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.
Necrosis is a form of cell death caused by an external factor of the cell, such as hypoxia. It is usually associated with rapidly growing malignancies in the breast, colon, brain, lungs, kidney, and pancreas. Multiphoton microscopy (MPM) based on intrinsic nonlinear optical signals were used to monitor the morphological changes of biological tissues and identify tumor tissue necrosis in breast cancer patients, as well as surrounding tumor cells and collagen. In this study, we performed MPM imaging of the breast tissue and found that there were two types of necrosis in the breast tissue, namely intraluminal necrosis and interstitial necrosis. Different types of necrosis may have different effects on the prognosis. It means MPM may provide a new assistant tool for pathologists to quickly and effectively identify tumor necrosis. It is expected that rapid identification of tumor necrotic areas can provide prognostic information for early recurrence or death, thus helping to diagnose and treat cancer.
Standard histopathology is well accepted as the gold standard for the diagnosis a wide range of diseases. Despite continuing advances in tissue staining automation, typical histological processing such as formalin-fixed paraffin-embedded are also labour- and time-intensive for treatment decisions in intraoperative histopathologic diagnosis. Multiphoton microscopy (MPM), based on second harmonic generation (SHG) and two-photon excited fluorescence (TPEF), can be a versatile tool that enables label-free mapping of endogenous fluorophores within a fresh specimen, which provides pathology-like images with cellular and subcellular details. Here, we describe the use of label-free MPM for visualizing rat and human ex vivo brain tissue without tissue fixation, processing, and staining. Moreover, MPM is able to identify 6 types of cells in rat cerebrum and cerebellum, including cortical neurons, glia cells, Purkinje cells, pyramidal neurons and granule neurons in hippocampus, as well as epithelial cells in lateral ventricle. In addition, we further demonstrate that MPM can provide definitive pathological features in cerebral ischemia and focal cortical dysplasia (FCD) for assisting pathologic diagnosis. Our work establishes the methodology and augments the diagnostic accuracy of traditional frozen section histopathology. With the development of the miniature two-photon microscope, MPM will show more potential as a practical clinical tool for providing intraoperative reference image guidance of resection in neurosurgery.