Tumor microenvironment (TME) is composed of tumor cells, stromal cells and the extracellular matrix (ECM), that it plays an important role in the occurrence and development of tumors, such as promoting local drug resistance, immune escape, and distal metastasis. Based on second harmonic generation (SHG) and two-photon excited fluorescence (TPEF), multiphoton microscopy (MPM) has the ability to label-freely visualize extracellular matrix and cells in the TME. In addition, combined SHG and TPEF imaging can be used to generate similar pathological images, providing additional information for pathologists and even surgeons. Cancer cells, adipocytes, microvessel, collagen fibers, and tumor-infiltrating lymphocytes (TILs) which were the important components in the TME were imaged using MPM in this study. The results showed that MPM can clearly present the tissue structure and cell morphology in the microenvironment. With the development and widely used of MPM, in the future, MPM imaging may be able to perform clinical imaging of the tumor microenvironment without the need for invasive operations. MPM may become a novel imaging tool for imaging various prognostic factors in breast cancer.
Hepatic steatosis, the accumulation of lipids within hepatocytes, is defined as intrahepatic fat of at least 5% of liver weight and is an important histological feature. Steatosis may manifest in a variety of liver diseases, and its clinical significance depends on the degree of hepatic steatosis. Excess intrahepatic fat content is a risk factor for disease progression. Increased hepatic steatosis could trigger metabolic dysfunction leading to insulin resistance, dyslipidemia, cardiovascular disease, and progression to non-alcoholic steatohepatitis (NASH), cirrhosis, and hepatocellular carcinoma (HCC). In many chronic liver diseases, hepatic steatosis is associated with increased hepatic fibrosis. Clinical methods of quantifying hepatic steatosis remain semi-quantitative, with potential limitations in precision. Moreover, the evaluation of hepatic steatosis and fibrosis cannot be performed simultaneously. In this work, multiphoton microscopy (MPM) combined two-photon excited fluorescence with second harmonic generation imaging was used to identify the hepatic steatosis and fibrosis in chronic liver disease. The result showed that MPM has the potential to be a pathological diagnostic tool for hepatic steatosis and fibrosis.
Breast tumor microenvironment is composed of tumor cells, tumor-related cells, blood vessels and a series of extracellular matrix fibers. Tumor-infiltrating lymphocytes (Tils) in the microenvironment can directly or indirectly influence other components in the microenvironment, thus promoting the occurrence and development of tumors. Multiphoton microscopy (MPM) is based on two-photon excited fluorescence (TPEF) and second harmonic generation (SHG). And it does not require the use of exogenous probes or staining of tissue. In this study, large-size images with subcellular resolution of the breast tumor tissue was performed using MPM. The results showed that the MPM could clearly distinguish intraepithelial Tils (iTils) and stromal compartments Tils (sTils) by comparing the signal strength and morphological difference. It demonstrated that MPM could be used as a means of pathological diagnosis and in clinical application.
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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