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Laser differential confocal Raman – Brillouin spectroscopy


Research background

There are obvious differences between cancerous cells and normal cells in terms of morphology, chemical properties and mechanical properties, and the detection of chemical and mechanical properties of tumor tissue cells can provide multi-dimensional information for the process of cell and human tissue lesions. Among the existing detection methods of tissue cell morphology, mechanical properties and chemical properties, confocal Raman spectroscopy microscopy can detect the chemical properties of sample microregions without contact and labels, confocal Brillouin spectroscopy can perform non-contact and non-destructive detection of the mechanical properties of sample microregions, and combine confocal Raman spectroscopy with Brillouin spectroscopy detection technology to simultaneously and isotopic detect the three-dimensional morphology, chemical properties and mechanical mechanical properties of micro-regions of tissue and even subcellular structures. It is expected to provide a new means for the detection of multidimensional lesion information of histiocytes.

Innovative research

Due to the lack of high-precision real-time fixed focus ability, the size of the spot focused on the sample during the scanning process changes with the fluctuation of the sample, which restricts the realization of the theoretical spatial resolution of the confocal spectral microscopy system. Secondly, due to the weak scattering spectral intensity of Raman and Brillouin and the long imaging integration time, the confocal spectral microscopy system is very susceptible to the influence of system drift, resulting in defocus, which in turn affects the spatial resolution and imaging quality. In addition, when imaging biological tissue section samples, the fluorescence signal generated by vertical incidence will reduce the signal-to-noise ratio of the Raman spectrum of the sample, thereby affecting the accuracy of Raman spectroscopy and Brillouin spectroscopy detection, and reducing the detection accuracy.

In view of this, with the support of the National Natural Science Foundation of China’s key project “Mechanical morphological performance laser spectropupil differential confocal Brilouin-Raman spectroscopy measurement principle and sensing system (51535002)”, Professor Zhao Weiqian’s team of Beijing Institute of Technology invented the highly stable, high-resolution, anti-scattering spectropupil laser differential confocal Raman (Divided-aperture Laser Differential Confocal) shown in Figure 1 Raman-Brillouin, DLDCRB) new method of map imaging (authorized Chinese invention patent ZL 201410086366.5 and European invention patent EP 3118608 B1), this method combines spectropupil laser differential confocal microscopy with Raman spectroscopy and Brillouin spectroscopy detection technology, and performs nanometer-precision sample fixed focus through differential confocal measurement technology to improve the spatial resolution and stability of the system; The spectral detection signal-to-noise ratio of the system is improved by the spectroscopy oblique excitation and detection technology to suppress the interfering light such as reflected light and interlayer scattered light. Through the homologous laser excitation and high-resolution separation detection of Raman spectrum and Brillouin spectrum, high-stability and high-resolution in situ pattern imaging of micro-geometry, Raman spectroscopy and Brillouin spectra are realized.

Figure 1. Principles of DLDCRB spectroscopic microscopic imaging

Based on this method, the DLDCRB spectral microscope with high spatial resolution and three-dimensional imaging focus tracking ability shown in Figure 2 was developed, with axial fixed-focus resolution of 1nm, spectral imaging lateral resolution of 400nm, Raman spectral resolution of 0.7cm-1, and Brillouin spectral detection resolution of 0.5GHz.

Figure 2. DLDCRB spectroscopy

The developed DLDCRB spectroscopy was used to clearly image the bar samples, and the results were shown in Figure 3, which verified the drift resistance of the proposed method. PMMA/SiO2 bilayer samples were detected, and the results are shown in Figure 4, which verifies the ability of the proposed method to suppress the interference of scattered light in the defocus layer.

Figure 3. Comparison of traditional confocal spectroscopy system and DLDCRB spectroscopy microscopy results (a) Classical confocal spectroscopy system imaging (blurred) (b) DLDCRB spectroscopic system imaging (clear)

Figure 4. The system anti-focus noise interference mechanism (a) oblique excitation and collection optical path (b) compresses the scatterer axial size

The Raman-Brillouin spectrogram analysis of gastric cancer tissues and adjacent normal tissues was carried out using the developed DLDCRB spectroscopy, which confirmed the previous hypothesis that the changes in protein substances in cancerous tissues and the changes in viscoelasticity of tissues led to increased infiltration.

Figure 5 shows the chemical imaging results of gastric cancer tissue and adjacent normal tissue by DLDCRB spectroscopy, the concentration of which is characterized by the intensity of the characteristic peak of Raman spectroscopy. Compared with normal histochemical imaging results next to cancer, gastric cancer tissue: collagen concentration is low and distribution is discrete; Gastric cancer cells have a high concentration and wide distribution of DNA substances; Low concentration of proteins in the tissue matrix of gastric cancer; Lipids in gastric cancer tissues have high concentrations within the matrix, while lipid distribution in normal tissues is relatively uniform.

Figure 5.Chemical imaging of gastric cancer tissue and adjacent normal tissues

Figure 6 shows the mechanical properties of gastric cancer tissue and adjacent normal tissue by DLDCRB spectroscopy, the frequency shift of Brillouin spectrum to characterize the storage modulus (elastic performance) of the species, and the half-height width of the Brillouin spectrum to characterize the loss modulus (viscosity performance) of the species. Compared with the mechanical imaging results of gastric cancer tissues and adjacent normal tissues, the elasticity of gastric cancer cells and interstitium was lower than that of normal cells and interstitium, and the elasticity of cancer nuclei was higher than that of normal cells. Gastric cancer cells and interstitium are less viscous than normal cells and interstitium, and the nucleus of cancer cells is more viscous than normal cells.

Figure 6. Comparison of mechanical properties of gastric cancer tissue and adjacent normal tissue

In this study, a spectroscopic pupil differential Raman – Brillouin mapping method with high stability, high resolution and anti-scattering was proposed, and the corresponding instrument was successfully developed, and the multi-dimensional information detection of three-dimensional morphology, mechanical properties and chemical components of the sample was realized, and the application was verified in the characterization and analysis of tumor tissue, which can provide a new means for the research of carcinogenesis process and cancer treatment.

The article was recently published in the top international academic journal Light: Science & Applications, entitled “A high-precision multi-dimensional microspectroscopic technique for morphological and properties analysis of cancer cell”. (Source: LightScience Applications WeChat public account)

Related paper information:https://www.nature.com/articles/s41377‍-023-0‍1153-y

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