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Super-resolution imaging of non-fluorescent molecules by photothermal relaxation localization microscopy

Nature Photonics (2023)Cite this article

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Abstract

Optical imaging beyond the diffraction limit has led to tremendous breakthroughs in recent years. Compared with widely employed fluorescence-based approaches, label-free imaging avoids the need for labelling and associated potential issues of cytotoxicity; however, non-fluorescent methods often rely on saturation effects or nonlinearity, which limit applicability to specific molecules and require high laser peak powers. Here we develop photothermal relaxation localization (PEARL) microscopy, a label-free super-resolution approach that overcomes these limitations. PEARL microscopy extracts subdiffraction features from the location-dependent modulation of the probe beam in photothermal microscopy and does not require special absorbers as it relies on general absorption processes such as electronic (E) and vibrational (V) absorption. We demonstrate label-free bond-selective E- and V-PEARL imaging of living cells 280 nm and 120 nm resolution, respectively, and show spectral and spatial imaging of individual lipid droplets and their distributions in mammalian and yeast cells. We believe PEARL may open new avenues for super-resolution imaging of non-fluorescent molecules, promising exciting and broad applications in biology, medicine and materials science.

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Fig. 1: The concept of PEARL imaging.
Fig. 2: E-PEARL imaging performance and cellular applications.
Fig. 3: Performance of V-PEARL imaging at higher harmonic demodulation.
Fig. 4: V-PEARL imaging of chondroblasts.
Fig. 5: V-PEARL imaging of live S. cerevisiae yeast cells.

Data availability

All data are available from the corresponding author on reasonable request.

Code availability

All codes used to produce the findings of this study are available from the corresponding author on reasonable request.

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Acknowledgements

We thank C. Ye and M. Yu for providing biological samples; Y. Zhang, Y. Wu and X. Ye for cell culture; and X. Xu for support on objective lens. This work was supported by the National Natural Science Foundation of China (grant nos. 12074339, 32050410293 and 11934011), Innovation Program for Quantum Science and Technology (grant no. 2021ZD0303200), MOE Frontier Science Center for Brain Science and Brain-Machine Integration of Zhejiang University and the Fundamental Research Fund for the Central Universities of China.

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Authors and Affiliations

  1. Interdisciplinary Center for Quantum Information, Zhejiang Province Key Laboratory of Quantum Technology and Device, and Department of Physics, Zhejiang University, Hangzhou, China

    Pengcheng Fu, Wanlin Cao, Taoran Le, Shiyao Zhu, Da-Wei Wang & Delong Zhang

  2. College of Computer Science and Technology, Zhejiang University, Hangzhou, China

    Tianrun Chen

  3. College of Biomedical Engineering and Instrument Science, Key Laboratory for Biomedical Engineering of Ministry of Education, Zhejiang University, Hangzhou, China

    Xiangjie Huang & Hyeon Jeong Lee

  4. Hefei National Laboratory, Hefei, China

    Shiyao Zhu, Da-Wei Wang & Delong Zhang

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Contributions

P.F. and D.Z. conceived the idea and designed the experiments. D.Z., P.F. and T.C. wrote the control program. P.F. performed the experiments. W.C. and T.L. performed the simulation. X.H. synthesized AuNPs and cultured cells with AuNPs uptake. P.F. and D.Z. analysed the data. P.F., S.Z., D.W.W., H.J.L. and D.Z. wrote the manuscript with input from all authors.

Corresponding author

Correspondence to Delong Zhang.

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Competing interests

D.Z., P.F. and H.J.L. have filed a patent (application no. 2022114119148) based on some key aspects described in the article. The other authors declare no competing interests.

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Nature Photonics thanks Wei Min and Ji-Xin Cheng for their contribution to the peer review of this work.

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Supplementary Method, Table 1 and Fig. 1–8.

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Fu, P., Cao, W., Chen, T. et al. Super-resolution imaging of non-fluorescent molecules by photothermal relaxation localization microscopy. Nat. Photon. (2023). https://doi.org/10.1038/s41566-022-01143-3

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