Direct imaging of molecular symmetry by coherent anti-stokes Raman scattering

Obtaining information on the organization of matter on the micrometer-scale with non-destructive methods still remains a challenge in chemical physics and biology. One well-established method for extracting matter organization information is fluorescence microscopy, which uses fluorescent molecules or proteins to tag the sample. However, this technique is limited to the observation of the probe itself, which may differ from the sample organization. Coherent Raman scattering (CRS) microscopy technique has proven to be powerful due to its label-free, three-dimensional, chemical selective and real-time imaging capabilities1,2,3,4,5,6. In coherent anti-Stokes Raman scattering (CARS), two beams of different frequencies interact with the sample to excite a vibrational resonance. A probe beam is used to probe the vibrational excitation by generating a new anti-Stokes frequency shifted beam. Unfortunately, CARS is limited by a non-resonant four wave mixing (FWM) background resulting in reduced image contrast and chemical selectivity. Recently, it has been shown that stimulated Raman scattering (SRS) can provide vibrational spectra without non-resonant background allowing to report vibrational information with high fidelity and high efficiency7,8,9,10 and CARS can benefit from non-resonant background to heterodyne-amplify weak Raman signals11. It is well-known that the symmetry properties of matter have a strong influence on its physical properties, for example, in crystalline samples. Similarly, it has been found that in biological environments anisotropic and symmetry properties of tissue are often related to specialized biological functionality. Nevertheless, the organization of molecular bonds in the focal volume, which is of particular interest in numerous situations where the medium is organized, is not contained in the spectral information. However, seminal works from nonlinear optics pioneers have shown that the Cartesian components of the nonlinear susceptibility tensor express the vibrational symmetry properties12,13. To read the tensor elements, polarization-resolved schemes have been proposed decades ago14, stimulating more recent developments in microscopy15,16,17,18,19,20. The molecular organization from a sample is usually retrieved by acquiring a stack of images from different polarization angles of the excitation or detection light fields, requiring long acquisition times and time consuming post-processing21. In this article, we introduce a label-free microscopy technique that is able to retrieve the individual symmetry orders of molecular organization in a single image acquisition. The symmetry-resolved CARS (SR-CARS) signal not only depends on the presence of molecular bonds, but also on their organization within the focal volume. By switching between combination of left- and right-handed circular polarization states for the involved fields, it is possible to directly image individual symmetry contributions of the sample. Beyond this organization selectivity, we show that our technique can (1) suppress the isotropic background in CARS images and spectra, thus enhancing the contrast by 1–2 orders of magnitude, and (2) retrieve quantitative information without pre-knowledge information on the molecular organization, without post-processing and independently of sample orientation in the transverse plane. SR-CARS provides higher chemical selectivity based on different symmetry characteristics, which are not accessible with regular spontaneous Raman or SRS microscopy.