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

Nature Photonics volume 17, pages 330–337 (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.

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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.

References

  1. Willig, K. I., Rizzoli, S. O., Westphal, V., Jahn, R. & Hell, S. W. STED microscopy reveals that synaptotagmin remains clustered after synaptic vesicle exocytosis. Nature 440, 935–939 (2006).

    Article  ADS  Google Scholar 

  2. Lee, S. H., Shin, J. Y., Lee, A. & Bustamante, C. Counting single photoactivatable fluorescent molecules by photoactivated localization microscopy (PALM). Proc. Natl Acad. Sci. USA 109, 17436–17441 (2012).

    Article  ADS  Google Scholar 

  3. Rust, M. J., Bates, M. & Zhuang, X. Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM). Nat. Methods 3, 793–796 (2006).

    Article  Google Scholar 

  4. Fujita, K., Kobayashi, M., Kawano, S., Yamanaka, M. & Kawata, S. High-resolution confocal microscopy by saturated excitation of fluorescence. Phys. Rev. Lett. 99, 228105 (2007).

    Article  ADS  Google Scholar 

  5. Yamanaka, M. et al. SAX microscopy with fluorescent nanodiamond probes for high-resolution fluorescence imaging. Biomed. Opt. Express 2, 1946–1954 (2011).

    Article  Google Scholar 

  6. Wang, P. et al. Far-field imaging of non-fluorescent species with subdiffraction resolution. Nat. Photon. 7, 449–453 (2013).

    Article  ADS  Google Scholar 

  7. Bi, Y. et al. Far-field transient absorption nanoscopy with sub-50 nm optical super-resolution. Optica 7, 1402–1407 (2020).

    Article  ADS  Google Scholar 

  8. Yao, J., Wang, L., Li, C., Zhang, C. & Wang, L. V. Photoimprint photoacoustic microscopy for three-dimensional label-free subdiffraction imaging. Phys. Rev. Lett. 112, 014302 (2014).

    Article  ADS  Google Scholar 

  9. Massaro, E. S., Hill, A. H. & Grumstrup, E. M. Super-resolution structured pump–probe microscopy. ACS Photon. 3, 501–506 (2016).

    Article  Google Scholar 

  10. Danielli, A. et al. Label-free photoacoustic nanoscopy. J. Biomed. Opt. 19, 086006 (2014).

    Article  ADS  Google Scholar 

  11. Goy, A. S. & Fleischer, J. W. Resolution enhancement in nonlinear photoacoustic imaging. Appl. Phys. Lett. 107, 211102 (2015).

    Article  ADS  Google Scholar 

  12. Zhang, Z., Shi, Y., Yang, S. & Xing, D. Subdiffraction-limited second harmonic photoacoustic microscopy based on nonlinear thermal diffusion. Opt. Lett. 43, 2336–2339 (2018).

    Article  ADS  Google Scholar 

  13. Rajakarunanayake, Y. N. & Wickramasinghe, H. K. Nonlinear photothermal imaging. Appl. Phys. Lett. 48, 218–220 (1986).

    Article  ADS  Google Scholar 

  14. Nedosekin, D. A., Galanzha, E. I., Dervishi, E., Biris, A. S. & Zharov, V. P. Super-resolution nonlinear photothermal microscopy. Small 10, 135–142 (2014).

    Article  Google Scholar 

  15. He, J., Miyazaki, J., Wang, N., Tsurui, H. & Kobayashi, T. Label-free imaging of melanoma with nonlinear photothermal microscopy. Opt. Lett. 40, 1141–1144 (2015).

    Article  ADS  Google Scholar 

  16. He, J., Miyazaki, J., Wang, N., Tsurui, H. & Kobayashi, T. Biological imaging with nonlinear photothermal microscopy using a compact supercontinuum fiber laser source. Opt. Express 23, 9762–9771 (2015).

    Article  ADS  Google Scholar 

  17. Tzang, O., Pevzner, A., Marvel, R. E., Haglund, R. F. & Cheshnovsky, O. Super-resolution in label-free photomodulated reflectivity. Nano Lett. 15, 1362–1367 (2015).

    Article  ADS  Google Scholar 

  18. Nakata, K., Tsurui, H. & Kobayashi, T. Further resolution enhancement of high-sensitivity laser scanning photothermal microscopy applied to mouse endogenous. J. Appl. Phys. 120, 214901 (2016).

    Article  ADS  Google Scholar 

  19. Samolis, P. D. & Sander, M. Y. Phase-sensitive lock-in detection for high-contrast mid-infrared photothermal imaging with sub-diffraction limited resolution. Opt. Express 27, 2643–2655 (2019).

    Article  ADS  Google Scholar 

  20. Levin, I. W. & Bhargava, R. Fourier transform infrared vibrational spectroscopic imaging: integrating microscopy and molecular recognition. Annu. Rev. Phys. Chem. 56, 429–474 (2004).

    Article  ADS  Google Scholar 

  21. Stewart, S., Priore, R. J., Nelson, M. P. & Treado, P. J. Raman imaging. Annu. Rev. Anal. Chem. 5, 337–360 (2012).

    Article  Google Scholar 

  22. Watanabe, K. et al. Structured line illumination Raman microscopy. Nat. Commun. 6, 1–8 (2015).

    Article  Google Scholar 

  23. Gong, L., Zheng, W., Ma, Y. & Huang, Z. Higher-order coherent anti-Stokes Raman scattering microscopy realizes label-free super-resolution vibrational imaging. Nat. Photon. 14, 115–122 (2020).

    Article  ADS  Google Scholar 

  24. Yonemaru, Y. et al. Super-spatial- and -spectral-resolution in vibrational imaging via saturated coherent anti-Stokes raman scattering. Phys. Rev. Appl. 4, 014010 (2015).

    Article  ADS  Google Scholar 

  25. Singh, A. K., Santra, K., Song, X., Petrich, J. W. & Smith, E. A. Spectral narrowing accompanies enhanced spatial resolution in saturated coherent anti-Stokes Raman scattering (CARS): comparisons of experiment and theory. J. Phys. Chem. A 124, 4305–4313 (2020).

    Article  Google Scholar 

  26. Gong, L., Zheng, W., Ma, Y. & Huang, Z. Saturated stimulated-Raman-scattering microscopy for far-field superresolution vibrational imaging. Phys. Rev. Appl. 11, 034041 (2019).

    Article  ADS  Google Scholar 

  27. Wurthwein, T., Irwin, N. & Fallnich, C. Saturated Raman scattering for sub-diffraction-limited imaging. J. Chem. Phys. 151, 194201 (2019).

    Article  ADS  Google Scholar 

  28. Zhang, D. et al. Depth-resolved mid-infrared photothermal imaging of living cells and organisms with submicrometer spatial resolution. Sci. Adv. 2, e1600521 (2016).

    Article  ADS  Google Scholar 

  29. Bai, Y., Zhang, D., Li, C., Liu, C. & Cheng, J. X. Bond-selective imaging of cells by mid-infrared photothermal microscopy in high wavenumber. Region. J. Phys. Chem. B 121, 10249–10255 (2017).

    Article  Google Scholar 

  30. Li, Z., Aleshire, K., Kuno, M. & Hartland, G. V. Super-resolution far-field infrared imaging by photothermal heterodyne imaging. J. Phys. Chem. B 121, 8838–8846 (2017).

    Article  Google Scholar 

  31. Li, X. et al. Fingerprinting a living cell by Raman integrated mid-infrared photothermal microscopy. Anal. Chem. 91, 10750–10756 (2019).

    Article  Google Scholar 

  32. Zhang, D. et al. Bond-selective transient phase imaging via sensing of the infrared photothermal effect. Light Sci. Appl. 8, 116 (2019).

    Article  ADS  Google Scholar 

  33. Klementieva, O. et al. Super-resolution infrared imaging of polymorphic amyloid aggregates directly in neurons. Adv. Sci. 7, 1903004 (2020).

    Article  Google Scholar 

  34. Xu, J., Li, X., Guo, Z., Huang, W. E. & Cheng, J. X. Fingerprinting bacterial metabolic response to erythromycin by Raman-integrated mid-infrared photothermal microscopy. Anal. Chem. 92, 14459–14465 (2020).

    Article  Google Scholar 

  35. Bai, Y., Yin, J. & Cheng, J.-X. Bond-selective imaging by optically sensing the mid-infrared photothermal effect. Sci. Adv. 7, eabg1559 (2021).

    Article  ADS  Google Scholar 

  36. Dazzi, A. & Prater, C. B. AFM-IR: technology and applications in nanoscale infrared spectroscopy and chemical imaging. Chem. Rev. 117, 5146–5173 (2017).

    Article  Google Scholar 

  37. Astrath, N. G., Bialkowski, S. E. & Proskurnin, M. Photothermal Spectroscopy Methods (Wiley, 2019).

  38. Boyer, D. Photothermal imaging of nanometer-sized metal particles among scatterers. Science 297, 1160–1163 (2002).

    Article  ADS  Google Scholar 

  39. Adhikari, S. et al. Photothermal microscopy: imaging the optical absorption of single nanoparticles and single molecules. ACS Nano 14, 16414–16445 (2020).

    Article  Google Scholar 

  40. Gaiduk, A., Yorulmaz, M., Ruijgrok, P. V. & Orrit, M. Room-temperature detection of a single molecule’s absorption by photothermal contrast. Science 330, 353 (2010).

    Article  ADS  Google Scholar 

  41. Olzmann, J. A. & Carvalho, P. Dynamics and functions of lipid droplets. Nat. Rev. Mol. Cell Biol. 20, 137–155 (2019).

    Article  Google Scholar 

  42. Liao, P. C. et al. Touch and go: membrane contact sites between lipid droplets and other organelles. Front Cell Dev Biol 10, 852021 (2022).

    Article  Google Scholar 

  43. Botstein, D. & Fink, G. R. Yeast: an experimental organism for 21st century biology. Genetics 189, 695–704 (2011).

    Article  Google Scholar 

  44. Yin, J. et al. Nanosecond-resolution photothermal dynamic imaging via MHZ digitization and match filtering. Nat. Commun. 12, 7097 (2021).

    Article  ADS  Google Scholar 

  45. de Andrade, R. B. et al. Quantum-enhanced continuous-wave stimulated Raman scattering spectroscopy. Optica 7, 470–475 (2020).

    Article  ADS  Google Scholar 

  46. Casacio, C. A. et al. Quantum-enhanced nonlinear microscopy. Nature 594, 201–206 (2021).

    Article  ADS  Google Scholar 

  47. Shi, J. et al. High-resolution, high-contrast mid-infrared imaging of fresh biological samples with ultraviolet-localized photoacoustic microscopy. Nat. Photon. 13, 609–615 (2019).

    Article  ADS  Google Scholar 

  48. Kozawa, Y., Matsunaga, D. & Sato, S. Superresolution imaging via superoscillation focusing of a radially polarized beam. Optica 5, 86–92 (2018).

    Article  ADS  Google Scholar 

  49. Bai, Y. et al. Ultrafast chemical imaging by widefield photothermal sensing of infrared absorption. Sci. Adv. 5, eaav7127 (2019).

    Article  ADS  Google Scholar 

  50. Tamamitsu, M. et al. Label-free biochemical quantitative phase imaging with mid-infrared photothermal effect. Optica 7, 359–366 (2020).

    Article  ADS  Google Scholar 

  51. Willomitzer, F. et al. Fast non-line-of-sight imaging with high-resolution and wide field of view using synthetic wavelength holography. Nat. Commun. 12, 6647 (2021).

    Article  ADS  Google Scholar 

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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. 17, 330–337 (2023). https://doi.org/10.1038/s41566-022-01143-3

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