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.
Necrosis is a form of cell death which is histologically characterized by homogeneous clusters and sheets of dead cells. Although several studies have indicated that the presence of tumor necrosis in pathological specimens may provide adverse prognostic information in solid tumor, the mechanism of necrosis is still unclear. Based on two-photon excited fluorescence (TPEF) and second-harmonic generation (SHG), multiphoton microscopy (MPM) is commonly used to monitor the morphological changes of biological tissues. In this study, we performed MPM imaging of the breast tissue and found that MPM can be used to rapidly classify the early and late-stage tumor necrosis in invasive breast cancer according to the changes of intracellular proteins. It demonstrated that MPM may provide a new assistant tool for pathologists to quickly and effectively classify the early and late-stage tumor necrosis.
The presence of blood vessel invasion (BVI) in breast tumor microenvironment has been recognized as an unfavorable prognostic factor. Invasion of cancer cells into vessels is one of the critical steps for metastasis. Therefore, visualization of BVI is vital for comprehending the progress of tumor. Multiphoton microscopy (MPM) based on second harmonic generation (SHG) and two-photon excited fluorescence (TPEF) can monitor morphological changes in biological tissues. In this study, we found significant differences in morphology between normal breast blood vessel and abnormal blood vessel encountered with tumor invasion using label-free MPM. Our study demonstrated that MPM has the ability to not only identify BVI in breast tumor environment but also reveal the morphological changes of breast blood vessel. By comparing with the hematoxylin and eosin (HE) stained image, it was confirmed that MPM provides a new assistant tool for pathologists to identify BVI effectively. Keywords:
Fibroblasts are important players in proliferation, invasion, migration and metastasis of cancer cells. Until now, histological methods such as hematoxylin and eosin (H and E) staining are still a gold standard to assess fibroblasts in traditional histopathology. Multiphoton microscopy (MPM) has become a key technology for biological tissues imaging without staining at the cellular level. In this study, MPM based on two-photon excited fluorescence (TPEF) and second harmonic generation (SHG) was used to identify the fibroblasts in breast cancer tissue. Normal fibroblasts with spindle shape and abnormal fibroblasts with stellate shape could be clearly obtained by MPM.
Florid type is a kind of morphological variation of lobular carcinoma in situ (LCIS). Florid lobular carcinoma in situ (FLCIS) has the same cytological features as LCIS, often associated with comedo-type necrosis. Unlike classic lobular carcinoma in situ (CLCIS), which is often managed with close observation or chemoprevention, the treatment guidelines recommend that FLCIS be managed in the same way as ductal carcinoma in situ (DCIS). Therefore, it is critical to accurately identify FLCIS for management purposes. Recently, multiphoton microscopy (MPM) has become a powerful imaging tool for label-free detection of biological tissue. In this work, we obtain high-resolution images of unstained normal and diseased breast specimens by MPM. In order to verify the imaging details, we also obtained hematoxylin-eosin (H and E) stained images of corresponding tissues to compare with the MPM images. Our results indicate that MPM can identify FLCIS and CLCIS through histological characteristics, including cell morphology and collagen structure. With the further improvement of MPM, its diagnostic capabilities of real-time and non-invasive may provide a new option for early detection of breast tumor
Pleomorphic invasive lobular carcinoma (pILC) is a distinct morphological variant of invasive lobular carcinoma (ILC), which is more aggressive than classic invasive lobular carcinoma (cILC) with worse overall prognosis. It is critical to distinguish between pILC and cILC for management and treatment of patients. Multiphoton microscopy (MPM),based on two-photon excited fluorescence (TPEF) and second harmonic generation (SHG), produces subcellular resolution images to show detailed information on structure and cell morphology of tissue sections without staining. In this work, MPM was introduced for label-free identification of normal breast lobules, pILC and cILC, which is consistent with haematoxylin and eosin (H&E) staining. Furthermore, analysis of nuclear area is used to quantitatively reflect their cytological characteristic. These results demonstrate that, MPM has the ability to distinguish between normal breast lobules, cILC, and pILC by histopathological features, which can provide additional indicators to determine adequate treatment strategies of patients with pILC for pathologists and surgeons. In the future, MPM may be used as a diagnostic tool of lobular cancer into routine clinical use.
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