Abstract Although genetically encoded fluorescent sensors are fundamental tools to gain an understanding of intracellular nitric oxide (NO) dynamics, their development is still limited. We recently introduced the blue-emitting fluorescent protein (FP) mTagBFP2 as a NO sensor, where the chromophoric group formed upon S-nitrosylation of Cys residues leads to fluorescence quenching through excitation energy transfer. In this work, we further elaborated on this concept and devised a simple, two-domain, ratiometric NO sensor. mTagBFP2 was fused, with a suitable linker, to mCherry, a red-emitting fluorescent protein. As mCherry lacks cysteine residues, it is insensitive to NO and provides a convenient reference signal to normalize the response of mTagBFP2 fluorescence emission in a ratiometric detection. We demonstrate the capability of the construct to sense NO in the micromolar range in solution (Kd ca. 1 μM) and within mammalian (HeLa) and bacterial (Escherichia coli (E. coli)) cells.
Abstract Particulate anode materials with high theoretical capacity show great promise for lithium-ion batteries (LIBs). However, understanding their dynamic behavior during electrochemical reactions requires characterizing single nanoparticles, a task where existing techniques struggle to capture multidimensional evolution in real time. Here, we develop a Cr-film-enabled in situ high-contrast reflectance interference imaging method for battery-relevant anode potentials that enables five-dimensional (5D) real-time observation, seamlessly integrating lateral spatial mapping (X–Y), interferometrically model-based reconstructed height (Z), temporal evolution (t), and chemically sensitive optical contrast. Using single Co3O4 nanoplates as a model, we investigate single-particle optical contrast changes associated with electrochemical evolution, which are interpreted through complementary ensemble and ex situ characterizations. Our 5D tracking uncovers a unique two-stage lithiation process: the initial Li insertion induces a previously unobserved reversible warping deformation that may partly arise from nonuniform lattice expansion, which is followed by a largely irreversible projected area expansion resulting from conversion reactions that form Li2O and metallic Co. These in situ optical observations are strongly corroborated by ex situ X-ray diffraction and transmission electron microscopy. Ultimately, this method provides multidimensional insights into the dynamic degradation mechanisms of single battery particles, offering a powerful tool for the rational design of robust electrode materials.
Abstract Pyridine nucleotide coenzymes, including NAD+/NADH and NADP+/NADPH redox couples, form a highly interconnected metabolic and signaling network that links cellular bioenergetics, biosynthesis, redox homeostasis, and stress responses. These cofactors are compartmentalized across the cytosol, mitochondria, and nucleus and are continuously remodeled by multiple biosynthetic, recycling, interconversion, and consumption pathways. Their spatiotemporal dynamics are further rewired in cancer, aging, neurodegeneration, and inflammation, making them informative reporters of pathway activity and attractive pharmacological targets. In this review, we discuss fluorescent biosensors for pyridine nucleotide imaging, including small-molecule probes, genetically encoded indicators, and chemigenetic platforms. We compare these approaches in terms of analyte definition, selectivity, dynamic range, reversibility, spatiotemporal resolution, and suitability for subcellular imaging. We then summarize what these tools have revealed about the networked regulation of pyridine nucleotide coenzymes in physiology and disease and highlight their growing utility in drug discovery, mechanism-of-action studies, and phenotypic screening. Finally, we outline the technical challenges that must be addressed to make pyridine nucleotide imaging more quantitative, interoperable, and predictive in chemical biology and biomedicine.
Abstract Many fluorescent recognition-based copper probes have been developed, but they generally suffer from a weak fluorescence change upon detection of low-abundance labile copper in biological systems. We have addressed this issue by developing an activity-based copper probe that recycles a portion of copper after detection, thereby amplifying the fluorescence signal. The probe showed good sensitivity and could be applied to in-cell experiments. We expect that similar systems will also be useful for the detection of other bioactive targets that exist at low concentrations.
Abstract Invadosome-mediated matrix degradation is a hallmark of cancer metastasis, yet the architecture of these invasive structures, which mainly consist of actin cores, has not been well characterized. Here, we visualized the actin core of the invadosome of MDA-MB-231 cells, a highly invasive breast cancer cell line, by confocal microscopy and showed that the actin cores had a rosette-like arrangement near the nucleus. Then, high-resolution light microscopy was utilized to characterize the actin cores of the invadosome induced by TGF-β, which may resemble the conditions in metastasis. The resulting three-dimensional views of the actin cores showed that they were circularly arranged and assembled into a barrel-like structure elongating toward the interface of the cells with an extracellular matrix, suggesting that they might be correlated with the invasion of the cancer cells. Statistics were also made to evaluate the structural features of the actin core assemblies by measuring their distribution, area, and quantity. Furthermore, inhibitors targeting actin assemblies and the corresponding pathways were applied to investigate the structural changes of actin rosettes, which showed that different inhibitors had different impacts on the actin core arrangement and cell invasiveness. Overall, these results suggest that the actin cores of the invadosome have specific structural features that may correlate with the invasiveness of cancer cells, and the structural information may provide mechanistic insights for tumor cell invasion and facilitate therapeutic strategies against cancer.
Leishmaniasis remains a neglected tropical disease with limited and highly toxic therapeutic options. In search of safer and more effective alternatives, bithiophene derivatives have emerged as promising scaffolds. In this study, we evaluated the antileishmanial potential of BT-Ac, a bithiophene previously shown to exert antitrypanosomal activity. BT-Ac displayed selective antiproliferative activity with IC50 for 28.9 and 50 mu M against promastigotes and amastigotes of Leishmania amazonensis at nontoxic concentrations for host cells, achieving a selectivity index of 4.69 against intracellular amastigotes. Electron microscopy revealed early morphological damage in promastigotes, including unprecedented changes at the plasma membrane and cytoskeletal interface, mitochondrial swelling, lipid body accumulation, and autophagic vacuole formation. Three-dimensional reconstructions further validated these findings, confirming that the ultrastructural alterations were intrinsic to BT-Ac treatment rather than technical artifacts. Biochemical analyses demonstrated reactive oxygen and nitrogen species overproduction, lipid peroxidation, and ATP fluctuations, indicating oxidative stress as a central mechanism. These events culminated in apoptosis-like and autophagic death in promastigotes, while amastigote death was primarily mediated by macrophage activation, evidenced by nitric oxide release. Together, our findings identify BT-Ac as a selective leishmanicidal agent that disrupts parasite homeostasis through oxidative damage, highlighting its potential as a lead compound for therapeutic strategies against leishmaniasis.
Abstract Pentamethine cyanine (Cy5) fluorophores are widely used in biological imaging but are often limited by rapid photobleaching under prolonged illumination and oxidative conditions. Here, we report a general strategy to enhance the photostability of pentamethine cyanines through electronic modulation of the indolenine scaffold. Specifically, we introduce electron-withdrawing substituents, including sulfonate groups on the benzene ring for aqueous solubility and difluoro- or trifluoroethyl groups at the N-alkyl position, to obtain H1 and H2. Importantly, the incorporation of these electron-withdrawing groups preserves the photophysical properties of the dyes while significantly reducing photooxidative degradation under continuous irradiation and in reactive oxygen and sulfur species-rich environments compared to commercial standards, including Alexa Fluor 647. Additionally, our strategy can be modulated to incorporate bioconjugation handles for biomolecule labeling. We demonstrate that bioconjugatable versions of H1 and H2 can be conjugated to antibodies (mAbs) at a range of labeling densities and retain their enhanced photostability. Overall, this work demonstrates that tuning the electronic properties of indolenine building blocks provides a general and effective approach to designing pentamethine cyanine fluorophores with improved photostability for advanced biological imaging applications.
The temperature distribution within cells, especially the debate on "how hot are mitochondria?", has recently attracted widespread attention. Several studies have reported that mitochondrial temperature may be 10-15 degrees C higher than the ambient temperature, which poses a significant challenge to the understanding of cellular thermal signaling and metabolic regulation mechanisms. Yet, other studies have raised concerns regarding the origin of the measurement signal. To address this controversy, we evaluated the impact of the intracellular environment on mitochondrial temperature measurement and found that microenvironmental factors such as viscosity can significantly interfere with the accuracy of traditional one-dimensional response curves, leading to temperature reading deviations. Here, we propose a universal multidimensional calibration framework that eliminates confounding factors by leveraging an information-matrix-based correction, without any modification to the probe material, enabling high-precision temperature reconstruction across complex environments. We investigated the dynamic temperature changes in different organelles within the cell under stimulation and observed a distinct temperature gradient within the cell. In our experiments, no mitochondrial temperature exceeding 50 degrees C, and the upper range was approximately 42-43 degrees C, which is consistent with the inactivation temperature of enzymes. Our results help to reassess the underlying logic of metrology and promote a better understanding on cellular thermodynamics.
Brine shrimp (Artemia sp.) are model organisms used in ecotoxicity studies and other important biological testing. These mesoscale (l & times; w & times; h = 100s - 1000s mu m) organisms mature rapidly and are ideal candidates for whole-organism imaging due to their ease of culture, definable anatomy, and well-documented development. We employ electrochemiluminescence (ECL) microscopy to examine both the chemical environment and morphology of these developing lower-level crustaceans on an electrified interface. We survey the tris(2,2 '-bipyridyl)ruthenium(II) ([Ru(bpy)3]2+) and luminol luminophores, highlighting important trade-offs across developmental stages. In the luminol system, positive ECL emission occurs from the cyst (egg) despite no addition of coreactant, thus pointing toward the potential location of reactive oxygen species in the local chemical environment near the cyst. We find that the kinetic regime (lower potentials) of the [Ru(bpy)3]2+ to be confined to the electrode surface and the Artemia. In contrast, in the mass-transfer-limited regime (higher potentials), shadow ECL dominates as Artemia acts as a blocking entity. Furthermore, we sum ECL micrographs and analyze the resulting composite images to evaluate trade-offs between temporal resolution and ECL intensity. Overall, this work establishes a foundation for mesoscale imaging of multicellular organisms and highlights the challenges and opportunities associated with ECL microscopy and luminophore choice.
Stimulated Raman scattering (SRS) as a nonparametric process is immune to the nonresonant background, but it is accompanied by other competing nonlinear effects, especially cross-phase modulation (XPM). Here, we present an XPM suppression method based on balanced detection of spatially segmented central and peripheral regions of the pump beam in stimulated Raman loss microscopy. This donut-shaped detection scheme harnesses the principle that the SRS signal in central and peripheral regions possesses the same phase as it measures vibrational absorption, whereas the XPM signal of the two regions shows an opposite phase as it measures energy redistribution induced by the refractive index change. By separately detecting the signals in the two regions and compensating with a scaling factor, XPM is effectively suppressed. This easy-to-implement method enhances SRS image contrast and spectral specificity by flattening the XPM contribution during hyperspectral imaging, enabling the extraction of a weak SRS signal from an overwhelming background.
Fluorescence-guided laparoscopic surgery has emerged as a transformative technology in minimally invasive surgery, fundamentally enhancing visualization and precision by providing real-time, high-contrast molecular information beyond traditional anatomical views. This paradigm shift is driven by synergistic innovations in two key areas: the rational design of target-specific fluorescent probes with improved optical properties and biorecognition capabilities, and significant advancements in imaging systems, including seamless hardware integration, multimodal data fusion, and AI-assisted image analysis. This comprehensive review critically summarizes the latest progress across this interdisciplinary spectrum, detailing breakthroughs in probe molecule engineering, technological evolution of imaging platforms, and expanding clinical applications in both oncologic and nononcologic fields. By synthesizing these developments and analyzing current challenges, this article outlines the trajectory of the field and underscores the integrated efforts required to translate optical precision into surgical outcomes and personalized patient care.
The urgent need to mitigate global energy and environmental crises underscores the importance of developing clean and sustainable energy technologies. Microbial electrochemical systems (MESs) have emerged as a promising platform, exploiting the ability of electroactive microorganisms to exchange electrons with electrodes through extracellular electron transfer (EET). While conventional studies have primarily focused on community-level processes, they often overlook the intrinsic heterogeneity and dynamic behaviors of individual cells that ultimately govern MES performance. To overcome these limitations, single-cell analytical approaches are being developed, though their application to EET studies remains in its infancy. This review provides a comprehensive summary of current single-cell microbial electrochemistry studying techniques, beginning with the fundamental mechanisms of EET and progressing to electrochemical microelectrode methods and optical imaging strategies. Particular attention is given to both label-free and probe-labeled imaging approaches, each offering distinct advantages. Finally, we briefly discuss the interplay between EET and microbial behaviors at single-cell scale. These insights highlight emerging methodologies for probing single-cell EET, their applications in uncovering microbial physiology and behaviors, and the future opportunities they open for advancing microbial electrochemistry and the rational design of MESs.
Electrochemiluminescence (ECL) imaging has progressed from a sensitive-intensity-based detection method to a powerful analytical platform capable of resolving biological and chemical heterogeneity at the single-entity level. This review maps the evolution of ECL imaging along three major axes: (i) spatiotemporal resolution, enabled by advanced luminophores, confined and built-in coreactant pathways, nanozyme catalysis, and 3D emissive-layer control; (ii) digitalization, in which analog luminescence is converted into discrete, statistically robust events for ultrasensitive quantification; and (iii) intelligent analysis, where artificial intelligence enhances denoising, emitter localization, dynamic tracking, multiplexed decoding, and kinetic inference. Mechanistic innovationssuch as nanoscale confinement, intramolecular coreactant design, in situ conversion of endogenous metabolites, and asymmetric nanostructuresprovide brighter, more localized, and biocompatible emission sources. Parallel advances in materials (metal nanoclusters, quantum dots, perovskites, AIE luminogens, Janus particles), imaging platforms (SECL/PECL modes, super-resolution strategies, microfluidics, smartphone/Raspberry-Pi devices, and large-scale bipolar-electrode arrays), and digital/AI pipelines collectively enable the high-fidelity capture of dynamic processes from single molecules to complex 3D spheroids. Together, these developments establish a coherent spatiotemporal-digital-intelligent framework and position ECL imaging as a versatile, scalable, and excitation-free modality for next-generation biosensing, clinical diagnostics, drug screening, environmental monitoring, and functional material characterization.
