Search Results (68)

Machine learning (ML) is transforming the analysis of biomolecular data, holding significant promise for improving the efficiency and accuracy of microscopy image analysis and for studying the dynamics of molecules in live cells. As data-driven approaches continue to evolve, they may eventually replace traditional statistical methods that rely on conventional analytical methods. This review examines and critically analyses the state of the art of ML techniques as applied to various levels of data supervision in the analysis of dynamic single-molecule datasets obtained using superresolution optical microscopy. Collectively encompassed under the umbrella of “nanoscopy”, these methods currently comprise targeted techniques such as stimulated emission depletion (STED) microscopy and stochastic techniques like single-molecule localization microscopies (SMLMs), comprising photoactivated localization microscopy (PALM), DNA points accumulation for imaging in nanoscale topography (DNA-PAINT) microscopy, and minimal fluorescence photon flux (MINFLUX) microscopy. These techniques all enable the imaging of subcellular components and molecules beyond the diffraction limit, and some are additionally capable of studying their dynamics in real time, as reviewed here, using several ML techniques that facilitate motion analysis in two or three dimensions with qualitative and quantitative characterisation in the live cell. It is expected that the growing use of learning-based approaches in biological microscopy data processing will dramatically increase throughput and accelerate progress in this rapidly developing field. Full article

Carbon dots (CDs), a class of fluorescent nanomaterials, have emerged as powerful tools for biological applications, particularly in the targeting, imaging, and therapeutic modulation of mitochondria. Due to their small size, simplicity of synthesis, biocompatibility, and tunable optical properties, CDs can be engineered to selectively accumulate in mitochondria, enabling real-time imaging of mitochondrial function and dynamics in live cells. Moreover, their ability to carry therapeutic agents, such as antioxidants, drugs, and gene delivery vectors, offers potential in treating mitochondrial dysfunction, which is central to various diseases, including neurodegenerative disorders, cancer, and metabolic diseases. Recent advancements in surface functionalization have enhanced mitochondrial targeting and specificity, while ongoing research aims to optimize the safety, efficiency, and clinical translation of CDs for therapeutic applications. This review highlights the latest developments in the use of carbon dots for mitochondrial imaging, therapeutic delivery, and disease intervention, offering promising avenues for future research and clinical applications. Full article

Background: Zebrafish-based drug discovery systems provide significant advantages over mammalian models for high-throughput in vivo screening. Among these, the NF-κB eGFP reporter system significantly enhances drug discovery in zebrafish by enabling real-time, high-resolution monitoring of pathway activity in live organisms, thereby streamlining mechanistic studies and high-throughput screening. Methods: We developed a novel AI (Quantifish and Orange software)-based zebrafish precision individualized 96-well ZF plates (0–7 dpf) and individualized MT tanks (8 dpf–4 mpf) protocol for the transposon-mediated transgenesis of the NFκB eGFP reporter system. Results: One-cell stage embryos were administered NFκB reporter construct and Tol2 transposase mRNA via microinjection and transferred to separate wells of a 96-well ZF plate. Bright-field and fluorescence images of each well were captured at 5 dpf in the F0, F1, and F2 generations using the automated confocal high-content imager CQ1. The Quantifish software was used for the automated detection and segmentation of zebrafish larval fluorescence intensity in specific regions of interest. Quantitative data on the fluorescence intensity and distribution patterns were measured in Quantifish, and advanced statistical and machine learning methods were applied using Orange. Imaging data with eGFP expression results were assessed to evaluate the efficiency of the transgenic protocol. Discussion: This AI-enhanced precision protocol allows for high-throughput screening and quantitative analysis of NFκB reporter transgenesis in zebrafish, enabling the efficient identification and characterization of stable transgenic lines that exhibit tissue-specific expression of the NF-κB reporter, such as lines with induced expression restricted to the retina following LPS stimulation. This approach streamlines the evaluation of regulatory elements, enhances data consistency, and reduces animal use, making it a valuable tool for zebrafish drug discovery. Full article

The detection of aliphatic and aromatic biogenic amines (BAs) is important in food spoilage, environmental monitoring, and disease diagnosis and treatment. Existing fluorescent probes predominantly detect aliphatic BAs with single signal variation and low sensitivity, impairing the adaptability of discriminative sensing platforms. Herein, we present a visual chemosensor (galactose-functionalized pyrrolopyrrole aza-BODIPY, PPAB-Gal) that simultaneously detects eight aliphatic and aromatic BAs in a real-time and intuitive way based on their unique electronic and structural features. Our findings reveal that the dual colorimetric and ratiometric emission changes are rapidly produced in presence of eight BAs through a noncovalent interaction (π–π stacking and hydrogen bond)-assisted chromophore reaction. Specifically, other lone-pair electrons containing compounds, such as secondary amines, tertiary amines, NH₃, and thiol, fail to exhibit these changes. As a result, superior sensing performances with distinctly dual signals (Δλ_ab = 130 nm, Δλ_em = 150 nm), a low LOD (~25 nM), and fast response time (<2 min) were obtained. Based on these advantages, a qualitative and smartphone-assisted sensing platform with a PPAB-Gal-loaded TLC plate is developed for visual detection of putrescine and cadaverine vapor. More importantly, we construct a connection between a standard quantitative index for the TVBN value and fluorescence signals to quantitatively determine the freshness of tuna and shrimp, and the method is facile and convenient for real-time and on-site detection in practical application. Furthermore, since the overexpressed spermine is an important biomarker of cancer diagnosis and treatment, PPAB-Gal NPs can be used to ratiometrically image spermine in living cells. This work provides a promising sensing method for BAs with a novel fluorescent material in food safety fields and biomedical assays. Full article

The ability of a virus to be propagated within a host cell is dependent on a multitude of dynamic virus–host interactions. Live-cell imaging is an invaluable approach in the study of virus replication cycles and virus–host interactions as it can allow for the direct visualisation of key events and interactions in real time. These details can provide unique insights into many aspects of viral infections including the cellular pathways that are exploited by viruses, the evasion of host immune defences, and viral pathogenesis. This review summarises the live-cell fluorescence imaging approaches that have been developed and applied to study Flaviviridae virus family members that are responsible for significant public health burdens and outbreaks which, in many instances, are increasing in frequency and severity. We discuss how these approaches have expanded our understanding of fundamental stages of viral replication cycles by enabling the direct visualisation of the localisation, trafficking, and interactions of virus particles, proteins, and genomes at distinct stages. The strategies that can be employed to enhance the biological relevance of live-cell fluorescence imaging acquisitions are discussed, along with how live-cell imaging approaches can be further developed to increase resolution, enable multi-colour imaging, and support the long-term visualisation of multiple stages of a viral replication cycle. Full article

Telomeres play a key role in maintaining chromosome stability and cellular aging. They consist of repetitive DNA sequences that protect chromosome ends and regulate cell division. Telomerase is a reverse transcriptase enzyme counteracts the natural shortening of telomeres during cell division by extending them. Its activity is pivotal in stem cells and cancer cells but absent in most normal somatic cells. Recent advances in biosensor technologies have facilitated the in situ detection of telomerase activity, which is essential for understanding its role in aging and cancer. Techniques such as fluorescence, electrochemistry, and DNA nanotechnology are now being employed to monitor telomerase activity in living cells, providing real-time insights into cellular processes. DNA-based biosensors, especially those incorporating molecular beacons, DNA walkers, and logic gates, have shown promise for enhancing sensitivity and specificity in telomerase imaging. These approaches also facilitate the simultaneous analysis of related cellular pathways, offering potential applications in early cancer detection and precision therapies. This review explores recent developments in intracellular telomerase imaging, highlighting innovative approaches such as DNA-functionalized nanoparticles and multi-channel logic systems, which offer non-invasive, real-time detection of telomerase activity in complex cellular environments. Full article

In the human body, carboxylesterases (CEs) play crucial roles in xenobiotic metabolism and lipid homeostasis. But abnormal expression of CEs is highly associated with some diseases, such as hyperlipidemia, diabetes, and liver cancer. Therefore, it is of great importance to develop an efficient tool for the accurate detection of CEs in living organisms. Herein, an innovative near-infrared (NIR) fluorescent probe, TTAP−AB, was designed for CE detection based on the aggregation-induced emission (AIE) mechanism. This probe exhibits rapid response (2 min), excellent sensitivity (limit of detection = 8.14 × 10⁻⁶ U/mL), and high selectivity to CEs. Additionally, owing to its good biocompatibility, the TTAP−AB probe enables the monitoring of dynamic changes in CE levels under drug-induced modulation in living cells and zebrafish. More importantly, the TTAP−AB probe was successfully employed to image liver tumors and assist in tumor resection through the real-time monitoring of CEs, indicating that TTAP−AB is promising to guide liver cancer surgery. Therefore, the TTAP−AB probe can not only enrich the strategies for CE detection in biological systems but also has great potential for some clinical imaging applications, including medical diagnosis, preclinical research, and imaging-guided surgery. Full article

Cellular senescence is a recently emerged research topic in modern biology. Often described as a double-edged sword, it encompasses numerous essential biological processes, including beneficial effects such as wound healing and embryonic development, as well as detrimental contributions to chronic inflammation and tumor development. Consequently, there is an increasing need to unravel the intricate networks of senescence and develop reliable detection methods to distinguish it from related phenomena. To address these challenges, a variety of detection methods have been developed. In particular, small-molecule fluorescent probes offer distinct advantages such as suitability for real-time live cell monitoring and in vivo imaging, superior tunable properties, and versatile applications. In this review, we explored recent advancements in the development of small-molecule fluorescent probes toward monitoring cellular senescence by targeting various senescence-related biological phenomena. These phenomena include the upregulation of senescence-associated enzymes, perturbation of the subcellular environment, and increased endogenous ROS levels. Moreover, multi-senescence biomarker-targeting approaches are also discussed to improve their sensitivities and specificities for the detection of cellular senescence. With recent advances in senescence probe development, current challenges in this field are also discussed to facilitate further progress. Full article

To monitor the biological function of H₂S in real time, this investigation demonstrated the design and synthesis of a novel fluorescent probe integrated with cyanine and 2,4-dinitrophenol for the qualitative and quantitative detection of H₂S. An NIR sensitive sensor (FS-HS-1) was provided with a straightforward process. Spectroscopy experiments elucidated that FS-HS-1 could selectively detect H₂S in a PBS solution (containing 40% acetonitrile) with a 111-fold fluorescence enhancement at 715 nm (ex. 605 nm). The response towards NaHS occurred in less than 2 min, and the detection limit was confirmed to be as low as 4.47 ± 0.11 nmol/L. Furthermore, the probe is capable of monitoring changes in exogenous H₂S concentrations within living cells with confocal and 2P imaging. Full article

Zinc ions (Zn²⁺) play a key role in maintaining and regulating protein structures and functions. To better understand the intracellular Zn²⁺ homeostasis and signaling role, various fluorescent sensors have been developed that allow the monitoring of Zn²⁺ concentrations and bioimaging in live cells in real time. This review highlights the recent development of organic fluorescent probes for the detection and imaging of intracellular Zn²⁺, including the design and construction of the probes, fluorescent response mechanisms, and their applications to intracellular Zn²⁺ detection and imaging on-site. Finally, the current challenges and prospects are discussed. Full article

Molecular probes with the ability to differentiate between subcellular variations in acidity levels remain important for the investigation of dynamic cellular processes and functions. In this context, a series of cyclic peptide and PEG bio-conjugated dual near-infrared emissive BF₂-azadipyrromethene fluorophores with maxima emissions at 720 nm (at pH > 6) and 790 nm (at pH < 5) have been developed and their aqueous solution photophysical properties determined. Their inter-converting emissions and fluorescence lifetime characteristics were exploited to track their spatial and temporal progression from first contact with the plasma membrane to subcellular locales to their release within extracellular vesicles. A pH-dependent reversible phenolate/phenol interconversion on the fluorophore controlled the dynamic changes in dual emission responses and corresponding lifetime changes. Live-cell confocal microscopy experiments in the metastatic breast cancer cell line MDA-MB-231 confirmed the usability of the dual emissive properties for imaging over prolonged periods. All three derivatives performed as probes capable of real-time continuous imaging of fundamental cellular processes such as plasma membrane interaction, tracking endocytosis, lysosomal/large acidic vesicle accumulation, and efflux within extracellular vesicles without perturbing cellular function. Furthermore, fluorescence lifetime imaging microscopy provided valuable insights regarding fluorophore progression through intracellular microenvironments over time. Overall, the unique photophysical properties of these fluorophores show excellent potential for their use as information-rich probes. Full article

Precise DNA quantification and nuclear imaging are pivotal for clinical testing, pathological diagnosis, and drug development. The detection and localization of mitochondrial DNA serve as crucial indicators of cellular health. We introduce a novel conjugated oligoelectrolyte (COE) molecule, COE-S3, featuring a planar backbone composed of three benzene rings and terminal side chains. This unique amphiphilic structure endows COE-S3 with exceptional water solubility, a high quantum yield of 0.79, and a significant fluorescence Stokes shift (λ_ex = 366 nm, λ_em = 476 nm), alongside a specific fluorescence response to DNA. The fluorescence intensity correlates proportionally with DNA concentration. COE-S3 interacts with double-stranded DNA (dsDNA) through an intercalation binding mode, exhibiting a binding constant (K) of 1.32 × 10⁶ M⁻¹. Its amphiphilic nature and strong DNA affinity facilitate its localization within mitochondria in living cells and nuclei in apoptotic cells. Remarkably, within 30 min of COE-S3 staining, cell vitality can be discerned through real-time nuclear fluorescence imaging of apoptotic cells. COE-S3’s high DNA selectivity enables quantitative intracellular DNA analysis, providing insights into cell proliferation, differentiation, and growth. Our findings underscore COE-S3, with its strategically designed, shortened planar backbone, as a promising intercalative probe for DNA quantification and nuclear imaging. Full article

Peroxynitrite (ONOO⁻) has been revealed to play crucial roles in many physiological and pathological processes, and many diseases were proven to be associated with its misregulated production. The development of fluorescent probes meets the need for tracking ONOO⁻ and gives a better understanding of its diverse mechanisms. In this work, a red-emitting fluorescent probe BP-ONOO was synthesized via functionalization of the rhodol-like fluorophore with a reactive site of hydrazide. The probe BP-ONOO exhibited high sensitivity, excellent selectivity, and short response time (less than 4 s) towards ONOO⁻ under neutral or weak alkaline conditions. These attractive properties favor its application in real-time imaging of ONOO⁻ in living cells, and the probe has been successfully applied for imaging the concentration levels of ONOO⁻ in RAW 264.7 macrophage cells under drug stimulation. Full article

Hydrogen sulfide (H₂S) is an important gasotransmitter, but only a few methods are available for real-time detection. Fluorescent probes are attractive tools for biological applications because of their high sensitivity, convenience, rapid implementation, noninvasive monitoring capability, and simplicity in fluorescent imaging of living cells and tissues. Herein, we report on a pro-fluorescent probe, NAP-Py-N₃ based on naphthalimide derivative, which was found to show high selectivity toward H₂S over various other analytes, including biothiols, making it feasible to detect H₂S. After reaction with H₂S, this probe showed rapid and significant turn-on green fluorescent enhancement at 553 nm (about 54-fold, k₂ = 9.62 M⁻¹s⁻¹), high sensitivity (LOD: 15.5 nM), significant Stokes shift (118 nm), and it was found that the fluorescence quantum yield of fluorescence product can reach 0.36. Moreover, the probe has also been successfully applied to detect the gaseous H₂S and to confirm the presence of H₂S released from modern organic donors, which in recent years have been commonly used to investigate the role of H₂S in biological systems. All the results indicate that this probe is excellent and highly valuable. Full article

The well-known small-molecule biothiols have been used to maintain the normal metabolism of peroxy radicals, forming protein structures, resisting cell apoptosis, regulating metabolism, and protecting the homeostasis of cells in the organism. A large amount of research has found that abnormal levels of the above biothiols can cause some adverse diseases, such as changes in hair pigmentation, a slower growth rate, delayed response, excessive sleep and skin diseases. In order to further investigate the exact intracellular molecular mechanism of biothiols, it is imperative to explore effective strategies for real-time biothiol detection in living systems. In this work, a new near-infrared (NIR) emission fluorescence probe (probe 1) for sensitive and selective detection of biothiols was devised by combining dicyanoisophorone derivatives with the dinitrobenzenesulfonyl (DNBS) group. As expected, probe 1 could specifically detect biothiols (Cys, Hcy and GSH) through the dinitrobenzenesulfonyl group to form dye 2, which works as a signaling molecule for sensing biothiols in real samples. Surprisingly, probe 1 showed superior sensing characteristics and low-limit detection towards biothiols (36.0 nM for Cys, 39.0 nM for Hcy and 48.0 nM for GSH) with a large Stokes shift (134 nm). Additionally, the function of probe 1 as a platform for detecting biothiols was confirmed by confocal fluorescence imaging of biothiols in MCF-7 cells and zebrafish. More importantly, the capability of probe 1 in vivo has been further evaluated by imaging the overexpressed biothiols in tumor tissue. It is reasonable to believe that probe 1 can provide a valuable method to explore the relationship between biothiols and the genesis of tumor. Full article