trafficking. The molecular mechanisms associated with microtubules have been analyzed. However, little is known about when and where tubulin, a component of microtubules, is biosynthesized in multinuclear and multicellular filamentous fungi. In this study, we visualized microtubules based on the enhanced green fluorescence protein (EGFP)-labeled α-tubulin and β-tubulin mRNA tagged by the EGFP-mediated MS2 system in living yellow Koji mold Aspergillus oryzae cells in order to understand the spatiotemporal production mechanism of tubulin. We found that mRNA of btuA, encoding for β-tubulin, localized at dot-like structures through the apical, middle and basal regions of the hyphal cells. In addition, some btuA mRNA dots showed microtubule-dependent motor protein-like dynamics in the cells. Furthermore, it was found that btuA mRNA dots were decreased in the cytoplasm just before mitosis but increased immediately after mitosis, followed by a gradual decrease. In summary, the localization and abundance of β-tubulin mRNA is spatiotemporally regulated in living A. oryzae hyphal cells. [ABSTRACT FROM AUTHOR]

Diabetic corneal neuropathy (DCN) is a common diabetic ocular complication with limited treatment options. In this study, we investigated the effects of topical and oral fenofibrate, a peroxisome proliferator-activated receptor-α agonist, on the amelioration of DCN using diabetic mice (n = 120). Ocular surface assessments, corneal nerve and cell imaging analysis, tear proteomics and its associated biological pathways, immuno-histochemistry and western blot on PPARα expression, were studied before and 12 weeks after treatment. At 12 weeks, PPARα expression markedly restored after topical and oral fenofibrate. Topical fenofibrate significantly improved corneal nerve fibre density (CNFD) and tortuosity coefficient. Likewise, oral fenofibrate significantly improved CNFD. Both topical and oral forms significantly improved corneal sensitivity. Additionally, topical and oral fenofibrate significantly alleviated diabetic keratopathy, with fenofibrate eye drops demonstrating earlier therapeutic effects. Both topical and oral fenofibrate significantly increased corneal β-III tubulin expression. Topical fenofibrate reduced neuroinflammation by significantly increasing the levels of nerve growth factor and substance P. It also significantly increased β-III-tubulin and reduced CDC42 mRNA expression in trigeminal ganglions. Proteomic analysis showed that neurotrophin signalling and anti-inflammation reactions were significantly up-regulated after fenofibrate treatment, whether applied topically or orally. This study concluded that both topical and oral fenofibrate ameliorate DCN, while topical fenofibrate significantly reduces neuroinflammation. [ABSTRACT FROM AUTHOR]

There is a close relation between membrane receptor dynamics and their behavior. Several microscopy techniques have been developed to study protein dynamics in live cells such as the Fluorescence Recovery After Photobleaching (FRAP) or the Single Particle Tracking (SPT). These methodologies require expensive instruments, are time consuming, allow the analysis of small portion of the cell or an extremely small number of receptors at a time. Here we propose a time-saving approach that allows to visualize the entire receptor pool and its localization in time. This protocol requires an epifluorescence microscope equipped for structured illuminated sectioning and for live cell imaging. It can be applied to characterize membrane receptor and multi protein complex and their response to activators or inhibitors. Image acquisition and analysis can be performed in two days, while cells and substratum preparation require a few minutes a day for three days. [ABSTRACT FROM AUTHOR]

Genetically encoded Ca2+ indicators (GECIs) are versatile for live imaging of cellular activities. Besides the brightness and dynamic range of signal change of GECIs, Ca2+ affinity is another critical parameter for successful Ca2+ imaging, as the concentration range of Ca2+ dynamics differs from low nanomolar to sub-millimolar depending on the celltype and organism. However, ultrahigh-affinity GECIs, particularly the single fluorescent protein (1FP)-type, are lacking. Here, we report a simple strategy that increases Ca2+ affinity through the linker length optimization in topology mutants of existing 1FP-type GECIs. The resulting ultrahigh-affinity GECIs, CaMPARI-nano, BGECO-nano, and RCaMP-nano (Kd = 17–25 nM), enable unique biological applications, including the detection of low nanomolar Ca2+ dynamics, highlighting active signaling cells, and multi-functional imaging with other second messengers. The linker length optimization in topology mutants could be applied to other 1FP-type indicators of glutamate and potassium, rendering it a widely applicable technique for modulating indicator affinity. A strategy is developed to obtain ultrahigh-affinity, single fluorescent protein-type calcium indicators (GECIs), based on the optimization of linkers in topology mutants of previously-characterized GECIs. [ABSTRACT FROM AUTHOR]

Polymorphism‐dependent cytotoxicity and cellular uptake of drug molecules have been studied for the past two decades. However, the visualization of polymorph‐dependent cellular uptake and cytotoxicity using microscopy imaging techniques has not yet been reported. The luminescent polymorph is an ideal candidate to validate the above hypothesis. Herein, we report the polymorph‐dependent cellular uptake, cytotoxicity, and bio‐imaging functions of polymorphs 1Y and 1R of a naphthalimide‐phenothiazine dyad. These polymorphs show different luminescence colors in the solid state and exhibit aggregation‐induced enhanced emission (AIEE) in the DMSO‐Water mixture. Bioimaging, cytotoxicity assay, and fluorescence‐activated cell sorting (FACS) studies revealed that these polymorphs show different levels of cytotoxicity, cellular uptake, localization, and imaging potential. Detailed photophysical, morphological, and biological studies revealed that the difference in molecular conformation in these polymorphs enables them to form aggregates of different sizes and morphology, which leads to the differential uptake of these into the cells and consequently shows different cytotoxicity and imaging potentials. [ABSTRACT FROM AUTHOR]

Shear wave elasticity imaging (SWEI) has long been used to quantify tissue stiffness in clinical diagnoses. In comparison with conventional bulk-based measurement methods, SWEI offers the distinct advantage of nondestructive sampling, thereby enabling the spatiotemporal monitoring of stiffness variations. However, applying SWEI to assessing millimeter-scale three-dimensional (3D) cell environments has faced limitations despite its potential in mechanobiology, and the existing techniques are insufficient for imaging inhomogeneous media environments. In this study, we investigated a computed tomography technique specifically designed for reflected SWEI (called R-SWCT) by rotational scanning in a sample using a 20-MHz ultrasound single-element transducer. We focused on samples containing a single inclusion with higher stiffness than the surrounding medium, mimicking the situation of a tumor sphere embedded in a 3D gel. Our method reconstructs the stiffness distribution using a curve-fitting method, wherein coefficients of Gaussian curves are fitted to the wavefronts of simulated signals. This reconstruction method yielded coefficients that closely approximated the wavefronts obtained experimentally, with a maximum difference between the measured and predicted shear wave speeds of only 0.1 m/s for phantom samples and 0.2 m/s for cell samples. The system and methodologies proposed in this research have demonstrated the feasibility of employing R-SWCT to study the remodeling dynamics of a cell group within its surrounding matrix in an in vitro setting. This noninvasive method also facilitates an exploration of how irradiation dosage used in radiation therapy can induce temporal alterations in the shear wave speed in 3D cancer cell cultures. [ABSTRACT FROM AUTHOR]

A sensitive and biocompatible N-rich probe for rapid visual uranium detection was constructed by grafting two trianiline groups to 2,6-bis(aminomethyl)pyridine. Possessing excellent aggregation-induced emission (AIE) property and the advantages to form multidentate chelate with U selectively, the probe has been applied successfully to visualize uranium in complex environmental water samples and living cells, demonstrating outstanding anti-interference ability against large equivalent of different ions over a wide effective pH range. A large linear range (1.0 × 10−7–9.0 × 10−7 mol/L) and low detection limit (72.6 nmol/L, 17.28 ppb) were achieved for the visual determination of uranium. The recognition mechanism, photophysical properties, analytical performance and cytotoxicity were systematically investigated, demonstrating high potential for fast risk assessment of uranium pollution in field and in vivo. [ABSTRACT FROM AUTHOR]

Discovering new modifications for oligonucleotide therapeutics is essential for expanding its application to new targets and diseases. In this project, we focus on conjugating metaled ligands to short interfering RNAs (siRNAs) to investigate robust and simple conjugation methods for adding new properties such as real‐time imaging to the siRNA. Here we report the chemical synthesis of novel Ga‐(III)‐corroles for their direct conjugation to siRNAs. Ga‐(III)‐corrole‐siRNAs showed promising results when evaluated for gene silencing and live cell imaging. The knockdown activity of the firefly luciferase reporter gene was measured to evaluate gene silencing activity. Gene silencing studies from two 5'‐Ga‐(III)‐labeled‐siRNAs exhibited dose‐dependent knockdown with IC50s of 812.7 and 451.4 pM, which is comparable to wild‐type (IC50=439.7 pM) in the absence of red light, and IC50s of 562.9 and 354.5 pM, which is also comparable to wild‐type (IC50=337.4 pM), in the presence of red light. In addition, imaging studies with Ga‐(III)‐corrole‐modified siRNAs showed intense fluorescence in HeLa cells, highlighting that the Ga‐(III)‐corrole modification is an effective fluorophore for siRNA tracing and imaging. Moreover, the photodynamic activity of free base corrole vs the Ga‐(III)‐corrole was evaluated. Results show an increase of light cytotoxicity of the corrole ligand upon the addition of Ga‐(III); however, no phototoxicity was observed when Ga‐(III) ligands were linked to siRNA. In conclusion, Ga‐(III)‐corrole‐siRNAs show promising results for applications in simultaneous real‐time imaging and gene silencing. [ABSTRACT FROM AUTHOR]

Presynaptic Ca2+ influx through voltage-gated Ca2+ channels (VGCCs) is a key signal for synaptic vesicle release. Synaptic neurexins can partially determine the strength of transmission by regulating VGCCs. However, it is unknown whether neurexins modulate Ca2+ influx via all VGCC subtypes similarly. Here, we performed live cell imaging of synaptic boutons from primary hippocampal neurons with a Ca2+ indicator. We used the expression of inactive and active Cre recombinase to compare control to conditional knockout neurons lacking either all or selected neurexin variants. We found that reduced total presynaptic Ca2+ transients caused by the deletion of all neurexins were primarily due to the reduced contribution of P/Q-type VGCCs. The deletion of neurexin1α alone also reduced the total presynaptic Ca2+ influx but increased Ca2+ influx via N-type VGCCs. Moreover, we tested whether the decrease in Ca2+ influx induced by activation of cannabinoid receptor 1 (CB1-receptor) is modulated by neurexins. Unlike earlier observations emphasizing a role for β-neurexins, we found that the decrease in presynaptic Ca2+ transients induced by CB1-receptor activation depended more strongly on the presence of α-neurexins in hippocampal neurons. Together, our results suggest that neurexins have unique roles in the modulation of presynaptic Ca2+ influx through VGCC subtypes and that different neurexin variants may affect specific VGCCs. [ABSTRACT FROM AUTHOR]

Akt is an important kinase in metabolism. Akt also phosphorylates and activates endothelial and neuronal nitric oxide (NO) synthases (eNOS and nNOS, respectively) expressed in M0 (unpolarized) macrophages. We showed that e/nNOS NO production downstream of bitter taste receptors enhances macrophage phagocytosis. In airway epithelial cells, we also showed that the activation of Akt by a small molecule (SC79) enhances NO production and increases levels of nuclear Nrf2, which reduces IL-8 transcription during concomitant stimulation with Toll-like receptor (TLR) 5 agonist flagellin. We hypothesized that SC79's production of NO in macrophages might likewise enhance phagocytosis and reduce the transcription of some pro-inflammatory cytokines. Using live cell imaging of fluorescent biosensors and indicator dyes, we found that SC79 induces Akt activation, NO production, and downstream cGMP production in primary human M0 macrophages. This was accompanied by a reduction in IL-6, IL-8, and IL-12 production during concomitant stimulation with bacterial lipopolysaccharide, an agonist of pattern recognition receptors including TLR4. Pharmacological inhibitors suggested that this effect was dependent on Akt and Nrf2. Together, these data suggest that several macrophage immune pathways are regulated by SC79 via Akt. A small-molecule Akt activator may be useful in some infection settings, warranting future in vivo studies. [ABSTRACT FROM AUTHOR]

Organic and polymer fluorescent nanomaterials are a frontier research focus. Here in this work, a series of fluorinated zwitterionic random copolymers end‐attached with a quasi‐chromophoric group of pyrene or tetraphenylethylene (TPE) are well synthesized via atom transfer radical polymerization with activators regenerated by electron transfer (ARGET ATRP). Those random copolymers with total degree of polymerization 100 or 200 are able to produce fluorescent single‐chain nanoparticles (SCNPs) through intra‐chain self‐folding assembly with quite uniform diameters in the range of 10–20 nm as characterized by dynamic light scattering and transmission electron microscopy. By virtue of the segregation or confinement effect, both SCNPs functionalized with pyrene or TPE group are capable of emitting fluorescence, with pyrene tethered SCNPs exhibiting stronger fluorescence emission reaching the highest quantum yield ≈20%. Moreover, such kind of fluorescent SCNPs manifest low cytotoxicity and good cell imaging performance for Hela cells. The creation of fluorescent SCNPs through covalently attached one quasi‐chromophore to the end of one fluorinated zwitterionic random copolymer provides an alternative strategy for preparing polymeric luminescence nanomaterials, promisingly serving as a new type of fluorescent nanoprobes for biological imaging applications. [ABSTRACT FROM AUTHOR]

Carbon dots (CDs) are a new type of nanomaterial with great potential in bioimaging, biosensing, and drug delivery. In this study, novel CDs were prepared from cumin seeds (Cuminum cyminum L) by pyrolysis. The pyrolysis method and several extraction steps were optimized to fabricate CDs from cumin seed. The size, structure, chemical composition, and photoluminescence properties of the CDs were characterized using size analyzer, zeta sizer, TEM, SEM, EDX, UV-Vis, fluorescence spectroscopy, and FTIR. The influence of the CDs on cell viability was investigated in A549, 3T3, and MCF-7 cell lines using MTT assay. The cancer cell imaging potential of the CDs was assessed using fluorescence microscopy. The results showed that the CDs with a mean diameter of 13.4 ± 3.7 nm and zeta potential of -23.3 ± 7.5 mv can be obtained by the pyrolysis of cumin seed powder. The obtained CDs exhibited strong and unique fluorescence properties when they were excited at different wavelengths, particularly 350 nm. The CDs were highly stable over time, and their fluorescence properties remained unchanged after three months. Apart from the physicochemical properties of the CDs, cell viability assays showed that these CDs were non-toxic to A549, MCF-7, and 3T3 cells, over 90% of the cells were viable after 24 h, even at the highest concentration (1000 µg/ml). The CDs successfully penetrated the MCF-7 cells as revealed by fluorescent images. Taken together, the fluorescent CDs derived from cumin seeds through the presented facile technique indicated excellent and stable fluorescence properties. In addition, these CDs were highly biocompatible in vitro and could be a potential option for theragnostic applications. [ABSTRACT FROM AUTHOR]

SUMMARY: Capturing images of the nuclear dynamics within live cells is an essential technique for comprehending the intricate biological processes inherent to plant cell nuclei. While various methods exist for imaging nuclei, including combining fluorescent proteins and dyes with microscopy, there is a dearth of commercially available dyes for live‐cell imaging. In Arabidopsis thaliana, we discovered that nuclei emit autofluorescence in the near‐infrared (NIR) range of the spectrum and devised a non‐invasive technique for the visualization of live cell nuclei using this inherent NIR autofluorescence. Our studies demonstrated the capability of the NIR imaging technique to visualize the dynamic behavior of nuclei within primary roots, root hairs, and pollen tubes, which are tissues that harbor a limited number of other organelles displaying autofluorescence. We further demonstrated the applicability of NIR autofluorescence imaging in various other tissues by incorporating fluorescence lifetime imaging techniques. Nuclear autofluorescence was also detected across a wide range of plant species, enabling analyses without the need for transformation. The nuclear autofluorescence in the NIR wavelength range was not observed in animal or yeast cells. Genetic analysis revealed that this autofluorescence was caused by the phytochrome protein. Our studies demonstrated that nuclear autofluorescence imaging can be effectively employed not only in model plants but also for studying nuclei in non‐model plant species. [ABSTRACT FROM AUTHOR]

SUMMARY: Plant cell walls are essential for defining plant growth and development, providing structural support to the main body and responding to abiotic and biotic cues. Cellulose, the main structural polymer of plant cell walls, is synthesized at the plasma membrane by cellulose synthase complexes (CSCs). The construction and transport of CSCs to and from the plasma membrane is poorly understood but is known to rely on the coordinated activity of cellulose synthase‐interactive protein 1 (CSI1), a key regulator of CSC trafficking. In this study, we found that Trs85, a TRAPPIII complex subunit, interacted with CSI1 in vitro. Using functional genetics and live‐cell imaging, we have shown that trs85‐1 mutants have reduced cellulose content, stimulated CSC delivery, an increased population of static CSCs and deficient clathrin‐mediated endocytosis in the primary cell wall. Overall, our findings suggest that Trs85 has a dual role in the trafficking of CSCs, by negatively regulating the exocytosis and clathrin‐mediated endocytosis of CSCs. Significance Statement: The synthesis of cellulose requires the transportation of cellulose synthase complexes (CSCs) to and from the plasma membrane. Our study has identified that Trs85, a TRAPPIII subunit, is important in both the delivery and endocytosis of CSCs at the plasma membrane in Arabidopsis. [ABSTRACT FROM AUTHOR]

SUMMARY: Confocal microscopy has greatly aided our understanding of the major cellular processes and trafficking pathways responsible for plant growth and development. However, a drawback of these studies is that they often rely on the manual analysis of a vast number of images, which is time‐consuming, error‐prone, and subject to bias. To overcome these limitations, we developed Dot Scanner, a Python program for analyzing the densities, lifetimes, and displacements of fluorescently tagged particles in an unbiased, automated, and efficient manner. Dot Scanner was validated by performing side‐by‐side analysis in Fiji‐ImageJ of particles involved in cellulose biosynthesis. We found that the particle densities and lifetimes were comparable in both Dot Scanner and Fiji‐ImageJ, verifying the accuracy of Dot Scanner. Dot Scanner largely outperforms Fiji‐ImageJ, since it suffers far less selection bias when calculating particle lifetimes and is much more efficient at distinguishing between weak signals and background signal caused by bleaching. Not only does Dot Scanner obtain much more robust results, but it is a highly efficient program, since it automates much of the analyses, shortening workflow durations from weeks to minutes. This free and accessible program will be a highly advantageous tool for analyzing live‐cell imaging in plants. Significance Statement: Live‐cell imaging analyses of fluorescently tagged particles in biological systems often require significant manual input that is time‐consuming and subject to bias. We have developed a free Python program, Dot Scanner, to allow users without any programming experience to analyze large datasets rapidly, efficiently, and with reduced bias. [ABSTRACT FROM AUTHOR]

Non-aromatic fluorescent and multi-responsive materials, exhibiting inherent fluorescence emission and controlled phase change, have garnered significant attention in recent years. However, the underlying interaction between their fluorescent properties and phase transition remains unclear. In this study, we synthesized a series of catalyst-free aza-Michael addition-based polyethyleneimine (RFPEI) materials by reacting polyethyleneimine (PEI) with N-isopropyl acrylamide (NIPAM). The resulting RFPEI was comprehensively characterized, and demonstrated dual-phase transition behavior (LCST and UCST) in water, which could be finely tuned by adjusting its composition or external factors such as pH. Notably, upon UV irradiation (365 nm), RFPEI exhibited strong fluorescence emission. We further investigated the effects of NIPAM grafting percentage to PEI, polymer concentration, and pH on the LCST/UCST and fluorescent properties of RFPEI aqueous solutions. Moreover, we showcased the great potential of RFPEI as a versatile tool for physiological cell imaging, trace detection, and controlled release of doxorubicin. Our study presents a novel class of stimuli-responsive fluorescent materials with promising applications in the field of biomedicine. [ABSTRACT FROM AUTHOR]

Ferric ion (Fe3+) serves as an essential and indispensable inorganic element in various biological process. Excess Fe3+ is harmful for biological systems and human health. Herein, highly sensitive carbon quantum dots (Fe-CQDs) for ferric ions were synthesized by a high-efficiency, one-step hydrothermal route utilizing L-arginine and citric acid. The prepared Fe-CQDs exhibited good water solubility, high photostability, and bright blue florescence with relatively high quantum yields (QYs) over 21.33% and a Stokes shift exceeding 100 nm. The functionalized Fe-CQDs offer rapid, sensitive, and selective recognition of Fe3+ with a linear relationship from 0.6 to 80 μM and a low detection limit of 0.2 μM. Moreover, the synthesized Fe-CQDs illustrated strong anti-interference properties in samples and buffer solutions. The developed sensor demonstrated high biocompatibility and was successfully applied for intracellular Fe3+ determination in SHSY5Y and HT22 cells, illustrating excellent imaging application. The present work provides a promising platform for the early clinical diagnosis of Fe3+-associated diseases. [ABSTRACT FROM AUTHOR]

In recent years, near‐infrared quantum dots (NIR QDs) have emerged as a promising candidate for biological imaging owing to their strong fluorescence penetrating into biological tissues and high imaging signal‐to‐noise ratio. Among various materials, InP QDs are environmentally friendly and have a relatively narrow bandgap of 1.35 eV, which provides a possibility for their emission wavelength to extend to the near‐infrared region. However, the strong reactivity of the precursor of phosphorus makes it challenging to synthesize NIR InP QDs, as it leads to rapid nucleation of the InP core. Herein, a method of epitaxial growth is reported to synthesize NIR InP QDs. Through high‐temperature nucleation and low‐temperature epitaxial growth, NIR InP QDs larger than 15 nm in size and with an emission wavelength of 807 nm are successfully synthesized. Furthermore, by removing InPOx defects on the surface of the core through HF etching, the quantum yield (QY) is increased from 6% to 12%. Ligand exchange successfully converted oil‐soluble ligands into water‐soluble ones, leading to excellent performance in cell imaging. The study provides a promising approach to the synthesis of desirable NIR InP QDs for use in biomedical imaging applications. [ABSTRACT FROM AUTHOR]

PEGylated 2-(2-(6-(dimethylamino)benzooxazol-2-yl)vinyl)quinolin-8-ol (BQ-mPEG) was synthesized, which exhibited strong fluorescence at 610 nm (φf = 0.11) and a large Stoke's shift (Δλ = 202 nm) in the aqueous solution. The fluorophore displayed a two-photon absorption characteristic and a strong red emission (λem = 625 nm) was observed with NIR (950 nm) irradiation. The feasibility of using BQ-mPEG as a fluorescent imaging reagent suggested that the fluorophore targets mitochondria, and with 950 nm excitation, bright fluorescence images in HeLa live cells were observed. Furthermore, the fluorophore exhibited some advantages of high photostability and pH stability, as well as low cytotoxicity. [ABSTRACT FROM AUTHOR]

Most animal cell types are classified as non‐excitable because they do not generate action potentials observed in excitable cells, such as neurons and muscle cells. Thus, resolving voltage signals in non‐excitable cells demands sensors with exceptionally high voltage sensitivity. In this study, the ultrabright, ultrasensitive, and calibratable genetically encoded voltage sensor rEstus is developed using structure‐guided engineering. rEstus is most sensitive in the resting voltage range of non‐excitable cells and offers a 3.6‐fold improvement in brightness change for fast voltage spikes over its precursor ASAP3. Using rEstus, it is uncovered that the membrane voltage in several non‐excitable cell lines (A375, HEK293T, MCF7) undergoes spontaneous endogenous alterations on a second to millisecond timescale. Correlation analysis of these optically recorded voltage alterations provides a direct, real‐time readout of electrical cell–cell coupling, showing that visually connected A375 and HEK293T cells are also largely electrically connected, while MCF7 cells are only weakly coupled. The presented work provides enhanced tools and methods for non‐invasive voltage imaging in living cells and demonstrates that spontaneous endogenous membrane voltage alterations are not limited to excitable cells but also occur in a variety of non‐excitable cell types. [ABSTRACT FROM AUTHOR]

Controlling 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. [ABSTRACT FROM AUTHOR]

Metal–organic self-assembly with flexible moieties is a budding field of research due to the possibility of the formation of unique architectures. Tetrazole, characterised by four nitrogen atoms in a five-member ring, exhibits immense potential as a component. Tetrazole offers four coordination sites for binding to the metal centre with nine distinct binding modes, leading to various assemblies. This review highlights different polymeric and discrete tetrazole-based assemblies and their functions. The meticulous manipulation of stoichiometry, ligands, and metal ions required for constructing discrete assemblies has also been discussed. The different applications of these architectures in separation, catalysis and detection have also been accentuated. The latter section of the review consolidates tetrazole-based cage composites, highlighting their applications in cell imaging and photocatalytic applications. [ABSTRACT FROM AUTHOR]

Synthesis and photophysical properties of a fluorescent probe HBD is described. Probe upon interaction with metal ions, anions and nucleoside pyrophosphates (NPPs) showed fluorescence quenching with Cu2+ due to chelation enhanced quenching effect (CHEQ). Moreover, interaction of ensemble HBD.Cu2+ with anions and NPPs showed fluorescence “turn‐On” response with ATP selectively. “On‐Off‐On” responses observed with Cu2+ and ATP is attributed to an interplay between ESIPT and TICT processes. Cyclic voltammogram of probe exhibited quasi‐reversible redox behaviour with three oxidation and two reduction potentials and the change in band gaps of probe suggested the interaction with Cu2+ and ATP. The 2 : 1 and 1 : 1 binding stoichiometry for an interaction between probe and Cu2+ (LOD, 62 nM) and ensemble, HBD.Cu2+ with ATP (LOD, 0.4 μM) respectively are realised by Job's plot and HRMS data. Cell imaging studies carried out to detect Cu2+ and ATP in HeLa cells. Also, the output emission observed with Cu2+ and ATP is utilized to construct an implication (IMP) logic gate. Test paper strips showed naked‐eye visible color responses to detect Cu2+ and ATP. In real water samples probe successfully detected copper (0.03 μM) between 5–6.5 ppb level (ICP‐MS method). [ABSTRACT FROM AUTHOR]

Cell states were traditionally defined by how they looked, where they were located, and what functions they performed. In this post-genomic era, the field is largely focused on a molecular view of cell state. Moving forward, we anticipate that the observables used to define cell states will evolve again as single-cell imaging and analytics are advancing at a breakneck pace via the collection of large-scale, systematic cell image datasets and the application of quantitative image-based data science methods. This is, therefore, a key moment in the arc of cell biological research to develop approaches that integrate the spatiotemporal observables of the physical structure and organization of the cell with molecular observables toward the concept of a holistic cell state. In this perspective, we propose a conceptual framework for holistic cell states and state transitions that is data-driven, practical, and useful to enable integrative analyses and modeling across many data types. Technological advances have brought "big data" to the cellular level. Rafelski and Theriot share their vision of defining a cell's state in a holistic way that reflects all molecular, organizational, functional, and environmental observables and spatiotemporal dynamics. The proposed view aims to provide a conceptual framework for quantitative modeling. [ABSTRACT FROM AUTHOR]

Sensitive detection of microRNA (miRNA), one of the most promising biomarkers, plays crucial roles in cancer diagnosis. However, the low expression level of miRNA makes it extremely urgent to develop ultrasensitive and highly selective strategies for quantification of miRNA. Herein, a DNA machine is rationally constructed for amplified detection and imaging of low-abundance miRNA in living cells based on the toehold-mediated strand displacement reaction (TMSDR). The isothermal and enzyme-free DNA machine with low background leakage is fabricated by integrating two DNA circuits into a cascade system, in which the output of one circuit serves as the input of the other one. Once the DNA machine is transfected into breast cancer cells, the overexpressed miRNA-203 initiates the first-layer circuit through TMSDR, leading to the concentration variation of fuel strands, which further influences the assembly of hairpin DNA in the second-layer circuit and the occurrence of fluorescence resonance energy transfer (FRET) for fluorescence imaging. Benefiting from the cascade of the two-layer amplification reaction, the proposed DNA machine acquires a detection limit down to 4 fM for quantification of miR-203 and a 10 000-fold improvement in amplification efficiency over the single circuit. Therefore, the two-layer circuit cascade-based DNA machine provides an effective platform for amplified analysis of low-abundance miRNA with high sensitivity, which holds great promise in biomedical and clinical research. [ABSTRACT FROM AUTHOR]

Optical imaging through dynamic and complex scattering media has attracted various applications, e.g., ranging from scene imaging to cell imaging. Nevertheless, imaging through complex media is full of challenges attributed to the inhomogeneous scattering, leading to nonlinear effects. Although ghost imaging (GI) has proven effective in solving some scattering problems, dynamic and complex scattering still requires an efficient solution. In this Letter, we report a model based on adaptive moving average (AMA) to correct the influence of dynamic scattering media from a statistical perspective for high-resolution GI. The developed AMA correction method selects an appropriate time window based on the changing trend of measured single-pixel light intensities to accurately correct a series of dynamic scaling factors. Then, the corrected single-pixel light intensities are used for ghost reconstruction using a second-order correlation algorithm. A series of optical experiments are conducted to verify superiority of the proposed method. Moreover, the proposed method can be applied with other algorithms to enhance the quality of the reconstructed ghost images. By leveraging a statistical model based on the measured data, the proposed scheme offers an enhanced solution to solving dynamic and complex scattering problems in GI. [ABSTRACT FROM AUTHOR]

The therapeutic targeting of ferroptosis requires full understanding of the molecular mechanism of this regulated cell death pathway. While lipid-derived electrophiles (LDEs), including 4-hydroxy-2-nonenal (4-HNE), are important biomarkers of ferroptosis, a functional role for these highly reactive species in ferroptotic cell death execution has not been established. Here, through mechanistic characterization of LDE-detoxification impairment, we demonstrate that LDEs mediate altered protein function during ferroptosis. Applying live cell fluorescence imaging, we first identified that export of glutathione-LDE-adducts through multidrug resistance-associated protein (MRP) channels is inhibited following exposure to a panel of ferroptosis inducers (FINs) with different modes of action (type I-IV FINs erastin, RSL3, FIN56, and FINO2). This channel inhibition was recreated by both initiation of lipid peroxidation and treatment with 4-HNE. Importantly, treatment with radical-trapping antioxidants prevented impaired LDE-adduct export when working with both FINs and lipid peroxidation initiators but not 4-HNE, pinpointing LDEs as the cause of this inhibited MRP activity observed during ferroptosis. Our findings, when combined with reports of widespread LDE alkylation of key proteins following ferroptosis induction, including MRP1, set a precedent for LDEs as critical mediators of ferroptotic cell damage. Lipid hydroperoxide breakdown to form truncated phospholipids and LDEs may fully explain membrane permeabilization and modified protein function downstream of lipid peroxidation, offering a unified explanation of the molecular cell death mechanism of ferroptosis. [ABSTRACT FROM AUTHOR]

AbstractBackgroundPurposeMethodsResultsIndocyanine Green (ICG) as an agent for photodynamic therapy (PDT) of melanoma cancer has low quantum yield, short circulation half-life, poor photo-stability, and tendency to aggregation.N-doped carbon quantum dot (CQD) nanoparticle was applied to encapsulate ICG and overcome ICG obstacle in PDT with simultaneous cell imaging property.CQD was prepared using hydrothermal method. Cell culture study and In vivo assessments on C57BL/6 mice containing melanoma cancer cells was performed.Results showed that CQD size slightly enhanced from 24.55 nm to 42.67 nm after ICG loading. Detection of reactive oxygen species (ROS) demonstrated that CQD improved ICG photo-stability and ROS generation capacity upon laser irradiation. Cell culture study illustrated that ICG@CQD could decrease survival rate of melanoma cancer cells of B16F10 cell line from 48% for pure ICG to 28% for ICG@CQD. Confocal microscopy images approved more cellular uptake and more qualified cell imaging ability of ICG@CQD. In vivo assessments displayed obvious inhibitory effect of tumor growth for ICG@CQD in comparison to free ICG on the C57BL/6 mice. In vivo fluorescence images confirmed that ICG@CQD accumulates remarkably more than free ICG in tumor region. Finally, ICG@CQD was proposed as an innovative nanocarrier for PDT and diagnosis. [ABSTRACT FROM AUTHOR]

Unraveling the cellular and molecular mechanisms underlying tumoral processes is fundamental for the diagnosis and treatment of cancer. In this regard, three-dimensional (3D) cancer cell models more realistically mimic tumors compared to conventional 2D cell cultures and are more attractive for performing such studies. Nonetheless, the analysis of such architectures is challenging because most available techniques are destructive, resulting in the loss of biochemical information. On the contrary, surface-enhanced Raman spectroscopy (SERS) is a non-invasive analytical tool that can record the structural fingerprint of molecules present in complex biological environments. The implementation of SERS in 3D cancer models can be leveraged to track therapeutics, the production of cancer-related metabolites, different signaling and communication pathways, and to image the different cellular components and structural features. In this review, we highlight recent progress in the use of SERS for the evaluation of cancer diagnosis and therapy in 3D tumoral models. We outline strategies for the delivery and design of SERS tags and shed light on the possibilities this technique offers for studying different cellular processes, through either biosensing or bioimaging modalities. Finally, we address current challenges and future directions, such as overcoming the limitations of SERS and the need for the development of user-friendly and robust data analysis methods. Continued development of SERS 3D bioimaging and biosensing systems, techniques, and analytical strategies, can provide significant contributions for early disease detection, novel cancer therapies, and the realization of patient-tailored medicine. [ABSTRACT FROM AUTHOR]

An AIE-based fluorescent probe was designed to evaluate peroxynitrite levels in complex biological samples. The newly synthesized hydrazone-conjugated probe fluoresces strongly in the presence of peroxynitrite. Clinically, the peroxynitrite levels can be measured in human serum and cellular mitochondria with an LOD of 6.5 nM by fluorescence imaging in vitro. [ABSTRACT FROM AUTHOR]

Many clinical procedures and biomedical research workflows rely on microscopy, including diagnosis of cancer, genetic disorders, autoimmune diseases, infections, and quantification of cell culture. Despite its widespread use, traditional image acquisition and review by trained microscopists is often lengthy and expensive, limited to large hospitals or laboratories, precluding use in point‐of‐care settings. In contrast, lensless or lensfree holographic microscopy (LHM) is inexpensive and widely deployable because it can achieve performance comparable to expensive and bulky objective‐based benchtop microscopes while relying on components that cost only a few hundred dollars or less. Lab‐on‐a‐chip integration is practical and enables LHM to be combined with single‐cell isolation, sample mixing, and in‐incubator imaging. Additionally, many manual tasks in conventional microscopy are instead computational in LHM, including image focusing, stitching, and classification. Furthermore, LHM offers a field of view hundreds of times greater than that of conventional microscopy without sacrificing resolution. Here, the basic LHM principles are summarized, as well as recent advances in artificial intelligence integration and enhanced resolution. How LHM is applied to the above clinical and biomedical applications is discussed in detail. Finally, emerging clinical applications, high‐impact areas for future research, and some current challenges facing widespread adoption are identified. [ABSTRACT FROM AUTHOR]

The process of image fusion is the process of enriching an image and improving the image's quality, so as to facilitate the subsequent image processing and analysis. With the increasing importance of image fusion technology, the fusion of infrared and visible images has received extensive attention. In today's deep learning environment, deep learning is widely used in the field of image fusion. However, in some applications, it is not possible to obtain a large amount of training data. Because some special organs of snakes can receive and process infrared information and visible information, the fusion method of infrared and visible light to simulate the visual mechanism of snakes came into being. Therefore, this paper takes into account the perspective of visual bionics to achieve image fusion; such methods do not need to obtain a significant amount of training data. However, most of the fusion methods for simulating snakes face the problem of unclear details, so this paper combines this method with a pulse coupled neural network (PCNN). By studying two receptive field models of retinal nerve cells, six dual-mode cell imaging mechanisms of rattlesnakes and their mathematical models and the PCNN model, an improved fusion method of infrared and visible images was proposed. For the proposed fusion method, eleven groups of source images were used, and three non-reference image quality evaluation indexes were compared with seven other fusion methods. The experimental results show that the improved algorithm proposed in this paper is better overall than the comparison method for the three evaluation indexes. [ABSTRACT FROM AUTHOR]

Rhenium(I) tricarbonyl complexes are widely studied for their cell imaging properties and anti-cancer and anti-microbial activities, but the complexes with S-donor ligands remain relatively unexplored. A series of six fac-[Re(NN)(CO)3(SR)] complexes, where (NN) is 2,2′-bipyridyl (bipy) or 1,10-phenanthroline (phen), and RSH is a series of thiocarboxylic acid methyl esters, have been synthesized and characterized. Cellular uptake and anti-proliferative activities of these complexes in human breast cancer cell lines (MDA-MB-231 and MCF-7) were generally lower than those of the previously described fac-[Re(NN)(CO)3(OH2)]+ complexes; however, one of the complexes, fac-[Re(CO)3(phen)(SC(Ph)CH2C(O)OMe)] (3b), was active (IC50 ∼ 10 μM at 72 h treatment) in thiol-depleted MDA-MB-231 cells. Moreover, unlike fac-[Re(CO)3(phen)(OH2)]+, this complex did not lose activity in the presence of extracellular glutathione. Taken together these properties show promise for further development of 3b and its analogues as potential anti-cancer drugs for co-treatment with thiol-depleting agents. Conversely, the stable and non-toxic complex, fac-[Re(bipy)(CO)3(SC(Me)C(O)OMe)] (1a), predominantly localized in the lysosomes of MDA-MB-231 cells, as shown by live cell confocal microscopy (λex = 405 nm, λem = 470–570 nm). It is strongly localized in a subset of lysosomes (25 μM Re, 4 h treatment), as shown by co-localization with a Lysotracker dye. Longer treatment times with 1a (25 μM Re for 48 h) resulted in partial migration of the probe into the mitochondria, as shown by co-localization with a Mitotracker dye. These properties make complex 1a an attractive target for further development as an organelle probe for multimodal imaging, including phosphorescence, carbonyl tag for vibrational spectroscopy, and Re tag for X-ray fluorescence microscopy. [ABSTRACT FROM AUTHOR]

Monitoring hydrogen sulfide (H2S) in living organisms is very important because H2S acts as a regulator in many physiological and pathological processes. Upregulation of endogenous H2S concentration has been shown to be closely related to the occurrence and development of tumors, atherosclerosis, neurodegenerative diseases and diabetes. Herin, a novel fluorescent probe HND with aggregation-induced emission was designed. Impressively, HND exhibited a high selectivity, fast response (1 min) and low detection limit (0.61 μM) for H2S in PBS buffer (10 mM, pH = 7.42). Moreover, the reaction mechanism between HND and H2S was conducted by Job's plot, HR-MS, and DFT. In particular, HND was successfully employed to detect H2S in HeLa cells. [ABSTRACT FROM AUTHOR]

Natural killer-like B (NKB) cells are unique innate immune cells expressing both natural killer (NK) and B cell receptors. As first responders to infection, they secrete IL-18 to induce a critical cascade of innate and adaptive immune cell infiltration and activation. However, limited research exists on the role of NKB cells in homeostasis and infection, largely due to incomplete and erroneous evaluations. To fill this knowledge gap, we investigated the expression of signaling and trafficking proteins, and the in situ localization and transcriptome of naïve NKB cells comparied to conventionally-defined NK and B cells, as well as modulations of these cells in SIV infection. Intracellular signaling proteins and trafficking markers were expressed differentially on naïve NKB cells, with high expression of CD62L and Syk, and low expression of CD69, α4β7, FcRg, Zap70, and CD3z, findings which were more similar to B cells than NK cells. CD20+NKG2a/c+ NKB cells were identified in spleen, mesenteric lymph nodes (MLN), colon, jejunum, and liver of naïve rhesus macaques (RM) via tissue imaging, with NKB cell counts concentrated in spleen and MLN. For the first time, single cell RNA sequencing (scRNAseq), including BCR sequencing, of sorted NKB cells confirmed that NKB cells are unique. Transcriptomic analysis of naïve splenic NKB cells by scRNAseq showed that NKB cells undergo somatic hypermutation and express Ig receptors, similar to B cells. While only 15% of sorted NKB cells showed transcript expression of both KLRC1 (NKG2A) and MS4A1 (CD20) genes, only 5% of cells expressed KLRC1, MS4A1, and IgH/IgL transcripts. We observed expanded NKB frequencies in RM gut and buccal mucosa as early as 14 and 35 days post-SIV infection, respectively. Further, mucosal and peripheral NKB cells were associated with colorectal cytokine mileu and oral microbiome changes, respectively. Our studies indicate that NKB cells gated on CD3-CD14-CD20+NKG2A/C+ cells were inclusive of transcriptomically conventional B and NK cells in addition to true NKB cells, confounding accurate phenotyping and frequency recordings that could only be resolved using genomic techniques. Although NKB cells were clearly elevated during SIV infection and associated with inflammatory changes during infection, further interrogation is necessary to acurately identify the true phenotype and significance of NKB cells in infection and inflammation. Author summary: Recently, our understanding of traditional innate immunity has become reshaped by the discovery of new innate immune cell subsets as well as newly identified functions/programming of previously described innate immune cell subsets. One such new and unique innate immune cell subset are the natural killer-like B (NKB) cells, which express both NK and B cell receptors. These cells have been described as first responder immune cells in infection and inflammation within mice, non-human primates (NHP), and humans. To clarify if the simple definition of CD3-NKG2A/C+CD20+ cells as NKB cells is sufficient to truly identify the unique NKB cell phenotype, we characterized these cells via imaging cytometry and single cell RNA sequencing for the first time in an NHP model. Our data suggests that the NKB cell phenotype and transcriptome, while unique and relevant in SIV infection, remains very infrequent. Therefore, using flow cytometry to identify NKB cells based on the commonly accepted marker expression could be misleading and cause the erroneous classification of conventional NK cells and/or B cells as NKB cells. [ABSTRACT FROM AUTHOR]

Sensitive detection of microRNA (miRNA), one of the most promising biomarkers, plays crucial roles in cancer diagnosis. However, the low expression level of miRNA makes it extremely urgent to develop ultrasensitive and highly selective strategies for quantification of miRNA. Herein, a DNA machine is rationally constructed for amplified detection and imaging of low-abundance miRNA in living cells based on the toehold-mediated strand displacement reaction (TMSDR). The isothermal and enzyme-free DNA machine with low background leakage is fabricated by integrating two DNA circuits into a cascade system, in which the output of one circuit serves as the input of the other one. Once the DNA machine is transfected into breast cancer cells, the overexpressed miRNA-203 initiates the first-layer circuit through TMSDR, leading to the concentration variation of fuel strands, which further influences the assembly of hairpin DNA in the second-layer circuit and the occurrence of fluorescence resonance energy transfer (FRET) for fluorescence imaging. Benefiting from the cascade of the two-layer amplification reaction, the proposed DNA machine acquires a detection limit down to 4 fM for quantification of miR-203 and a 10 000-fold improvement in amplification efficiency over the single circuit. Therefore, the two-layer circuit cascade-based DNA machine provides an effective platform for amplified analysis of low-abundance miRNA with high sensitivity, which holds great promise in biomedical and clinical research. [ABSTRACT FROM AUTHOR]

The deployment of small‐molecule fluorescent agents plays an ever‐growing role in medicine and drug development. Herein, we complement the portfolio of powerful fluorophores, reporting the serendipitous discovery and development of a novel class with an imidazo[1,2‐a]pyridinium triflate core, which we term PyrAtes. These fluorophores are synthesized in a single step from readily available materials (>60 examples) and display Stokes shifts as large as 240 nm, while also reaching NIR−I emissions at λmax as long as 720 nm. Computational studies allow the development of a platform for the prediction of λmax and λEm. Furthermore, we demonstrate the compatibility of these novel fluorophores with live cell imaging in HEK293 cells, suggesting PyrAtes as potent intracellular markers. [ABSTRACT FROM AUTHOR]

To avoid the unexpected aggregation and reduce the cytotoxicity of nanomaterials as optical probes in cell imaging applications, we propose a programmed DNA-cube as a carrier for silver nanoparticles (Ag NPs) to construct a specific hydrogen sulfide (H2S) responsive platform (Ag NP@DNA-cube) for diagnosing colorectal cancer (CRC) in this study. The DNA-cube maintains good dispersion of Ag NPs while providing excellent biocompatibility. Based on the characteristic overexpression of endogenous H2S in CRC cells, the Ag NPs are etched by H2S within target cells into silver sulfide quantum dots, thereby selectively illuminating the target cells. The Ag NP@DNA-cube exhibits a specific fluorescence response to CRC cells and achieves satisfactory imaging. [ABSTRACT FROM AUTHOR]

Anthracene carboximides (ACIs) conjugated with gluco‐, galacto‐ and mannopyranosides are synthesized, by glycosylation of N‐hydroxyethylanthracene carboximide acceptor with glycosyl donors. Glycoconjugation of anthracene carboximide increases the aq. solubility by more than 3‐fold. The glycoconjugates display red‐shifted absorption and emission, as compared to anthracene. Large Stokes shift (λabs/λem=445/525 nm) and high fluorescence quantum yields (Φ) of 0.86 and 0.5 occur in THF and water, respectively. The ACI‐glycosides undergo facile photodimerization in aqueous solutions, leading to the formation of the head‐to‐tail dimer, as a mixture of syn and anti‐isomers. Solution phase and solid‐state characterizations by dynamic light scattering (DLS), microscopic imaging by atomic force (AFM) and transmission electron (TEM) microscopies reveal self‐assembled vesicle structures of ACI glycosides. These self‐assembled structures act as multivalent glycoclusters for ligand‐specific lectin binding, as evidenced by the binding of Man‐ACI to Con A, by fluorescence and turbidity assays. The conjugates do not show cellular cytotoxicity (IC50) till concentrations of 50 μM with HeLa and HepG2 cell lines and are cell‐permeable, showing strong fluorescence inside the cells. These properties enable the glycoconjugates to be used in cell imaging. The non‐selective cellular uptake of the glycoconjugates suggests a passive diffusion through the membrane. [ABSTRACT FROM AUTHOR]

Macroautophagy/autophagy is a catabolic process crucial for degrading cytosolic components and damaged organelles to maintain cellular homeostasis, enabling cells to survive in extreme extracellular environments. ENAH/MENA, a member of the Ena/VASP protein family, functions as a highly efficient actin elongation factor. In this study, our objective was to explore the role of ENAH in the autophagy process. Initially, we demonstrated that depleting ENAH in cancer cells inhibits autophagosome formation. Subsequently, we observed ENAH’s colocalization with MAP1LC3/LC3 during tumor cell starvation, dependent on actin cytoskeleton polymerization and the interaction between ENAH and BECN1 (beclin 1). Additionally, mammalian ATG9A formed a ring-like structure around ENAH-LC3 puncta during starvation, relying on actin cytoskeleton polymerization. Furthermore, ENAH’s EVH1 and EVH2 domains were found to be indispensable for its colocalization with LC3 and BECN1, while the PRD domain played a crucial role in the formation of the ATG9A ring. Finally, our study revealed ENAH-led actin comet tails in autophagosome trafficking. In conclusion, our findings provide initial insights into the regulatory role of the mammalian actin elongation factor ENAH in autophagy.Abbreviations: 3-MA 3-methyladenine; ABPs actin-binding proteins; ATG autophagy related; ATG9A autophagy related 9A; Baf A1 bafilomycin A1; CM complete medium; CytERM endoplasmic reticulum signal-anchor membrane protein; Cyto D cytochalasin D; EBSS Earl’s balanced salt solution; ENAH/MENA ENAH actin regulator; EVH1 Ena/VASP homology 1 domain; EVH2 Ena/VASP homology 2 domain; GAPDH glyceraldehyde-3-phosphate dehydrogenase; Lat B latrunculin B; LC3-I unlipidated form of LC3; LC3-II phosphatidylethanolamine-conjugated form of LC3; MAP1LC3/LC3 microtubule associated protein 1 light chain 3; mEGFP monomeric enhanced green fluorescent protein; mTagBFP2 monomeric Tag blue fluorescent protein 2; OSER organized smooth endoplasmic reticulum; PRD proline-rich domain; PtdIns3K class III phosphatidylinositol 3-kinase; WM wortmannin. [ABSTRACT FROM AUTHOR]

Imaging artifacts often hinder the accuracy of microscopy images, affecting sample analyses and their interpretations. In this article, we explore five prominent imaging artifacts that commonly plague microscopy: blooming/saturation, pixelation, temporal/time varying noise, phototoxicity/photobleaching, and compression. Effective mitigation of these imaging artifacts hinges on careful consideration of key camera parameters, including quantum efficiency, bit-depth, resolution, and dynamic range, each crucial for optimizing image quality and fidelity. A wide range of camera solutions offered by e-con Systems provides consistent support to the various types of microscopy techniques from fluorescence microscopy to live cell imaging. [ABSTRACT FROM AUTHOR]

Molecular beacons (MBs) have been used on surfaces for detecting oligonucleotides. Attempts to use them intracellularly for monitoring mRNA content have been made, however, without any clear conclusion regarding the reliability of the method, mainly due to false positive signals. To reach an understanding of the intracellular fate of MBs, a critical question remains: how long after MB delivery and where in the cell does a false positive signal appear? To answer that question, the MB delivery method should allow for a time‐stamped synchronized delivery of MBs to multiple cells, resulting in MBs being distributed in the cytosol immediately after delivery. Herein, nanostraws are used to inject MBs targeting insulin (Ins1) mRNA directly in the cytosol of clonal beta‐cells, and the evolution of the MB fluorescence in time and space is monitored. The results show an MB translocation to the nucleus, where MBs are degraded or where they open nonspecifically, before the fluorophore alone is expelled back from the nucleus to the cytosol. The signal translocation to the nucleus and back to the cytosol is faster when scrambled MBs are used. The results shed light on the intracellular fate of MBs and highlight the short time scales before false positive signals become predominant. [ABSTRACT FROM AUTHOR]

Fluorescence microscopy is a powerful tool used in scientific and medical research, but it is inextricably linked to phototoxicity. Neglecting phototoxicity can lead to erroneous or inconclusive results. Recently, several reports have addressed this issue, but it is still underestimated by many researchers, even though it can lead to cell death. Phototoxicity can be reduced by appropriate microscopic techniques and carefully designed experiments. This review focuses on recent strategies to reduce phototoxicity in microscopic imaging of living cells and tissues. We describe digital image processing and new hardware solutions. We point out new modifications of microscopy methods and hope that this review will interest microscopy hardware engineers. Our aim is to underscore the challenges and potential solutions integral to the design of microscopy systems. Simultaneously, we intend to engage biologists, offering insight into the latest technological advancements in imaging that can enhance their understanding and practice. [ABSTRACT FROM AUTHOR]

Iron, as the most prevalent critical trace element in the human body, is crucial for many basic physiological functions and has many environmental applications. Therefore, excellent chemosensors with high sensitivity and good selectivity toward iron are highly desired. Three new fluorescent colorimetric chemosensors (CS1, CS2, and CS3) were synthesized and prepared through a one-step reaction to selectively detect Fe3+ in aqueous media. To effectively coordinate metal ions, the sensors contain rhodamine derivatives with different "N, O" donor atoms. Of these sensors, CS2 and CS3 demonstrated sensing behavior and colorimetric "off–on" sensing for Fe3+ over various biologically important metal ions, rendering good selectivity. Fluorescent imaging was performed to confirm the possibility of applying the sensors in living cells. [ABSTRACT FROM AUTHOR]

Owing to their high significance in fundamental study and diverse applications, stimuli-responsive and fluorescent polymers, particularly those with cluster-triggered emission (CTE) featured by non-conjugated chromophores, have drawn tremendous attention in recent years. In this work, fluorescent and multi-responsive polysiloxane (FRPS) was synthesized by hydrolytic condensation polymerization of 3-aminopropyl methyl diethoxysilane (APMS) with 3-(N-isopropyl propionamide) iminopropyl methyl diethoxysilane (APMS-NIP), which was formed in situ through aza-Michael addition between APMS and N-isopropyl acrylamide. FRPS was not only highly sensitive to temperature, pH and CO2 in water, but also showed an enhanced and stimuli-adjustable fluorescence emission. The effects of monomer feeding, pH and CO2 on its lower critical solution temperature and fluorescent property were investigated. FRPS fluorescence emission was ascribed to CTE mechanism. In addition, FRPS was shown to be highly potential as physiological indicator for cell imaging, and for controlled release and trace detection of doxorubicin. This study provides therefore a type of stimuli-responsive and fluorescent material for potential applications in biomedical fields, and it is also of great significance for understanding of the fluorescence mechanism of polysiloxane-based stimuli-responsive polymers. [ABSTRACT FROM AUTHOR]

Carbon dots have been widely focused on the field of sensing and detection due to their excellent optical property. Herein, novel orange fluorescent nitrogen and chlorine co-doped carbon dots (N,Cl-CDs) are obtained by one-pot hydrothermal method using o-phenylenediamine and neutral red. Based on the inner filter effect, the prepared N,Cl-CDs can be innovatively developed as an effective "signal-off" multifunctional sensing platform for sensitive determination of tetracycline. The proposed sensor was utilized to realize the determination of tetracycline in Rirver water samples/milk samples (λex = 390 nm, λem = 606 nm) with satisfactory recoveries and relative standard deviations. The linear range of are 0.05 to 45 μM and 45 to135 μM, and detection limit is 3.9 nM (3σ/m). Meanwhile, the luminescent intensity of N,Cl-CDs was reduced gradually when pH changed continuously from 12 to 2, showing a pH-responsive fluorescence property with two linear ranges of pH 3–7 and pH 7–10. In addition, due to the characteristics of low toxicity and excellent biocompatibility, the N, Cl-CDs were also used in the imaging of oocystis cells, which is hopeful to realize the detection of tetracycline in living cells. [ABSTRACT FROM AUTHOR]