Macrophages are at the center of the invasion and play a protumoral role in the tumor microenvironment. They canfacilitate angiogenesis, extracellular matrix remodeling, invasion and migration of cancer cells, while inhibit theantitumoral immune surveillance. Tumor-associated macrophages are a major component of malignant tumors, whichcan stimulate the progress of tumors. In many human cancers, the presence of macrophages in tumor microenvironmenthas been correlated with unfavorable prognosis. Therefore, visual diagnosis of macrophages in tumor microenvironmentis great significance for us to comprehend the occurrence and progress of tumor. Multiphoton microscopy (MPM) withsubcellular resolution based on second harmonic generation (SHG) and two-photon excited fluorescence (TPEF) is verysuitable for real-time detecting morphological and structural changes in biological tissues without tissue staining andexogenous probe molecule. In this study, we describe the use of label-free MPM for analyzing the intratumoraldistribution and morphological changes of macrophages in breast tumor microenvironment. Our results indicated thatMPM can accurately identify macrophages in breast tumor tissues. MPM images were well consistent hematoxylin andeosin (H&E) stained images. This work would provide the basis for further quantifying these changes using MPM. Withthe development of miniaturized imaging devices, MPM may be a promising imaging technique for clinicians to studyvarious structural features in tumor microenvironment.
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