Breaking the diffraction limit of label-free chemical imaging

Super-resolution optical imaging plays an important role in exploring the microscopic mechanisms of biological processes. Based on super-resolution imaging technology of fluorescent staining, live cell imaging of macromolecules such as proteins has been successfully achieved at the nanoscale. However, fluorescent labeling requires complex preparation and cannot effectively label small molecule metabolites (such as sugars, lipids, etc.). Stimulated Raman scattering (SRS) is a label-free imaging technology that can achieve chemical imaging by capturing the intrinsic chemical bond vibrations of cell molecules. However, the spatial resolution of SRS is only about 300 nanometers, which is not enough to capture the fine nanostructures in cells. Therefore, how to achieve super-resolution label-free chemical imaging of biological samples is still a major issue in academia.

Recently, Hilton B. de Aguiar’s group at Kastler-Brossel Laboratory in France reported a super-resolution technology called Blind-S3 in Advanced Imaging, which successfully combined SRS with structured illumination microscopy (SIM). Compared with traditional wide-field SIM, the authors designed an imaging method that combines wide-field and point-scanning seconds based on the characteristics of SRS. The research team used speckle illumination for the pump light (Pump) and captured SRS images with a single detector by laser scanning the Stokes beam. Subsequently, a series of spot illumination images were reconstructed using advanced computational methods. This “instrument + computation” collaborative strategy not only overcomes the problem that SRS signals cannot be detected by cameras, but also enhances the penetration depth of SRS by using speckle illumination. Blind-S3 achieved a two-fold resolution improvement and demonstrated its effect by imaging HeLa cells and mouse brain tissue slices. This technology does not require high-power laser irradiation or special sample processing and has broad application prospects in biological cytology.
Despite the significant progress made by Blind-S3, its resolution is still far behind the capabilities of fluorescence super-resolution imaging. This is not only limited by the poor resolution of near-infrared excitation, but also stems from the limited sensitivity of SRS. Therefore, new vibration imaging technologies that can achieve sub-100 nanometer resolution while maintaining sensitivity to non-fluorescent metabolites remain a hot research area.