Dechen Jiang

Electrochemiluminescence (ECL) microscopy is an emerging imaging technology that combines electrochemistry with optical microscopy. It operates without external light excitation, effectively eliminating photobleaching, phototoxicity, and scattered light noise, and thus enables high-contrast, low-background visualization of interfacial processes. This tutorial review first systematically outlines the instrumentation of ECL microscopy, covering the optical module such as objective lenses, cameras, and optical configurations, the electrochemical module (electrode architectures and electrolyte composition), as well as the software control for imaging acquisition, synchronization, and data analysis. Representative applications of ECL microscopy are then summarized in two major fields: bioanalysis, where imaging analysis of single cells and organelles, single biomolecules detection, biocoreactant-enhanced ECL imaging, and multiplexed high-throughput assays are highlighted; and single-particle catalysis, where activity mapping for noble metals, quantum dots, and single-atom catalysts is reviewed. Finally, we discuss current challenges and future directions of ECL microscopy, aiming to provide a comprehensive reference for advancing ECL microscopy toward broader applications in biomedicine, energy, and environment.

Background/Objectives: Human islet amyloid polypeptide (IAPP) aggregation plays a critical role in the pathogenesis of type 2 diabetes mellitus (T2DM). Although metabolic alterations are a hallmark of T2DM, the functional roles of differential metabolites in regulating disease-associated molecular processes remain largely unexplored. This study aimed to establish a metabolomics-guided strategy for identifying endogenous metabolites with anti-amyloid activity and to investigate their underlying chemical interactions with IAPP. Methods: Untargeted metabolomic profiling of clinical samples from T2DM patients, obesity patients and healthy controls was performed to identify differential metabolites. Candidate metabolites were subsequently screened for their ability to modulate IAPP aggregation. Transmission electron microscopy (TEM), thioflavin T (ThT) fluorescence assays, and cell viability measurements were employed to evaluate their effects on fibril formation and cytotoxicity. Mass spectrometry was further used to characterize metabolite–IAPP interactions. Results: Metabolomic analysis identified 3-hydroxypyruvic acid (also known as β-hydroxypyruvic acid, hereafter referred to as HPA) as a significantly altered endogenous metabolite associated with T2DM and a candidate regulator of IAPP aggregation. Functional assays demonstrated that HPA effectively inhibited amyloid fibril formation, as evidenced by the absence of typical fibrillar structures and a prolonged lag phase during aggregation. HPA also significantly alleviated IAPP-induced cytotoxicity. Mass spectrometric analysis revealed the formation of HPA–IAPP oligomer complexes (n < 4), suggesting that HPA directly interacts with early oligomeric intermediates and interferes with their progression toward mature fibrils. Conclusions: This work demonstrates that untargeted metabolomics of clinical samples can serve as an effective strategy for discovering bioactive endogenous metabolites involved in disease-related molecular processes. The identification of HPA as a potential endogenous inhibitor of IAPP aggregation provides new chemical insight into the relationship between metabolic dysregulation and amyloidogenesis and highlights endogenous metabolites as a valuable source of potential therapeutic lead compounds.

Here, an electrochemiluminescence (ECL) imaging technique in gel is established to visualize single ruthenium-labeled brain tissue sections using a coreactant (e.g., tripropylamine, TPA) filled agarose hydrogel, instead of the coreactant in solution. The confined space between the gel and the tissue section restricts the diffusion of intermediates during the ECL reaction, resulting in enhanced and localized ECL emission, thereby improving the microstructural resolution. Accordingly, spatial heterogeneity in the ECL signals on the mouse midbrain tissue sections was observed, which primarily results from the selective impact of intrinsic tissue structural differences on the local diffusion-limited microenvironment under gel confinement. Consequently, this gel-confined ECL imaging technique can directly translate the intrinsic biophysical differences in neuroanatomy into quantifiable imaging signals without relying on complex immunohistochemical staining or high-resolution optical systems. Furthermore, the imaging method in gel offers relatively good stability over days and repeated imaging. These advantages lay the groundwork for future long-term sample preservation and potential clinical applications.

Ferroptosis, driven by iron-dependent lipid peroxidation, represents a promising therapeutic strategy for hepatocellular carcinoma (HCC). However, reactive oxygen species (ROS) released from ferroptotic HCC cells may activate hepatic stellate cells (HSCs), potentially exacerbating liver fibrosis, an unexpected risk in HCC treatment. Resolving this rapid intercellular crosstalk requires methods that can track extracellular ROS release and key parameters of HSC activation (i.e., intracellular redox homeostasis and mitochondrial activity) with high spatiotemporal resolution. Here, we established an in vitro coculture model of HuH7 (HCC) and LX-2 (HSCs) cells and employed scanning electrochemical microscopy (SECM) to spatiotemporally resolve extracellular ROS fluxes from ferroptotic HuH7 cells, while subsequent intracellular ROS generation, mitochondrial respiratory activity, and intracellular temperature changes in LX-2 cells. We found that ROS released from ferroptotic HuH7 cells increased by ∼2.44 μM, and was temporally followed by NOX2-associated intracellular ROS bursts in LX-2 cells, with a maximum generation rate of approximately 5.44 × 10^-18 mol min^-1. This intracellular ROS burst was coupled to mitochondrial hypermetabolism in LX-2 cells, reflected by a ∼1.27-fold elevation in oxygen consumption and a ∼1.65 K rise in intracellular temperature. Finally, we also found that pirfenidone, an antifibrotic agent, could effectively suppress this ferroptosis-induced HSC activation. Our study introduces an SECM approach providing real-time single-cell evidence that ferroptotic HCC cells can trigger ROS-mediated activation of neighboring HSCs, and supports further evaluation of antifibrotic combination strategies for safer ferroptosis-based HCC therapy.

Galvanic replacement (GR) at solid-liquid interfaces enables nanoscale synthesis, yet its initiation and kinetics remain unclear. Here, scanning ion conductance microscopy enables noncontact, correlative mapping of morphology and apparent stiffness during Ag-Pt GR at single nanowires. We identify a mechanically defined initiation stage in which pronounced softening precedes detectable morphological change, establishing mechanical response as an early kinetic reporter. Quantitative extraction of effective mechanical rate constants provides a kinetic descriptor inaccessible to morphology alone. Stage-resolved maps further reveal nonmonotonic morphology-mechanics coupling and asynchronous activation of discrete surface sites that nevertheless follow a conserved sequence of mechanical and geometric transitions. This correlative approach mitigates morphology-only bias and makes otherwise hidden kinetic stages accessible. This strategy provides a general framework for tracking heterogeneous interfacial reactions, supporting nanoscale interpretation and control of reaction pathways.

Existing cellular heterogeneity necessitates continuous analysis in a single cell to capture dynamic information. However, current mass spectrometry-based single-cell metabolomics analysis is a destructive technique and cannot perform multiple samplings from the same cell, which complicates the accurate tracking of metabolite level alterations. In this study, we present a minimally invasive single-cell mass spectrometry technique that enables sampling of less than 10% of the total cell volume per iteration by using nanocapillaries. This minimally invasive process allows for repeated sampling from a single cell without compromising its essential physiological functions. The resulting time-resolved metabolomics analysis provides the alteration in metabolite levels in a single cell in a process of erastin-induced ferroptosis. As compared to the metabolite levels at certain time points, the alteration in metabolite levels exhibits a stronger correlation with the extent of mitochondrial lipid peroxidation. More importantly, by mapping the differentiation trajectory of individual cells during this process, two distinct groups─proliferating and quiescent cells─are identified. These two groups of cells exhibit different sensitivities to ferroptosis, with quiescent cells being more susceptible to ferroptosis, which could only be tracked by using the time-resolved single-cell metabolomics analysis. Ultimately, this technique overcomes the challenge in single-cell dynamic studies posed by cellular heterogeneity, providing more accurate biological insights into the diversity and complexity of cellular metabolic activities.