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.

相干拉曼散射（例如，相干反斯托克斯拉曼散射（CARS）和受激拉曼散射）显微镜已成为生物和生物医学系统中无标记生物分子成像的强大工具，但其空间分辨率受到衍射的限制。在这里，我们报告了更高阶的相干抗斯托克斯拉曼散射（HO-CARS）显微镜，打破了无标签，超分辨率振动成像的衍射极限。HO-CARS显微镜的分辨率提高已在生物样品（例如活HeLa和颊细胞）中进行了分析和证明。与常规CARS显微镜相比，HO-CARS技术提供了固有的高共振与非共振背景比。我们确认，在严格聚焦下，HO-CARS信号源自高阶非线性过程（χ ^（5），χ ^（7））在级联的低阶非线性过程（χ ^（3））中占主导地位，从而产生了更丰富的光谱信息。这项研究表明，HO-CARS显微镜可以成为具有高图像对比度的生物和生物医学系统中无标签，超分辨率成像的吸引人的工具。