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Mapping enzyme activity in living systems by real-time mid-infrared photothermal imaging of nitrile chameleons

Nature Methods volume 21, pages 342–352 (2024)Cite this article

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Abstract

Simultaneous spatial mapping of the activity of multiple enzymes in a living system can elucidate their functions in health and disease. However, methods based on monitoring fluorescent substrates are limited. Here, we report the development of nitrile (C≡N)-tagged enzyme activity reporters, named nitrile chameleons, for the peak shift between substrate and product. To image these reporters in real time, we developed a laser-scanning mid-infrared photothermal imaging system capable of imaging the enzymatic substrates and products at a resolution of 300 nm. We show that when combined, these tools can map the activity distribution of different enzymes and measure their relative catalytic efficiency in living systems such as cancer cells, Caenorhabditis elegans, and brain tissues, and can be used to directly visualize caspase–phosphatase interactions during apoptosis. Our method is generally applicable to a broad category of enzymes and will enable new analyses of enzymes in their native context.

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Fig. 1: Laser-scan MIP microscope for real-time bond-selective imaging of live cells at 300 nm spatial resolution.
Fig. 2: Development of nitrile chameleons for mapping specific enzyme activity.
Fig. 3: Real-time MIP imaging of nitrile chameleons generates the activity maps of caspase-3/7 and phosphatase in living cells.
Fig. 4: Multicolor MIP imaging of nitrile chameleons in live cancer cells provides evidence of caspase–phosphatase cooperation in apoptosis.
Fig. 5: Multicolor MIP imaging of nitrile chameleons generates activity maps of caspase and phosphatase inside C. elegans and brain tissues.

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Data availability

All data related to the work are available in the article and supplementary information in this paper. The original data for the figures are available at https://doi.org/10.5281/zenodo.10085096.

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Acknowledgements

This work was supported by R35GM136223 and R33CA261726 to J.-X.C., R01CA142746 to B.X. and grant 2023-321163 from the Chan Zuckerberg Initiative Donor-Advised Fund at the Silicon Valley Community Foundation. Source data are provided with this paper.

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Author notes

  1. These authors contributed equally: Hongjian He, Jiaze Yin.

Authors and Affiliations

  1. Department of Electrical and Computer Engineering, Boston University, Boston, MA, USA

    Hongjian He, Jiaze Yin, Mingsheng Li & Ji-Xin Cheng

  2. Photonics Center, Boston University, Boston, MA, USA

    Hongjian He, Jiaze Yin, Mingsheng Li, Chinmayee Vallabh Prabhu Dessai, Xinyan Teng, Meng Zhang, Yueming Li, Zhiyi Du & Ji-Xin Cheng

  3. Department of Biomedical Engineering, Boston University, Boston, MA, USA

    Chinmayee Vallabh Prabhu Dessai, Meng Zhang & Ji-Xin Cheng

  4. Department of Chemistry, Brandeis University, Waltham, MA, USA

    Meihui Yi & Bing Xu

  5. Department of Chemistry, Boston University, Boston, MA, USA

    Xinyan Teng, Zhiyi Du & Ji-Xin Cheng

  6. Department of Mechanical Engineering, Boston University, Boston, MA, USA

    Yueming Li

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Contributions

J.-X.C. and H.H. conceived the study. H.H. synthesized the probes, performed the experiments and drafted the paper. J.Y. developed the laser-scanning MIP microscope. J.Y. and M.L. helped with the MIP imaging. M.Y. and X.T. helped in the probe synthesis. M.Z. helped in the culturing and MIP imaging of C. elegans. C.V.P.D., Y.L. and Z.D. carried out the extraction of mice brains, tissue sectioning and tissue imaging. B.X. provided material support on synthesis and made intellectual contributions to experiment design.

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Correspondence to Ji-Xin Cheng.

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Nature Methods thanks Lingyan Shi, Stephen Weber and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available. Primary Handling Editor: Rita Strack, in collaboration with the Nature Methods team.

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Supplementary information

Supplementary Information

Molecular synthesis and identification, Supplementary Figs. 1–21, Supplementary Text and Supplementary Schemes 1–3.

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Supplementary Video 1

Live cell MIP imaging of the dynamic cellular movements at amide II.

Supplementary Video 2

3D reconstruction of phosphatase activity in C. elegans.

Supplementary Video 3

3D reconstruction of caspase activity in C. elegans.

Source data

Source Data Fig. 3

Statistical source data.

Source Data Fig. 4

Statistical source data.

Source Data Fig. 5

Statistical source data.

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He, H., Yin, J., Li, M. et al. Mapping enzyme activity in living systems by real-time mid-infrared photothermal imaging of nitrile chameleons. Nat Methods 21, 342–352 (2024). https://doi.org/10.1038/s41592-023-02137-x

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