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Higher-order coherent anti-Stokes Raman scattering microscopy realizes label-free super-resolution vibrational imaging


Coherent Raman scattering (for example, coherent anti-Stokes Raman scattering (CARS) and stimulated Raman scattering) microscopy has emerged as a powerful tool for label-free biomolecular imaging in biological and biomedical systems, but its spatial resolution is diffraction limited. Here, we report a higher-order coherent anti-Stokes Raman scattering (HO-CARS) microscopy to break the diffraction limit for label-free, super-resolution vibrational imaging. The resolution enhancement of HO-CARS microscopy has been analysed and demonstrated in biological samples (for example, live HeLa and buccal cells). The HO-CARS technique provides an inherent high resonant to non-resonant background ratio compared with conventional CARS microscopy. We affirm that under a tight focusing, the HO-CARS signal originating from the higher-order nonlinear process (χ(5), χ(7)) dominates over the cascaded lower-order nonlinear process (χ(3)), yielding much richer spectroscopic information. This study illustrates that HO-CARS microscopy can be an appealing tool for label-free, super-resolution imaging in biological and biomedical systems with high image contrast.

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Fig. 1: Principle of HO-CARS.
Fig. 2: Experimental observation of HO-CARS.
Fig. 3: Characteristics of HO-CARS processes.
Fig. 4: Super-resolution HO-CARS images of a DPBD crystal.
Fig. 5: Super-resolution HO-CARS images of live unstained HeLa and buccal cells at 2,845 cm−1 (CH2 stretching of lipids), and buccal cells at 2,935 cm−1 (CH3 stretching of proteins and lipids).

Data availability

The data that support the plots within this paper and other findings of this study are available from the corresponding author upon reasonable request.


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This work was supported by the Academic Research Fund (AcRF)-Tier 1 and Tier 2 from Ministry of Education (MOE) (MOE2014-T2-1-010), and the National Medical Research Council (NMRC) (NMRC/TCR/016-NNI/2016), Singapore.

Author information




L.G. and Z.H. conceived the concept and designed the experiments. L.G. performed the experiments. Y.M. performed chemical synthesis. L.G., W.Z. and Z.H. performed the data analysis and wrote the manuscript. Z.H. finalized the manuscript.

Corresponding author

Correspondence to Zhiwei Huang.

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

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