Electrochemiluminescence Generation Research Articles

Bipolar electrochemical sensor with perylene diimide-based cathodic luminophore for dopamine detection and imaging

Bipolar electrochemical microscopy (BEM), which visualizes the concentration distribution of molecular species in biological systems by electrochemiluminescence (ECL), is expected to be applied to the high-spatiotemporal-resolution imaging of biomolecules, enabling the analysis of cellular functions. In the past, the molecular species that could be imaged by BEM were generally restricted to oxidized molecules due to the limitation derived from the ECL mechanism of the luminophore. Recently, the imaging of dopamine (DA), a reduced molecule, was achieved using Ru (bpy)32+/glutathione disulfide (GSSG) as a cathodic luminophore. However, a large driving voltage was required for ECL generation, resulting in a low S/N ratio. In this study, we employed N,N′-dimethyl-3,4,9,10-perylenetetracarboxylic diimide (PDI-CH3)/potassium peroxodisulfate (K2S2O8), which is a cathodic luminophore that can be reduced at a nobler potential to produce ECL than [Ru(bpy)3]2+/GSSG. First, the ECL mechanism of PDI-CH3/K2S2O8 was elucidated by using a PDI-CH3 drop-cast glassy carbon electrode (GCE) immersed in K2S2O8 solution as the working electrode in a 3-electrode system. The PDI-CH3 drop-casted GCE, a single closed bipolar electrode (c-BPE), was used as the cathode in the successful quantification of 50–500 μmol L−1 DA in a sample chamber in which a c-BPE anode was immersed, resulting in a high S/N. The selective detection of DA in the presence of ascorbic acid was achieved by modifying the anode with Nafion. Finally, DA imaging was demonstrated using a commercially available anisotropic conducting film with PDI-CH3 coating on the cathode surface as a c-BPE array. The change in the concentration distribution in the inflow of DA was successfully imaged based on the change in the ECL intensity at the c-BPE cathode. This BEM system is expected to be useful for DA imaging of the brain.

Bright luminol electrochemiluminescence mediated by a simple TEMPO radical for visualized multiplex detection

In this work, a series of 2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPO) radicals bearing different functional groups were exploited as a simple catalyst to promote electrochemiluminescence (ECL) generation in luminol/H2O2 system. These TEMPO radicals were found to facilitate the electrochemical oxidation of H2O2 and luminol through different catalytic mechanisms, as well as the subsequent ECL generation of luminol/H2O2 system. The electrochemical oxidation and luminol ECL generation could be tuned by the functional group on the para-position of TEMPO, for which the structure/activity relationship was revealed. Finally, with the combination of enzymatic system, luminol ECL enhancement up to 9.6-fold was obtained through the catalysis of 4-hydroxyl-TEMPO. The enhanced luminol ECL allows acquiring brighter ECL images in a single-electrochemical system (SEES) for multiplex detection of cholesterol, H2O2 and glucose.

Sensitive detection of uric acid based on low-triggering-potential cathodic luminol electrochemiluminescence achieved by ReS2 nanosheets.

The majority of previously reported cathodic electrochemiluminescence (ECL) systems often required very negative potential to be carried out, which hasgreatly limited their applications in the sensing field. Screening high-performance cathodic ECL systems with low triggering potential is a promising way to broaden their applications. In this work, rhenium disulfide nanosheets (ReS2 NS) have been revealed as an efficient co-promoter to realize low-triggering-potential cathodic luminol ECL. One strong cathodic ECL signal appeared at a potential of -0.3V and one anodic ECL peak was obtained at -0.15V under the reverse potential scan, which were caused by electrogenerated reactive oxygen species (ROS) from hydrogen peroxide. The generation of strong luminol ECL at low potential was the result of the electrocatalytic effect of ReS2 NS on the reduction of H2O2. The scavenging effect of uric acid (UA) on the ROS could significantly inhibit the cathodic ECL. As a result, an ECL sensor was proposed, which showed outstanding performance for the detection of UA in the range of 10nM to 0.1mM with a low detection limit of 1.53nM. Moreover, the ECL sensor was successfully applied in the sensitive detection of UA in real samples. This work provides anew avenue to establish a low-potential cathodic ECL system, which will sufficiently expand the potential application of cathodic ECL in the sensing field.

Electrocatalytic Effects of Au Nanoclusters in Its Electrochemiluminescence Enhanced by Coreactant Tertiary Amines

AbstractAtomically precise metal nanoclusters are increasingly exploited in electrocatalysis such as water electrolysis and CO2 reduction. The catalytic effects are generally evaluated by conventional electrochemical methods or end‐product analysis. Herein, near infrared electrochemiluminescence (ECL) of the nanoclusters is introduced as a new signaling mechanism to complement classical voltammetric analysis for elucidating essential electrocatalytic parameters. Coreactant ECL from an aqueous soluble Au22 nanocluster (NCs) enhanced by a common pH buffer materials 4‐(2‐hydroxyethyl)‐1‐piperazineethanesulfonic acid (HEPES) and by a piperazine drug hydroxyzine (HDZ) is studied under oxidative reduction pathways. Foot of the wave analysis on voltametric features and the kinetics profiles in potential‐step experiments are studied in different nanocluster and coreactant concentrations. Benchmark parameters such as rate constants are determined under EC (Electron‐transfer‐Chemical‐reaction) mechanism as feasibility validations. Because the luminescence properties of AuNCs are sensitive to the surface ligands or adsorbents, ECL is proposed as a new signal readout for operando studies providing insights for governing reaction mechanism and kinetics during catalytic reactions. Retrospectively, mechanistic understanding of the complex multi‐step reactions during ECL generation can provide guidance for the evaluation and optimization of ECL property for better quantitative applications.

Unraveling Mechanisms of Highly Efficient Yet Stable Electrochemiluminescence from Quantum Dots.

With CdSe/CdS/ZnS core/shell/shell quantum dots (QDs) as the model system, time- and potential-resolved spectroelectrochemical measurements are successfully applied for studying the general mechanisms and kinetics of electrochemiluminescence (ECL) generation. The rate constant of electron injection from the cathode into a QD to form a negatively charged QD (QD-) increases monotonically from -0.88 V to -1.2 V (vs Ag/AgCl). Mainly due to the deep LUMO of the QDs, the resulting QD- as the key intermediate for ECL generation is structurally stable and possesses very slow spontaneous deionization channels. The latter (the main non-ECL channels) are usually 3-4 orders of magnitude slower than the rate constant of the successive hole injection from an active co-reactant into a QD-. The kinetic studies quantify the internal ECL quantum yield of ideal QD ECL emitters to be nearly identical to that of photoluminescence, which is near unity for the current system. Identification of the key intermediate, discovery of the related elementary steps, and determination of all rate constants not only establish a general framework for understanding ECL generation but also offer basic design rules for ECL emitters.

Bright dual‐color electrochemiluminescence of a structurally determined Pt1Ag18 nanocluster

AbstractMetal nanoclusters possess excellent electrochemical, optical, and catalytic properties, but correlating these properties remains challenging, which is the foundation to generate electrochemiluminescence (ECL). Herein, we report for the first time that a structurally determined Pt1Ag18 nanocluster generates intense ECL and simultaneously enhances the ECL of carbon dots (CDs) via an electrocatalytic effect. Pt1Ag18 nanocluster show aggregation‐induced emission enhancement and aggregation‐induced ECL enhancement under light and electrochemical stimulation, respectively. In the presence of tripropylamine (TPrA) as a coreactant, solid Pt1Ag18 shows unprecedented ECL efficiency, which is more than nine times higher than that of 1 mM Ru(bpy)32+ with the same TPrA concentration. Potential‐resolved ECL spectra reveal two ECL emission bands in the presence of TPrA. The ECL emission centered at 650 nm is assigned to the solid Pt1Ag18 nanocluster, consistent with the peak wavelength in self‐annihilation ECL and photoluminescence of the solid state. The ECL emission centered at 820 nm is assigned to the CDs on the glassy carbon electrode. The electrocatalytic effect of the nanoclusters enhanced the ECL of the CDs by a factor of more than 180 in comparison to that without nanoclusters. Based on the combined optical and electrochemical results, the ECL generation pathways and mechanisms of Pt1Ag18 and CDs are proposed. These findings are extremely promising for designing multifunctional nanocluster luminophores with strong emissions and developing ratiometric sensing devices.

Gold Microbeads Enabled Proximity Electrochemiluminescence for Highly Sensitive and Size-Encoded Multiplex Immunoassays.

Developing highly sensitive multiplex immunoassays is urgently needed to guide medical research and improve clinical diagnosis. Here, we report the proximity electrochemiluminescence (ECL) generation enabled by gold microbeads (GMBs) for improving the detection sensitivity and multiplexing capacity of ECL immunoassays (ECLIAs). As demonstrated by microscopy and finite element simulation, GMBs can function as spherical ultramicroelectrodes for triggering ECL reactions in solutions. Employing GMBs as solid carriers in the bead-based ECLIA, the electrochemical oxidation of a coreactant can occur at both the GMB surface and the substrate electrode, allowing the coreactant radicals to diffuse only a short distance of ∼100 nm to react with ECL luminophores that are labeled on the GMB surface. The ECL generation via this proximity low oxidation potential (LOP) route results in a 21.7-fold increase in the turnover frequency of ECL generation compared with the non-conductive microbeads that rely exclusively on the conventional LOP route. Moreover, the proximity ECL generation is not restricted by the diffusion distance of short-lived coreactant radicals, which enables the simultaneous determination of multiple acute myocardial infarction biomarkers using size-encoded GMB-based multiplex ECLIAs. This work brings new insight into the understanding of ECL mechanisms and may advance the practical use of multiplex ECLIAs.

Energy Level Engineering in Gold Nanoclusters for Exceptionally Bright NIR Electrochemiluminescence at a Low Trigger Potential.

Electrochemiluminescence (ECL) is a widely used light output mechanism from electrochemical excitation. Understanding the intrinsic essence for ideal ECL generation remains a fundamental challenge. Here, based on the molecular orbital theory, we reported an energy level engineering strategy to regulate the ECL performance by using ligand-protected gold nanoclusters (AuNCs) as luminophores and N,N-diisopropylethylamine (DIPEA) as a coreactant. The energy level matching between the AuNCs and DIPEA effectively promoted their electron transfer reactions, thus improving the excitation efficiency and reducing the trigger potential. Simultaneously, the narrow band gap of the AuNCs further enabled enhanced emission efficiency. Using the energy level engineering theory developed here, a dual-enhanced strategy was proposed, and β-CD-AuNCs were designed to further verify this mechanism. The β-CD-AuNCs/DIPEA system resulted in highly stable near-infrared ECL with an unprecedented ECL efficiency (145-fold higher than that of the classic Ru(bpy)32+/tetra-n-butylammonium perchlorate system) and a low trigger potential of 0.48 V. A visual NIR-ECL based on this ECL system was successfully realized by an infrared camera. This work provides an original mechanistic understanding for designing efficient ECL systems, which promises to be a harbinger for broad applicability of this strategy for other ECL systems and ECL sensing platforms.

High electrochemical active Au-NP/2D zinc-metal organic frameworks heterostructure-based ECL sensor for the miRNA-522 detection in triple negative breast cancer

In this work, a novel electrochemiluminescence (ECL) sensor has been developed to detect the miRNA-522 in the tumor tissues of triple-negative breast cancer (TNBC) patients. Au NPs/Zn MOF heterostructure was obtained by in situ growth and used as novel luminescence probe. Firstly, zinc-metal organic framework nanosheets (Zn MOF NSs) were synthesized with Zn2+ as the central metal ion and 2-aminoterephthalic acid (NH2-BDC) as the ligand. 2D MOF nanosheets with ultra-thin layered structure and relatively large specific surface areas can enhance the catalytic activity in the ECL generation. Furthermore, the electron transfer capacity and the electrochemical active surface area of MOF were greatly improved by the growth of Au NPs. Therefore, Au NPs/Zn MOF heterostructure showed the significant electrochemical activity in the sensing process. In addition, the magnetic Fe3O4@SiO2@Au microspheres were used as capture units in the magnetic separation step. The magnetic spheres with hairpin aptamer H1 can capture target gene. Then the captured miRNA-522 triggered the target catalyzed hairpin assembly (CHA) sensing process and linked Au NPs/Zn MOF heterostructure. The concentration of miRNA-522 can be quantified by the ECL signal enhancement of the Au NPs/Zn MOF heterostructure. Due to the high catalytic activity of Au NPs/Zn MOF heterostructure and their unique structural and electrochemical properties, the prepared ECL sensor achieved high-sensitive detection of miRNA-522 in the range of 1 fM to 0.1 nM with the detection limit of 0.3 fM. This strategy can provide a potential alternative for miRNA detection in medical research and clinical diagnosis of triple negative breast cancer.

High-Density N-Vacancy-Induced Multipath Electrochemiluminescence Improvement of 3D g-C3N4 for Ultrasensitive MiRNA-222 Analysis

Using dissolved O2 as the cathodic co-reactant of three-dimensional (3D) g-C3N4 is a convenient method to improve the electrochemiluminescence (ECL) signal, but it still suffers the disadvantages of limited luminous efficiency of 3D g-C3N4 and low content, low reactivity, and instability of dissolved O2. Here, N vacancy with high density was first introduced into the structure of 3D g-C3N4 (3D g-C3N4-NV), which could conveniently realize multipath ECL improvement by simultaneously solving the above shortcomings effectively. Specifically, N vacancy could change the electronic structure of 3D g-C3N4 to broaden its band gap, increase fluorescence (FL) lifetime, and accelerate electron transfer rate, obviously improving the luminous efficiency of 3D g-C3N4. Meanwhile, N vacancy made the excitation potential of 3D g-C3N4-NV to shift from -1.3 to -0.6 V, effectively weakening the electrode passivation. Moreover, the adsorption capacity of 3D g-C3N4-NV was obviously enhanced, which could make the dissolved O2 enrich around 3D g-C3N4-NV. And massive active NV sites of 3D g-C3N4-NV could promote O2 to more efficiently convert to reactive oxygen species (ROS) that were key intermediates in ECL generation. Using the newly proposed 3D g-C3N4-NV-dissolved O2 system as an ECL emitter, an ultrasensitive target conversion biosensor was constructed for miRNA-222 detection. The fabricated ECL biosensor exhibited satisfactory analytical performance for miRNA-222 with a detection limit of 16.6 aM. The strategy achieved multipath ECL improvement by introducing high-density N vacancy simply in the 3D structure of g-C3N4 and could open a new horizon for developing a high-performance ECL system.

Electrochemiluminescence Distance and Reactivity of Coreactants Determine the Sensitivity of Bead-Based Immunoassays.

Herein we report the study of electrochemiluminescence (ECL) generation by tris(2,2'-bipyridyl)ruthenium (Ru(bpy)3 2+ ) and five tertiary amine coreactants. The ECL distance and lifetime of coreactant radical cations were measured by ECL self-interference spectroscopy. And the reactivity of coreactants was quantitatively evaluated in terms of integrated ECL intensity. By statistical analysis of ECL images of single Ru(bpy)3 2+ -labeled microbeads, we propose that ECL distance and reactivity of coreactant codetermine the emission intensity and thus the sensitivity of immunoassay. 2,2-bis(hydroxymethyl)-2,2',2''-nitrilotriethanol (BIS-TRIS) can well balance ECL distance-reactivity trade-off and enhance the sensitivity by 236 % compared with tri-n-propylamine (TPrA) in the bead-based immunoassay of carcinoembryonic antigen. The study brings an insightful understanding of ECL generation in bead-based immunoassay and a way of maximizing the analytical sensitivity from the aspect of coreactant.

Electrochemiluminescence Distance and Reactivity of Coreactants Determine the Sensitivity of Bead‐Based Immunoassays

AbstractHerein we report the study of electrochemiluminescence (ECL) generation by tris(2,2′‐bipyridyl)ruthenium (Ru(bpy)32+) and five tertiary amine coreactants. The ECL distance and lifetime of coreactant radical cations were measured by ECL self‐interference spectroscopy. And the reactivity of coreactants was quantitatively evaluated in terms of integrated ECL intensity. By statistical analysis of ECL images of single Ru(bpy)32+‐labeled microbeads, we propose that ECL distance and reactivity of coreactant codetermine the emission intensity and thus the sensitivity of immunoassay. 2,2‐bis(hydroxymethyl)‐2,2′,2′′‐nitrilotriethanol (BIS‐TRIS) can well balance ECL distance‐reactivity trade‐off and enhance the sensitivity by 236 % compared with tri‐n‐propylamine (TPrA) in the bead‐based immunoassay of carcinoembryonic antigen. The study brings an insightful understanding of ECL generation in bead‐based immunoassay and a way of maximizing the analytical sensitivity from the aspect of coreactant.

Review: Electrochemiluminescence of Perovskite-Related Nanostructures

Perovskite nanostructures are promising nanomaterials for their possible application in electrochemiluminescent (ECL) analytical systems due to their unique optical, electronic, and chemical properties. This review focuses on the most recent advances in the application of perovskite and perovskite-related nanostructures, with different chemical compositions and modifications, in ECL with various media, coreactants, and reaction types. The most optimal methods of perovskite nanoparticle synthesis and electrode modification methods were reviewed. Possibilities and perspectives of the use of perovskite-related nanostructures for the ECL generation were demonstrated.

Efficient annihilation electrochemiluminescence of [Ru(bpy)3]2+ within a deep eutectic solvent as green electrolyte

The generation of annihilation electrochemiluminescence (ECL) is typically limited to highly purified organic solvent-supporting electrolyte solutions or ionic liquids. Herein, we have prepared a deep eutectic solvent (DES) based on choline chloride (ChCl) and lactic acid (LA), which was directly used to investigate the annihilation ECL of [Ru(bpy)3]2+ for the first time. [Ru(bpy)3]2+ could be oxidized to stable [Ru(bpy)3]3+ radicals, while reduced to unstable [Ru(bpy)3]+ radicals in ChCl-LA DES. Moreover, [Ru(bpy)3]3+ was sufficiently stable on the time scale to react with [Ru(bpy)3]+ in ChCl-LA DES, resulting in the generation of intense annihilation ECL by a potential scanning or pulsing. This annihilation ECL system was further integrated into chitosan (CS) for the formation of homogenous and microporous ChCl-LA DES/CS/[Ru(bpy)3]2+ ionic gel, which was used as the emitting layer in ECL devices (ECLDs). The display performances for the ECLDs were demonstrated based on the efficient annihilation ECL in the ionic gel, giving bright light-emission visible to naked eye. Hence, this work confirmed the possibilities for studying annihilation ECL and its application in ECLDs using green DESs and CS materials.

Valence-State-Engineered Electrochemiluminescence from Au Nanoclusters.

To determine the intrinsic effects of body elements on the electrochemiluminescence (ECL) of metal nanoclusters (NCs), herein, a valence-state engineering strategy is developed to adjust the NCs' ECL with bovine serum albumin (BSA)-stabilized AuNCs as a model, in which engineering the valence state of the Au body element, i.e., Au(0) and Au(I), is performed via successively reducing the precursor AuCl4- to Au(I) and Au(0) with BSA. The obtained BSA-AuNCs/N2H4 system leads to three anodic ECL processes at 0.37 (ECL-1), 0.85 (ECL-2), and 1.45 V (ECL-3). ECL-1 is generated from the BSA-Au(0) section of BSA-AuNCs in a surface-defect-involved route and is much stronger and red-shifted compared to ECL-2 and ECL-3, which are generated from the BSA-Au(I) section of BSA-AuNCs in the band-gap-engineered route. Each of the anodic ECL processes can be selectively generated and/or suppressed via adjusting the Au(I)/Au(0) ratio of BSA-AuNCs, tunable ECL generation route, and triggering potential, and the emission intensity and waveband of metal NCs are conveniently achieved in body-element-involved valence-state engineering.

Toward mechanistic understanding of electrochemiluminescence generation by tris(2,2′-bipyridyl)ruthenium(II) and peroxydisulfate

Electrochemiluminescence (ECL) generation by tris(2,2’-bipyridyl)ruthenium (Ru(bpy)32+) and S2O82− is one of most frequently studied cathodic systems. However, the reaction mechanism is yet to be fully resolved, since the reaction involving highly reactive intermediates occurs at the negative potential limit of aqueous solutions. In this work we used bismuth microwire (BiMW) as the working electrode and ECL microscopy (ECLM) as the imaging tool to study the mechanism of cathodic ECL generation by Ru(bpy)32+/S2O82−. Thanks to its high hydrogen evolution overpotential, BiMW electrode could provide us with a wide negative potential window in aqueous solutions. While ECLM allowed us to determine the thickness of ECL layer (TEL), which was found to vary from ca. 3.4 μm to 3.0 μm with increasing the concentration of S2O82−. Given the chemical reduction of S2O82− by Ru(bpy)3+ is essential for extending the concentration profile of SO4•−, the slightly changed TEL is likely related to the instability of Ru(bpy)3+ in aqueous solutions. Finally, no ECL was detected for Ru(bpy)32+-labelled microbeads, implying that S2O82− could not work as the cathodic coreactant like tri-n-propylamine (TPrA) as the anodic one in bead-based immunoassays.

Interparticle Charge-Transport-Enhanced Electrochemiluminescence of Quantum-Dot Aerogels.

Electrochemiluminescence (ECL) represents a widely explored technique to generate light, in which the emission intensity relies critically on the charge-transfer reactions between electrogenerated radicals. Two types of charge-transfer mechanisms have been postulated for ECL generation, but the manipulation and effective probing of these routes remain a fundamental challenge. Here, we demonstrate the design of quantum dot (QD) aerogels as novel ECL luminophores via a versatile water-induced gelation strategy. The strong electronic coupling between adjacent QDs enables efficient charge transport within the aerogel network, leading to the generation of highly efficient ECL based on the selectively improved interparticle charge-transfer route. This mechanism is further verified by designing CdSe-CdTe mixed QD aerogels, where the two mechanistic routes are clearly decoupled for ECL generation. We anticipate our work will advance the fundamental understanding of ECL and prove useful for designing next-generation QD-based devices.

Arginine-modified black phosphorus quantum dots with dual excited states for enhanced electrochemiluminescence in bioanalysis

The electrochemiluminescence (ECL) is generally emitted via radiative transition of singlet or triplet excited state (S1 or T1). Herein, an ECL mechanism with the transitions of both S1 and T1 of black phosphorus quantum dots (BPQDs) is found, and an arginine (Arg) modification strategy is proposed to passivate the surface oxidation defects of BPQDs, which could modulate the excited states for enhancing the ECL efficiency of BPQDs. The Arg modification leads to greater spatial overlap of highest and lowest occupied molecular orbitals, and spectral shift of radiative transitions, and improves the stability of anion radical of BPQDs. To verify the application of the proposed mechanism, it is used to construct a sensitive method for conveniently evaluating the inhibiting efficiency of cyclo-arginine-glycine-aspartic acid-d-tyrosine-lysine to cell surface integrin by using Arg containing peptide modified BPQDs as signal tag. The dual excited states mediated ECL emitters provide a paradigm for adjustable ECL generation and extend the application of ECL analysis.

Benzothioxanthene Dicarboximide as a Tuneable Electrogenerated Chemiluminescence Dye

AbstractOverlooked for years, the promising benzothioxanthene dicarboximide (BTXI) structure is nonetheless known to be an excellent fluorophore. As a further step towards the exploration of this rylene structure, the generation of electrochemiluminescence (ECL) in acetonitrile solutions is investigated herein as well as its direct comparison with a diphenylamine (DPA) derivative. Electrochemical characterization shows that the bare BTXI can be reversibly reduced or oxidized, characteristics which suggest the possibility that the electrogenerated reactive species can be exploited in the context of ECL. As a result, the BTXI moiety was found to be ECL‐active in both annihilation and co‐reactant mechanisms. Tri‐n‐propylamine (TPA) and benzoyl peroxide (BPO) were selected as sacrificial co‐reactants for promoting ECL. An ECL efficiency of 13 % (BTXI) and 6 % (BTXI‐DPA) compared to that of Ru(bpy)32+ was found when using TPA in oxidative ECL. Moreover, the ECL efficiency increased to 75 % and 55 % of Ru(bpy)32+ with BPO (i. e. reductive‐oxidation ECL) for BTXI and BTXI‐DPA, respectively. These results can be understood by calculating the free enthalpy of the redox reactions associated with each ECL pathway. Although the BTXI‐DPA red emitter exhibits more suitable thermodynamic properties, the BTXI green emitter is nonetheless more effective in combination with a co‐reactant due to a much larger photoluminescence quantum yield. The ECL spectra of the two molecules are compared to their photoluminescence spectra, revealing the involvement of the same excited states in the two different photoemission processes. In turn, the chemical engineering of BTXI is shown to be an effective platform for achieving tuneable ECL generation.

Electrochemiluminescence microscopy: From single objects to living cells

Electrochemiluminescence (ECL) is a light-emitting phenomenon triggered by electrochemistry. The resulting optical signal enables spatiotemporal resolution of the luminescent signal that can be imaged with high accuracy. The field of ECL is evolving rapidly from an analytical method widely applied in biosensing to a novel microscopy technique. The efficient generation of ECL depends on the electrode materials, the luminophore/coreactant systems, and experimental configurations, focusing research on improving these parameters. Nowadays, ECL enables the analysis of transient events and micro/nanometric objects, surface boundaries, interfaces, and physical phenomena, as well as catalytic and biological processes. Since ECL microscopy offers many novel applications, this article will describe first the context and then the most recent advances in ECL imaging of single objects and living cells or subcellular structures.