Study On Pathological Characteristics Of Breast Cancer Using Confocal Raman Microspectral Imaging And Multiphoton Microscopy Imaging

Breast cancer is considered a highly heterogeneous disease due to the diversity of subtypes,which predisposes patients to death as the tumor invades and metastasizes,so it is crucial to study the pathological analysis and disease evolution of breast cancer.Although histopathology and biomedical imaging techniques are used as standard methods for breast cancer diagnosis,these techniques rely on the experience of pathologists,which can lead to misdiagnosis.Therefore,focused on practical needs for pathological diagnosis of breast cancer,new technology needs to be developed to achieve accurate identification and diagnosis of tumors at the molecular level.Confocal Raman Microspectral Imaging(CRMI)technology enables accurate and efficient pathological imaging of breast cancer based on label-free,rapid,and nondestructive detection of biochemical composition of tissues.In addition to visualizing the composition and distribution of specific molecules by CRMI,the structural differences of tissues or cells can also be described and evaluated by some other optical techniques.Based on the Two-Photon Excited Fluorescence(TPEF)and Second-Harmonic Generation(SHG)principles of nonlinear optical effects,the Multiphoton Microscopy(MPM)is well suited for human tissue imaging with advantages of high resolution,low photobleaching,low photodamage,and real-time imaging.The combination of the two techniques can provide complementary information to characterize the biochemical and structural of cancer tissue from multiple perspectives.In this study,CRMI and MPM techniques were used to characterize the pathological evolution of breast cancer from the perspective of compositional imaging combined with morphological imaging,as follows:Firstly,the pathological diagnostic ability of Raman spectroscopy technology for Formalin-Fixed Paraffin-Embedded(FFPE)breast sections was evaluated by comparing the Raman spectra of FFPE and frozen breast tissue.The experimental results showed that using frozen tissue sections as a reference,the preparation process of FFPE tissues would lead to compositional loss and could not reflect the complete biochemical composition information,but could retain the characteristics of compositional differences between normal and breast cancer tissues.The spectral features between the Healthy(H),Ductal Carcinoma in Situ(DCIS)and Invasive Ductal Carcinoma in Situ(IDC)tissue of FFPE are further identified and distinguished by multivariate analysis models.The above work confirms the feasibility of FFPE sections based on Raman spectroscopy for retrospective study of diseases,but for the pathological study of biochemical components,frozen sections are more advantageous.Secondly,based on ensuring the integrity of biochemical information,pathological analysis of frozen breast tissue sections in different TNM Stages was performed by the CRMI technology.In the experiment,Raman single spectra of different TNM Stages breast cancer tissues were obtained to explore the information of composition related to tumor evolution,and the results indicate that processes of tumor growth and evolution were accompanied by changes in carotenoid,protein,nucleic acid and lipid content and conformation.On this basis,the spectral discrimination model based on the Principal component analysis-Linear discriminant analysis(PCA-LDA)algorithm accurately distinguishes different tissue types.In addition,K-means cluster analysis(KCA)and univariate imaging methods were used to visualize changes in the distribution of biochemical composition and tissue structure during tumor evolution.This work contributes to the understanding of tumor evolution mechanisms at the molecular level and confirms the potential of CRMI combination with multivariate analysis for disease staging.However,the CRMI technology is still challenging for achieving fine structural imaging of tumor tissue at the cellular level.Finally,the combination of CRMI and MPM was used for the multi-angle pathological evaluation of IDC tissue sections.The MPM technique is more advantageous in imaging the fine structure of a large range of tissues,where the SHG signal shows the characteristics of the collagen structure around the IDC cancer and the TPEF signal shows the cancer cell structure.The imaging results demonstrated the loss of collagen bundles,malignant cell infiltration,basement membrane damage and blood vessel growth.Meanwhile,the CRMI technique was used to perform Raman imaging for IDC tissues in the same area,which visualizes the spatial distribution of tumor margin profiles and biological components.The combination of the two imaging methods provides comprehensive pathological information for the rapid diagnosis of breast cancer and provides data for supporting clinical applications of multimodal optical imaging.