ElectrochemiLuminescence nano-Imaging of Single Entities – ELISE

Electrochemiluminescence (ECL), also known as “electrogenerated chemiluminescence”, can be defined as the production of light following an initial electrochemical reaction on the surface of an electrode. It combines electrochemistry and photochemistry. At present, the method is based mainly on the reaction of a phosphor and a sacrificial co-reactant in aqueous electrolyte solutions. The excited state of the phosphor is generated by a highly exergonic electron transfer reaction between electrogenerated species. Subsequently, the phosphor returns to its ground state by emitting a photon (?ECL). The most efficient and widely used ECL system is composed of the tris(bipyridine) ruthenium(II) phosphor ([Ru(bpy)3]2+) and the sacrificial coreactant tri-n-propylamine (TPrA), which produces red light. The ECL light emission process does not require incident light to generate the excited state. ECL is characterized by a luminous signal with virtually no background noise, avoiding the effects of photobleaching, autofluorescence or phototoxicity. ECL is mainly used in (bio)analytical applications. This is also due to its remarkable intrinsic properties: high sensitivity, signal linearity, selectivity, in situ generation of reagents.
Imaging single nanosized objects or domains by ECL still constituted a major challenge in the field as well as the ultimate single molecule level imaging. Probably this is because it requires the joint efforts of experts at least from ECL and Super-Localization Microscopy (SLM) at the single object to single molecule level. Additional central challenges to combine ECL with SLM are the optimization of the ECL intensity, potentially at different wavelengths, and the precise control of the thickness of the ECL-emitting layer, which is confined at the electrode surface. The reactivity of the model ECL systems (typically, [Ru(bpy)3]2+ luminophore and tripropylamine, TPA, as a coreactant) is a very active research area. Finally, there is an urgent demand for analytical approaches that enable precise quantification of the transport of biologically active compounds across cellular membranes. Since ECL is based on an initial electrochemical step, the transport of the ECL reagents or biomolecules through membranes may allow to monitor membrane permeation kinetics. To approach the performances of fluorescence microscopies used in most biological studies, ECL needs to demonstrate single biological nanodomain (down to single protein/molecule) level imaging. In this context, ELISE aimed to combine ECL with SLM in order to develop novel ultrasensitive ECL-based nano-imaging methods for the analysis of single entities.

The project was based on a three-pronged approach: mechanistic study of ECL, instrumental and methodological development, and bio-imaging applications. The detailed understanding of ECL mechanisms achieved in collaboration with ITODYS has enabled us to amplify the ECL signal at the level of functionalized beads for immunoassay, using iridium complexes dissolved in solution. These studies have paved the way for new collaborations on the study of organometallic luminophores involving ruthenium and iridium complexes and their cross-ECL reactivities. This has enabled us to carry out bimodal imaging of cells using positive and negative ECL.
In collaboration with the Institut de la Vision, we investigated the central question of the minimum ECL luminophore concentration required to image single entities. We have demonstrated the possibility of recording ECL images of single cells and mitochondria at concentrations in the nM and pM range. These concentrations are 7 orders of magnitude lower than those conventionally used, and correspond to a few hundred luminophores scattering around the biological entities. The approach described is a simple, rapid and highly sensitive method, opening up new avenues for ultrasensitive ECL imaging and ECL reactivity at the single-molecule level. The latter has been demonstrated at the level of cell membranes. Micalis has carried out protein analysis of the Campylobacter jejuni total secretome to correlate ECL imaging of extracellular vesicles released by the bacterium with their composition.

Work on the ELISE project is continuing. However, we can identify two main directions for the perspectives opened up by this project. The first concerns the amplification of the ECL signal, which has been made possible by a detailed understanding of the ECL mechanism, in particular by studying the addition of a redox mediator in solution. The second concerns the development of different variants of ECL microscopy, providing complementary information to more conventional fluorescence microscopy.

The results of the ELISE project have been published in the following articles:

Truchet, S. et al. (2025). Visualization of the Biogenesis, Dynamics, and Host Interactions of Bacterial Extracellular Vesicles. Chem. Biomed. Imaging, DOI : 10.1021/cbmi.5c00002

Kneževic, S, et al. (2024). Electrochemiluminescence Microscopy. Angew. Chem. Int. Ed., e202407588

Kneževic, S, et al. (2024). Enhanced electrochemiluminescence at the gas/liquid interface of bubbles propelled into solution. J. Am. Chem. Soc., 146, 22724

Kneževic, S, et al. (2024). Electrocatalytic amplification of coreactant electrochemiluminescence using redox mediators. Electrochimica Acta, 499, 144677.

Adamson, N. S. et al (2024). Electrochemiluminescence Enhanced by a Non-Emissive Dual Redox Mediator. Angew. Chem. Int. Ed., 63, e202412097

Descamps, J, et al. (2023) Ultrasensitive imaging of cells and sub-cellular entities by electrochemiluminescence. Angew. Chem. Int. Ed. (135) e202218574

Kneževic, S, et al. (2023) Bimodal electrochemiluminescence microscopy of single cells. Anal. Chem. (95) 7372–7378

Kerr, E, et al. (2023) Electrochemiluminescence amplification in bead-based assays induced by a freely diffusing iridium(III) complex. ACS Sensors (8) 933–939

Kneževic, S, et al. (2022). Electrochemiluminescence microscopy: from single objects to living cells. Current Opinion in Electrochemistry, 35, 101096

Liu, Y, et al. (2021) Single biomolecule imaging by electrochemiluminescence. J. Am. Chem. Soc. (143) 17910–17914

Electrochemiluminescence (ECL) is the light emitted by the excited state of a luminophore upon an initial electrochemical reaction. It is a hybrid technique combining orthogonal modalities, in which electrochemical stimulation is coupled to optical detection. ECL is a powerful analytical technique, which is already appealing for medical diagnosis and, increasingly, for imaging. Our proposal aims to combine ECL with superlocalization microscopy for the 3D tracking of individual electrochemical or biological objects, and ultimately single molecules. We propose an original methodology to investigate the behavior of single entities in electrochemistry and biology using a nanometer-range precision optical readout, with all the advantages of a photoexcitation-free approach. In a first part, we will study the ECL mechanisms in order to improve the control of the reactivity of the process and tune the spatial distribution of the ECL-emitting layer in the vicinity of the electrode surface. This mechanistic insight, combined to optimized resonance energy transfer processes and the complete toolbox of molecular biology, will lead to an enhancement of ECL emission, thus allowing the ECL visualization of specific cell organelles. The development of original optical amplitude-and-phase imaging technique will enable the study of single nano-objects, biological entities and ECL-emitting molecules with unprecedented precision. The partners of the ELISE project gather complementary knowledge and recognized expertise in ECL, electrochemistry, mechanistic simulations, nano-imaging, molecular biology, and microbiology.