To improve the study of the pathological processes of cancer cells, researchers at Beijing Institute of Technology combined divided-aperture laser differential confocal microscopy with Raman spectroscopy and Brillouin spectroscopy. The researchers showed that their multichannel, microspectroscopic tool improves the spatial resolution of confocal microscopy and the detection accuracy of Raman and Brillouin spectroscopy for the study of human cells and tissues. The technique, which the researchers call divided-aperture laser differential confocal Raman-Brillouin spectrum microscopy (DLDCRBSM), simultaneously detects multidimensional information, including 3D geometrical morphology, Raman spectrum, and Brillouin spectrum, in situ, in real time. This information can be used to characterize the 3D spatial distribution of the topographic, chemical, and mechanical properties of tumor tissues. The team led by professor Weiqian Zhao used differential confocal microscopy to enable DLDCRBSM to focus on samples with nanometer-scale precision. The researchers achieved an axial focusing accuracy of 1 nm within the height range of 200 μm, ensuring that the multimodal technique could keep the focused spot size at its smallest and the system resolution at its highest when the sample height changed. This is critical when imaging biological tissue samples, because cancer cells are often larger and more variable in size than normal cells. High-precision focusing during scanning is essential to obtain high-resolution spectral mapping of living biological tissue, which typically deforms slowly over time. Other issues of the newly described method involve unwanted signal interference frequency shift. The fluorescence of slides can interfere with the Raman signal from cells, and Raman spectral signals between different layers of biological tissues are prone to crosstalk. In addition, the Brillouin frequency shift of biological tissues is typically low and is susceptible to interference from reflected light signals. (a) The test results of divided-aperture laser differential confocal Raman-Brillouin spectrum microscopy (DLDCRBSM) and confocal Raman system (CRM) when the sample is tilted. With focusing capability, mapping results of DLDCRBSM are clear (upper). Without focusing capability, mapping results of traditional CRM/confocal Brillouin microscopy are ambiguous (lower). (b) Compared with the conventional CRM, DLDCRBSM spatially separates the excitation light path and the collection light path of the Raman spectrum, and the size of the focal region in the axial direction is compressed. Therefore, the system can suppress the interference of stray light in the defocusing layer. Courtesy of L. Qiu et al. The researchers addressed these issues by designing the divided aperture to suppress the interference of defocused stray light during the detection of Raman spectroscopy, and the interference of reflected light during the detection of Brillouin spectroscopy. DLDCRBSM reduces the noise influence depth of Raman detection by 35.4%, and it increases the Brillouin extinction ratio by 22 dB. Using DLDCRBSM, the researchers realized high-spatial-resolution simultaneous imaging for geometrical topography, Raman, and Brillouin spectra from the same region. The group’s instrument demonstrates a Raman spectrum detection resolution of 0.7cm−1 and a Brillouin spectrum detection resolution of 0.5 GHz. The researchers demonstrated DLDCRBSM with a polymethyl methacrylate sample on a silicon substrate. Due to the system’s real-time axial focusing capabilities, the researchers obtained clear images of the sample and were able to verify that the microscope has anti-drift capability. Measurements from additional tests indicated that the method suppresses interference from defocused stray light. The researchers also used the new microscope to perform Raman and Brillouin mapping of gastric cancer tissues and adjacent normal tissues, and they achieved high-resolution imaging of the geometry, the various chemical components, and the viscoelasticity of the tissues. According to the researchers, the experimental results confirmed a previous hypothesis, that is, that changes in the protein substances in cancer tissues and changes in tissue viscoelasticity foretell increased invasiveness. According to the researchers, as an imaging technology to characterize the chemical and mechanical properties of cancer cells and tissues in real time and with high stability, DLDCRBSM could help advance emerging cancer therapies that are based on regulating the biochemical response and viscoelasticity of cancer tissues. This, they said, could be deployed to prevent metastasis in patients. The research was published in Light: Science & Applications (www.doi.org/10.1038/s41377-023-01153-y).