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Blood vessels are the important components of the circulatory systems that transport blood throughout the human body and maintain the homeostasis of physiological tissues. Pathologically, blood vessels are often affected by diseases, leading to the formation of unstable, irregular, and hyperpermeable blood vessels. In the tumor microenvironment, abnormal leakage of tumor blood vessels is related to the histological grade and malignant potential of tumors and may also facilitate metastasis of cancer. Visual diagnosis of blood vessels is very important for us to understand the occurrence and development of diseases. Multiphoton microscopy (MPM) is a potential label-free diagnostic tool based on second harmonic generation (SHG) and two-photon excited fluorescence (TPEF). MPM can effectively observe the morphological changes of biological tissues at the molecular and cellular levels. In this work, we demonstrate that label-free MPM can be used to visualize the microstructure of blood vessels in human normal breast and breast tumor tissue. Moreover, MPM can monitor the changes of blood vessels in tumor microenvironment. These results show that the MPM will become a promising technique for clinicians to study the properties of the microstructure of the blood vessels.

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Invasive micropapillary carcinoma of the breast (IMPC) is a rare form of breast cancer with unique histological features, and is associated with high axillary lymph node metastasis and poor clinical prognosis. Thus, IMPC should be diagnosed in time to improve the treatment and management of patients. In this study, multiphoton microscopy (MPM) is used to label‐free visualize the morphological features of IMPC. Our results demonstrate that MPM images are well in agreement with hematoxylin and eosin staining and epithelial membrane antigen staining, indicating MPM is comparable to traditional histological analysis in identifying the tissue structure and cell morphology. Statistical analysis shows significant differences in the circumference and area of the glandular lumen and cancer nest between the different IMPC cell clusters with complete glandular lumen morphology, and also shows difference in collagen length, width, and orientation, indicating the invasive ability of different morphologies of IMPC may be different.

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