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Direct imaging of single-molecule electrochemical reactions in solution

Abstract

Chemical reactions tend to be conceptualized in terms of individual molecules transforming into products, but are usually observed in experiments that probe the average behaviour of the ensemble. Single-molecule methods move beyond ensemble averages and reveal the statistical distribution of reaction positions, pathways and dynamics1,2,3. This has been shown with optical traps and scanning probe microscopy manipulating and observing individual reactions at defined locations with high spatial resolution4,5, and with modern optical methods using ultrasensitive photodetectors3,6,7 that enable high-throughput single-molecule measurements. However, effective probing of single-molecule solution chemistry remains challenging. Here we demonstrate optical imaging of single-molecule electrochemical reactions7 in aqueous solution and its use for super-resolution microscopy. The method utilizes a chemiluminescent reaction involving a ruthenium complex electrochemically generated at an electrode8, which ensures minimal background signal. This allows us to directly capture single photons of the electrochemiluminescence of individual reactions, and to develop super-resolved electrochemiluminescence microscopy for imaging the adhesion dynamics of live cells with high spatiotemporal resolution. We anticipate that our method will advance the fundamental understanding of electrochemical reactions and prove useful for bioassays and cell-imaging applications.

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Fig. 1: Single-molecule ECL imaging setup and observation of stochastic reactions.
Fig. 2: Observation of single-molecule reactions at different exposure times.
Fig. 3: Kinetic analysis of single-molecule ECL reactions.
Fig. 4: Single-molecule ECL imaging of ITO structures.
Fig. 5: Single-molecule ECL imaging of live cells.

Data availability

The data that support the findings of this study are included in the paper and its Supplementary Information and Supplementary Videos and are available from the corresponding author upon reasonable request.

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Acknowledgements

This work was funded by the National Natural Science Foundation of China (21974123), the Natural Science Foundation of Zhejiang Province (LR20B050002), the Fundamental Research Funds for the Central Universities (2019XZZX003-01) and the Hundreds Program of Zhejiang University. We thank G. Tang for advice with cell culture, the Micro and Nano Fabrication Centre at Zhejiang University for facility support and W. Wang at the State Key Laboratory of Modern Optical Instrumentation for assistance with FIB.

Author information

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Authors

Contributions

J.F. conceived the idea, designed and supervised all experiments, and wrote the manuscript. J.D. prepared the samples, performed the experiments and analysed the data. Y.L. performed super-resolution imaging and image analysis. Y.X. conducted data analysis and sample characterizations. F.C. prepared cell samples and performed labelling. J.Y. and Y.C. carried out experimental characterizations.

Corresponding author

Correspondence to Jiandong Feng.

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Competing interests

The authors declare no competing interests.

Additional information

Peer review information Nature thanks the anonymous reviewers for their contribution to the peer review of this work.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Supplementary Information

This file contains the Supplementary Methods, Supplementary Discussions, Supplementary Figs 1-31 and Supplementary References.

Video 1 Observed single-molecule ECL signals under applied voltage

Video data acquired from the EMCCD reveal the single-molecule ECL signals for the result presented in Fig. 1. Limited by the data size, only part of the raw data is shown.

Video 2 Influence of exposure time during the acquisition

With increasing exposure time, the appearance of the single-molecule ECL events changes from isolated to continuous signals (Fig. 2).

Video 3 Concentration dependence of stochastic observations

The video data correspond to results shown in Supplementary Fig. 15, which is also discussed in Fig. 3.

Video 4 The processing of super-resolved ECL imaging

The video data (for Fig. 4) reveal the processing of the single-molecule localizations of ECL reactions frame by frame. A ROI of Fig. 4 is used in the video for illustration. After localizing individual emitters, a super-resolved ECL image is reconstructed.

Video 5 Processed video from live cell single-molecule ECL imaging

The video data show the dynamic visualization of cell adhesions at different moments (36 s, 48 s, 60 s, 72 s) for two selected adhesive regions in Fig. 5.

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Dong, J., Lu, Y., Xu, Y. et al. Direct imaging of single-molecule electrochemical reactions in solution. Nature 596, 244–249 (2021). https://doi.org/10.1038/s41586-021-03715-9

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