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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All data are available from the corresponding author on reasonable request.
All codes used to produce the findings of this study are available from the corresponding author on reasonable request.
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).
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).
Rust, M. J., Bates, M. & Zhuang, X. Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM). Nat. Methods 3, 793–796 (2006).
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).
Yamanaka, M. et al. SAX microscopy with fluorescent nanodiamond probes for high-resolution fluorescence imaging. Biomed. Opt. Express 2, 1946–1954 (2011).
Wang, P. et al. Far-field imaging of non-fluorescent species with subdiffraction resolution. Nat. Photon. 7, 449–453 (2013).
Bi, Y. et al. Far-field transient absorption nanoscopy with sub-50 nm optical super-resolution. Optica 7, 1402–1407 (2020).
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).
Massaro, E. S., Hill, A. H. & Grumstrup, E. M. Super-resolution structured pump–probe microscopy. ACS Photon. 3, 501–506 (2016).
Danielli, A. et al. Label-free photoacoustic nanoscopy. J. Biomed. Opt. 19, 086006 (2014).
Goy, A. S. & Fleischer, J. W. Resolution enhancement in nonlinear photoacoustic imaging. Appl. Phys. Lett. 107, 211102 (2015).
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).
Rajakarunanayake, Y. N. & Wickramasinghe, H. K. Nonlinear photothermal imaging. Appl. Phys. Lett. 48, 218–220 (1986).
Nedosekin, D. A., Galanzha, E. I., Dervishi, E., Biris, A. S. & Zharov, V. P. Super-resolution nonlinear photothermal microscopy. Small 10, 135–142 (2014).
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).
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).
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).
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).
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).
Levin, I. W. & Bhargava, R. Fourier transform infrared vibrational spectroscopic imaging: integrating microscopy and molecular recognition. Annu. Rev. Phys. Chem. 56, 429–474 (2004).
Stewart, S., Priore, R. J., Nelson, M. P. & Treado, P. J. Raman imaging. Annu. Rev. Anal. Chem. 5, 337–360 (2012).
Watanabe, K. et al. Structured line illumination Raman microscopy. Nat. Commun. 6, 1–8 (2015).
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).
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).
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).
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).
Wurthwein, T., Irwin, N. & Fallnich, C. Saturated Raman scattering for sub-diffraction-limited imaging. J. Chem. Phys. 151, 194201 (2019).
Zhang, D. et al. Depth-resolved mid-infrared photothermal imaging of living cells and organisms with submicrometer spatial resolution. Sci. Adv. 2, e1600521 (2016).
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).
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).
Li, X. et al. Fingerprinting a living cell by Raman integrated mid-infrared photothermal microscopy. Anal. Chem. 91, 10750–10756 (2019).
Zhang, D. et al. Bond-selective transient phase imaging via sensing of the infrared photothermal effect. Light Sci. Appl. 8, 116 (2019).
Klementieva, O. et al. Super-resolution infrared imaging of polymorphic amyloid aggregates directly in neurons. Adv. Sci. 7, 1903004 (2020).
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).
Bai, Y., Yin, J. & Cheng, J.-X. Bond-selective imaging by optically sensing the mid-infrared photothermal effect. Sci. Adv. 7, eabg1559 (2021).
Dazzi, A. & Prater, C. B. AFM-IR: technology and applications in nanoscale infrared spectroscopy and chemical imaging. Chem. Rev. 117, 5146–5173 (2017).
Astrath, N. G., Bialkowski, S. E. & Proskurnin, M. Photothermal Spectroscopy Methods (Wiley, 2019).
Boyer, D. Photothermal imaging of nanometer-sized metal particles among scatterers. Science 297, 1160–1163 (2002).
Adhikari, S. et al. Photothermal microscopy: imaging the optical absorption of single nanoparticles and single molecules. ACS Nano 14, 16414–16445 (2020).
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).
Olzmann, J. A. & Carvalho, P. Dynamics and functions of lipid droplets. Nat. Rev. Mol. Cell Biol. 20, 137–155 (2019).
Liao, P. C. et al. Touch and go: membrane contact sites between lipid droplets and other organelles. Front Cell Dev Biol 10, 852021 (2022).
Botstein, D. & Fink, G. R. Yeast: an experimental organism for 21st century biology. Genetics 189, 695–704 (2011).
Yin, J. et al. Nanosecond-resolution photothermal dynamic imaging via MHZ digitization and match filtering. Nat. Commun. 12, 7097 (2021).
de Andrade, R. B. et al. Quantum-enhanced continuous-wave stimulated Raman scattering spectroscopy. Optica 7, 470–475 (2020).
Casacio, C. A. et al. Quantum-enhanced nonlinear microscopy. Nature 594, 201–206 (2021).
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).
Kozawa, Y., Matsunaga, D. & Sato, S. Superresolution imaging via superoscillation focusing of a radially polarized beam. Optica 5, 86–92 (2018).
Bai, Y. et al. Ultrafast chemical imaging by widefield photothermal sensing of infrared absorption. Sci. Adv. 5, eaav7127 (2019).
Tamamitsu, M. et al. Label-free biochemical quantitative phase imaging with mid-infrared photothermal effect. Optica 7, 359–366 (2020).
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).
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.
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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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