There are clear distinctions between cancerous cells and normal cells in terms of their morphology, chemical properties, and mechanical properties. The detection of cytochemical and mechanical characteristics of tumor tissues can provide valuable multidimensional information regarding the pathological processes of cells and human tissues.
Among the existing methods for assessing tissue and cell morphology, mechanics, and chemical properties, confocal Raman spectroscopy stands out. It can detect the chemical properties of micro-regions within samples without the need for contact or labeling. Similarly, confocal Brillouin spectroscopy can assess the mechanical properties of micro-regions within samples in a nondestructive and contactless manner.
By combining confocal Raman spectroscopy with Brillouin spectroscopy, it becomes possible to simultaneously and in situ examine the three-dimensional morphology, chemical properties, and mechanical properties of micro-regions within tissues, including subcellular structures. This approach holds promise as a novel method for obtaining multidimensional pathological information about tissues and cells.
However, current confocal Raman/Brillouin spectroscopic microscopy imaging technologies lack high-precision real-time focusing capability. As a result, the size of the focal spot on the sample changes with fluctuations in the sample during the scanning process, which hampers the realization of the theoretical spatial resolution of the confocal spectrum microscopy system.
Furthermore, confocal spectrum microscopy is susceptible to system drift due to weak Raman and Brillouin scattering spectra and long integration times. This drift leads to defocusing, thereby compromising the spatial resolution and imaging quality. Additionally, when used for imaging biological tissue slices, the fluorescence signal generated by vertical incidence reduces the signal-to-noise ratio of the sample’s Raman spectrum, thereby affecting the accuracy of Raman and Brillouin spectrum detection and diminishing overall detection precision.
In a recent publication in Light Science & Application, Professor Weiqian Zhao and a team of scientists from the Beijing Institute of Technology introduced a new technique called divided-aperture laser differential confocal Raman-Brillouin spectrum microscopy (DLDCRBSM). This approach overcomes the limitations mentioned earlier by offering high stability, high resolution, and anti-scattering capability.
DLDCRBSM combines divided-aperture laser differential confocal microscopy with Raman spectroscopy and Brillouin spectroscopy. It leverages differential confocal microscopy for nanometer-precision sample focusing, enhancing spatial resolution and stability. The divided-aperture technology effectively suppresses interference from stray light in defocused layers and reflected light, thereby improving the signal-to-noise ratio. Additionally, co-axial excitation and high-resolution separate detection enable simultaneous imaging of geometric topography, Raman spectra, and Brillouin spectra from the same region with high stability and spatial resolution.
The researchers successfully developed a divided-aperture differential confocal Raman-Brillouin spectrum microscope with exceptional spatial resolution and 3D imaging focusing and tracking capabilities. This microscope demonstrated an impressive axial focusing accuracy of 1nm, a lateral resolution for spectral imaging better than 400nm, a Raman spectrum detection resolution of 0.7cm-1, and a Brillouin spectrum detection resolution of 0.5GHz.
To validate the effectiveness of the proposed method, the microscope was utilized to image a strip-shaped polymethyl methacrylate (PMMA) sample on a silicon (Si) substrate. The resulting images were clear and well-defined, highlighting the microscope’s real-time axial focusing capability and confirming its anti-drift capability. Additionally, the microscope was employed to examine a double-layer transparent sample consisting of an upper layer of PMMA and a lower layer of SiO2. The measurements conducted on this sample demonstrated the microscope’s ability to suppress interference caused by defocused stray light.
The team further conducted Raman and Brillouin mapping of gastric cancer tissues and adjacent normal tissues using the developed differential confocal spectroscopy microscope. The results obtained from these experiments provided additional confirmation of the earlier hypothesis, indicating that alterations in protein substances within cancer tissues and changes in tissue viscoelasticity contribute to increased invasiveness.
In Figure 3(a), the chemical imaging results of gastric cancer tissues and adjacent normal tissues obtained using the differential confocal spectroscopy microscope are displayed. The concentration of different compounds is indicated by the intensity of characteristic peaks in the Raman spectra.
Compared to the adjacent normal tissues, gastric cancer tissues exhibited low and discrete collagen concentration. The DNA material concentration in gastric cancer cells was high and widely distributed. The protein concentration in the cell matrix of gastric cancer tissues was found to be low. In terms of lipid concentration, gastric cancer tissues showed higher levels within the matrix, while lipid distribution in normal tissues was relatively uniform.
Moving to Figure 3(b), the imaging results of the mechanical properties of gastric cancer tissue and adjacent normal tissues are presented. The frequency shift of the Brillouin spectrum reflects the energy storage modulus (elasticity) of a substance, while the full width at half maximum of the Brillouin spectrum represents the loss modulus (viscosity) of a substance. When compared to adjacent normal tissues, gastric cancer cells and the intercellular substance exhibited lower elasticity, while the cancer cell nucleus displayed higher elasticity. The stickiness of gastric cancer cells and the interstitial tissue was lower, while the stickiness of the cancer cell nucleus was higher.
The proposed laser differential confocal Raman-Brillouin spectroscopy imaging method, characterized by its high stability, high resolution, and anti-scattering capabilities, was successfully developed in this study. The corresponding instrument enabled the detection of three-dimensional morphology, mechanical properties, and multidimensional information of the samples. It was applied to the characterization and analysis of tumor tissues, validating its potential for cancer research and treatment.
This innovative approach offers a new avenue for investigating the cancer process and facilitating advancements in cancer treatment research.
Source: Chinese Academy of Sciences