Microscopy imaging is fundamental in analyzing bacterial morphology and dynamics, offering critical insights into bacterial physiology and pathogenicity. Image segmentation techniques enable quantitative analysis of bacterial structures, facilitating precise measurement of morphological variations and population behaviors at single-cell resolution. This paper reviews advancements in bacterial image segmentation, emphasizing the shift from traditional thresholding and watershed methods to deep learning-driven approaches. Convolutional neural networks (CNNs), U-Net architectures, and three-dimensional (3D) frameworks excel at segmenting dense biofilms and resolving antibiotic-induced morphological changes. These methods combine automated feature extraction with physics-informed postprocessing. Despite progress, challenges persist in computational efficiency, cross-species generalizability, and integration with multimodal experimental workflows. Future progress will depend on improving model robustness across species and imaging modalities, integrating multimodal data for phenotype-function mapping, and developing standard pipelines that link computational tools with clinical diagnostics. These innovations will expand microbial phenotyping beyond structural analysis, enabling deeper insights into bacterial physiology and ecological interactions.

The intricate pathological mechanisms of ischemia-reperfusion injury (IRI) are intimately associated with the imbalance of metabolic substance supply and demand. Investigation of the fluctuated molecules reveals the progression of reperfusion injury, facilitating earlier diagnosis and treatments. Fluorescence imaging is a powerful technique in fluorescent optical diagnosis, essential for detecting biomarker levels both in vitro and in vivo . By integrating multifunctional scaffolds with specific recognition groups, small-molecule fluorescent probes (SMFPs) effectively monitor biomarkers related to IRI, providing valuable insights into pathological mechanisms and enhancing early diagnostic capabilities. This review systemically summarizes the recent developments of SMFPs, focusing on design strategies and their applications in the main types of IRI. Furthermore, we discuss the challenges and propose prospects based on existing SMFP applications in this area. We aim to provide a comprehensive analysis of SMFPs for disease diagnosis and inspire researchers to further innovate and develop effective tools for clinical applications. (c) 2025 Published by Elsevier B.V. on behalf of Chinese Chemical Society and Institute of Materia Medica, Chinese Academy of Medical Sciences.

The ability to decode the relationship between mitochondrial morphology and function at the level of individual organelles is central to understanding cellular responses to stress, such as hypoxia. Herein, a comprehensive strategy is presented that integrates tailored fluorescent probes with artificial intelligence (AI) for single mitochondrion analysis. Focus is on three interrelated biomarkers, reactive oxygen species (ROS), viscosity, and mitochondrial membrane potential (MMP), that together form a pathophysiological axis indicative of mitochondrial state under hypoxic stress. A functional probe set is used to image these features simultaneously, including a newly developed dual-cationic probe, MitoVP, which enhances mitochondrial targeting and resolution for viscosity sensing. Mitochondrial morphological features are then extracted using a deep learning-based algorithm, which further classified individual mitochondria into dot, rod, and network morphotypes. This analysis enabled quantitative mapping between mitochondrial morphology and functional states, revealing significant heterogeneity across diverse physiological conditions. Based on this characterization, a random forest classifier trained on over 10,000 mitochondria accurately distinguished normoxic from hypoxic states and identified viscosity as a primary contributor to mitochondrial status under hypoxia. This integrated approach provides a powerful platform for single organelle investigations and advances the understanding of mitochondrial dysfunction in complex biological systems.

Organelle morphology and dynamics are closely linked to cellular function and fate, yet their relationships remain poorly defined across physiological and pathological contexts. Live-cell imaging enables the visualization of subcellular structures and dynamic processes but often requires extensive manual analysis, introducing variability and limiting reproducibility and throughput. Image segmentation partitions digital images into meaningful regions, facilitating the quantification of organelle morphology and molecular behavior for precise subcellular analysis. Herein, this review surveys recent advances in live-cell imaging segmentation algorithms across diverse organelles, from traditional thresholding-based methods to deep learning approaches that enhance accuracy and adaptability in complex biological environments. We discuss key challenges, including 3-dimensional imaging, multi-organelle segmentation, and generalization across diverse imaging modalities. We also highlight label-efficient strategies, synthetic data, and physics-guided modeling that reduce reliance on manual annotations and large annotated datasets. By advancing generalist models, these innovations improve quantitative cell biology, accelerate disease research, and drive therapeutic discovery, underscoring the transformative role of artificial intelligence in biomedical microscopy.

AbstractAs one of the major causes of antimicrobial resistance, β‐lactamase develops rapidly among bacteria. Detection of β‐lactamase in an efficient and low‐cost point‐of‐care testing (POCT) way is urgently needed. However, due to the volatile environmental factors, the quantitative measurement of current POCT is often inaccurate. Herein, we demonstrate an artificial intelligence (AI)‐assisted mobile health system that consists of a paper‐based β‐lactamase fluorogenic probe analytical device and a smartphone‐based AI cloud. An ultrafast broad‐spectrum fluorogenic probe (B1) that could respond to β‐lactamase within 20 s was first synthesized, and the detection limit was determined to be 0.13 nmol/L. Meanwhile, a three‐dimensional microfluidic paper‐based analytical device was fabricated for integration of B1. Also, a smartphone‐based AI cloud was developed to correct errors automatically and output results intelligently. This smart system could calibrate the temperature and pH in the β‐lactamase level detection in complex samples and mice infected with various bacteria, which shows the problem‐solving ability in interdisciplinary research, and demonstrates potential clinical benefits.

Hepatic ischemia-reperfusion injury (HIRI) is characterized by mitochondrial dysfunction and oxidative stress. Monitoring mitochondrial hydrogen peroxide (mtH2O2) levels in real-time through super-resolution imaging is crucial for elucidating its distribution in live cells and its mechanism of action during HIRI. However, low-background fluorogenic probes have been overlooked in the context of super-resolution imaging. In this study, we developed a low-background fluorogenic probe (Mito-WG) with the potential for super-resolution morphology-correlated mitochondrial identification to track the fluctuates of mtH2O2 in HIRI. Activation of the desirable fluorescence properties of the probe by mtH2O2 was confirmed using structural illumination microscopy (SIM), enabling high-quality mitochondrial imaging with exceptional specificity and sensitivity. Fluctuations in mtH2O2 levels were successfully observed in both cellular and rat models of HIRI. Furthermore, we associated the decline in mitochondrial redox homeostasis with accelerated mtH2O2 production during HIRI, which triggered mitophagy deficiency and led to cell death. In conclusion, Mito-WG possesses excellent photophysical and low-background properties for SIM imaging, making it a promising tool for mtH2O2 tracking in HIRI research and clinical diagnosis.

Nitroreductase (NTR), a common enzymatic biomarker of hypoxia, is widely used to evaluate tumor microenvironments. To date, numerous optical probes have been reported for NTRs detection. Approaches capable of concisely guiding the probe design of NTRs suitable for deep-tissue imaging, however, are still lacking. As such, direct optical imaging of endogenous NTR activities from tumors derived from cancer patients is thus far not possible. Herein, aided by computational calculations, the authors have successfully developed a series of two-photon (TP) small-molecule fluorogenic probes capable of sensitively detecting general NTR activities from various biological samples; by optimizing the distance between the recognition moiety and the reactive site of NTRs from different sources, the authors have discovered and experimentally proven that X4 displays the best performance in both sensitivity and selectivity. Furthermore, X4 shows excellent TP excited fluorescence properties capable of directly monitoring/imaging endogenous NTR activities from live mammalian cells, growing zebrafish, and tumor-bearing mice. Finally, with an outstanding TP tissue-penetrating imaging property, X4 is used, for the first time, to successfully detect endogenous NTR activities from the liver lysates and cardia tissues of a cancer patient. The work may provide a universal strategy to design novel TP small-molecule enzymatic probes in future clinical applications.

β-内酰胺类抗生素是20世纪的伟大发明之一,拯救了数以万计生命.然而,随着抗生素临床应用的普及,由β-内酰胺酶导致的致病菌耐药性问题也日益突出.耐药菌的进化和传播严重影响到人类健康和全球社会经济发展.因此,检测/抑制β-内酰胺酶的活性对合理使用抗生素、有效治疗感染类疾病具有重要意义.目前,特异性检测、抑制β-内酰胺酶的相关方法和临床应用已有报道.本综述主要阐述细菌产生β-内酰胺酶的耐药机理,总结近年来β-内酰胺酶荧光探针和抑制剂的发展,以期为今后设计特异性高、灵敏度强的该类荧光探针和抑制剂提供理论依据,解决抗生素耐药性问题.

Tyrosinase is an important enzyme in controlling the formation of melanin in melanosome, and plays a key role in the pigmentation of hair and skin. The abnormal expression or activation of tyrosinase is associated with several diseases such as albinism, vitiligo, melanoma and Parkinson disease. Excessive deposition of melanin could cause diseases such as freckles and brown spots in the human body, and it is also closely related to browning of fruits and vegetables and insect molting. Detecting and inhibiting the activity of tyrosinase is of extraordinary value in the progress of diagnosis and treatment of these diseases. Therefore, many selective optical detection probes and small molecular inhibitors have been developed, and have made significant contributions to the basic and clinical research on these diseases. In this paper, the detection and inhibition of tyrosinase and their application in whitening products are reviewed, with special emphasis on development of fluorescent probes and inhibitors. Hopefully, this review will help design more efficient and sensitive tyrosinase probes and inhibitors, as well as shed light on novel treatment of diseases such as melanoma.

β-Lactam antibiotics are generally perceived as one of the greatest inventions of the 20th century, and these small molecular compounds have saved millions of lives. However, upon clinical application of antibiotics, the β-lactamase secreted by pathogenic bacteria can lead to the gradual development of drug resistance. β-Lactamase is a hydrolase that can efficiently hydrolyze and destroy β-lactam antibiotics. It develops and spreads rapidly in pathogens, and the drug-resistant bacteria pose a severe threat to human health and development. As a result, detecting and inhibiting the activities of β-lactamase are of great value for the rational use of antibiotics and the treatment of infectious diseases. At present, many specific detection methods and inhibitors of β-lactamase have been developed and applied in clinical practice. In this Minireview, we describe the resistance mechanism of bacteria producing β-lactamase and further summarize the fluorogenic probes, inhibitors of β-lactamase, and their applications in the treatment of infectious diseases. It may be valuable to design fluorogenic probes with improved selectivity, sensitivity, and effectiveness to further identify the inhibitors for β-lactamases and eventually overcome bacterial resistance.

The cover feature picture shows a Chinese scientist standing on a survey ship with binoculars watching the meteors of the North Pole night. Under these extreme conditions, the meteors change from dull to dazzling yellow as they pass through the atmosphere in the night sky. This symbolizes the fast and visible fluorescence response of the fluorogenic probe in the environment of hypochlorite under extreme conditions. More information can be found in the full paper by C. Yu, L. Li, et al. on page 831 in Issue 6, 2019 (DOI: 10.1002/cbic.201800659).

A fast-response fluorogenic probe-compound D1-for monitoring hypochlorite (ClO- ), based on specific ClO- cleavage of a C=N bond and producing results observable to the naked eye, has been developed. The response of the probe to ClO- increases linearly, and the fluorescence intensity was heightened by a factor of about 25. D1 responses to ClO- , with high selectivity and sensitivity, were observable by naked eye within 10 s. D1 can not only detect levels of hypochlorite in vitro, such as in urine, but is also capable of monitoring hypochlorite content under extremely cold conditions, as low as -78 °C. Meanwhile, its good biocompatibility permitted the use of D1 to detect intracellular ClO- by confocal microscopy. Moreover, D1 was successfully applied to monitor exogenous and endogenous ClO- in zebrafish through fluorescence imaging.

Arene ruthenium(II) complexes have been widely investigated as one of the most promising candidates in chemotherapy because of their low toxicity and high inhibiting activity against the proliferation, invasion, migration, and angiogenesis of various tumors in vitro and in vivo. This review highlights the recent developments in different chemical types of arene Ru(II) complexes, as well as their biological activity and underlying mechanisms. The sustained efforts in this aspect of arene Ru(II) complexes would be essential in developing novel anti-tumor agents in the near future.

Arene Ru(II) complexes have long been extensively studied as potential inhibitors against the proliferation of tumor cells, but their behavior against the migration and invasion of tumor cells needs further research. In this work, a series of arene Ru(II) complexes, (n(6-)C(6)H(6))Ru(p-XPIP)Cl]Cl (X = H, 1; F, 2; Cl, 3; Br, 4; and I, 5), have been synthesized, and their inhibitory activity against the migration and invasion of MDA-MB-231 breast cancer cells have been investigated. It is found that all of these complexes exhibit excellent inhibitory activity (IC50) against the growth of MDA-MB-231 breast cancer cells, and the value of IC50 for 1, 2, 3, 4, and 5 is about >300, 52.6, 11.4, 45.5, and 59.1 mu M, respectively. Further studies by wound-healing assay, FITC-geltain assay, and flow cytometry assay showed that 3 can apparently suppress the migration and invasion of MDA-MB-231 cells via the joint action of S-phase arrest and apoptosis. Moreover, the binding behavior of these arene Ru(II) complexes with c-myc G-quadruplex DNA has also been studied, and the results showed that these complexes can bind and stabilize c-myc Gquadruplex DNA in groove binding mode. Also, the low toxicity of 3 was confirmed by its low inhibitory activity against the growth of normal MCF-10A breast cells in vitro and the development of zebrafish embryos in vivo. In other words, these results indicated that synthetic arene Ru(II) complexes can be developed as low-toxicity agents against the proliferation, migration, and invasion of breast cancer cells.

Gene therapy has long been limited in the clinic, due in part to the lack of safety and efficacy of the gene carrier. Herein, a single enantiomer ruthenium(II) complex, Λ-[Ru(bpy)2(p-BEPIP)](ClO4)2 (Λ-RM0627, bpy = 4,4′-bipyridine, p-BEPIP = 2-(4-phenylacetylenephenyl)imidazole [4,5f][1, 10] phenanthroline), has been synthesized and investigated as a potential gene carrier that targets the nucleus. In this report, it is shown that Λ-RM0627 promotes self-assembly of c-myc DNA to form a nanowire structure. Further studies showed that the nano-assembly of c-myc DNA that induced Λ-RM0627 could be efficiently taken up and enriched in the nuclei of HepG2 cells. After treatment of the nano-assembly of c-myc DNA with Λ-RM0627, over-expression of c-myc in HepG2 cells was observed. In summary, Λ-RM0627 played a key role in the transfer and release of c-myc into cells, which strongly indicates Λ-RM0627 as a potent carrier of c-myc DNA that targets the nucleus of tumor cells.

Novel ruthenium(ii) complex 1 can be developed as a low toxicity fluorescence probe for living cell nuclei in future.

Fibrinogen is a plasma glycoprotein that is an established cardiovascular risk and it participates in the blood-clotting mechanism. Nitrated fibrinogen has been shown to inhibit platelet aggregation and thrombus formation. However, there are only a few reports relating to the activity and structural changes of nitrified fibrinogen when metal ions are present in the reaction. Mn (II) ion plays an important physiological role in the nervous system and cardiac function. In this study, we use UV-Vis, 3D-fluorescence, SDS-PAGE electrophoresis and Von-Clauss to detect 3-nitrotyrosine (3-NT) production and the activity changes of fibrinogen after nitration and oxidation damage caused by ONOO- in the presence of Mn (II). Results showed that Mn (II) can enhance the production of 3-NT in fibrinogen, promote fluorescence quenching of fibrinogen, and increase the injury to gamma and A alpha chains of fibrinogen in the presence of peroxynitrite. Consequently, Mn (II) promotes concentration dependent fibrinogen nitrification damage and significantly reduces the biological activity of nitrified fibrinogen.