Macrophages and collagen fibers are important components of the tumor microenvironment. Macrophages would secrete extracellular matrix degrading enzymes to degrade collagen, which is conducive to the formation of local infiltration and distant metastasis of tumor cells. During tumor progression, macrophages are actively recruited into tumors where they alter the tumor microenvironment to accelerate tumor progression. A high density of these tumor-associated macrophages may correlates with poor prognosis. In this work, multiphoton microscopy (MPM) using two-photon excited fluorescence combined with second harmonic generation imaging was used to monitor the changes in collagen fibers around macrophages. The experimental results show that this microscope has the ability to directly monitor the collagen changes induced by the invasion of macrophages in the absence of labels. Moreover, collagen content around macrophages in the matrix can be quantitatively calculated by image processing, and quantitative results show that the collagen content in the tumor microenvironment will significantly reduce with the appearance of macrophages. Therefore, MPM has the potential to be used as a new auxiliary tool for pathologists to quickly and effectively evaluate collagen changes in breast tumor microenvironment.
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