Label-free Nonlinear Optical Microscopic Imaging Of Fresh Pancreatic Tissues

Pancreatic cancer and pancreatitis are the most common human diseases in exocrine pancreas. Pancreatic cancer is a malignant tumor that is the fourth leading cause of cancer-related mortality in the worldwide, with an overall5-year survival rate of1-5%。 Chronic pancreatitis is a high risk factor of pancreatic cancer, and acute pancreatitis also shows a relatively high death rate. It is hard to detect tiny neoplastic lesions in abnormal pancreatic tissues using traditional medical imaging methods. Meanwhile, the current studies on exocrine pancreatic diseases are limited to cells in culture or tissue sections. The research methods based on cells or tissue sections do not show the ability to identify multiple components involved in the diseases simultaneously, and thus provide information on the relationships between these components indirectly. During the past two decades, the label-free nonlinear optical microscopic imaging technique based on two-photon absorption and second harmonic generation emerged as a novel intravital imaging mordality. It can be used to obtain the real-time and high-resolution3-D images of thick tissues without the need of staining or the other processing procedures of the tissues. In this study, we applied the label-free nonlinear optical microscopic imaging technique to the visualization of exocrine pancreatic tissues for the first time and evaluated its potential for the investigations of pancreatic cancer and pancreatitis according to the challenges metioned above.The histological morphology of the fresh normal pancreas, pancreatic cancer tissues and subcutaneous pancreatic tumor xenografts harvested at different stages were observed and compared using the label-free nonlinear optical microscopic imaging technique. In the optical images of the normal pancreas, the detaild structural morphology of the exocrine pancreatic tissues such as the acini and the collagen fibers were observed. In comparison with the normal pancreas, the optical images of the pancreatic cancer tissues show great differences in cell shape and the content of collagen fibers. The subcutaneous pancreatic tumor xenografts harvested at different stages were further imaged, and the changes of cell size, the content and the structure of the collagen fibers were observed during the growth of the tumors. In addition, we developed the orientation-dependent gray level co-occurrence matrix method for the quantitative analysis of the morphology of the collagen fibers. Our results show that the label-free nonlinear optical microscopic imaging technique is effective for the characterization of the high-resolution3-D structures of the fresh exocrine pancreatic tissues and the differences of histological between normal and abnormal tissues. It represents a powerful tool for the real-time and accurate detection of neoplastic lesions and the investigations of the pathological process during pancreatic cancer and pancreatitis.Based on the multichannel detection of the intrinsic optical signals and the cell shapes, we observed and characterized multiple components that are involved in the diseases simultaneously during the onset and development of chronic pancreatitis. The pancreatic tissues harvested at1day,7days, and28days after the induction of chronic pancreatitis were imaged respectively. Multiple components such as the pancreatic Stellate Cells (PSCs), the inflammatory cells, and the collagen fibers were identified. The changes of the above components during the chronic pancreatitis were analyzed, including the infiltration of the inflammatory cells, fibrosis, morphological alteration and activation of PSCs. The above results indicate that the label-free nonlinear optical microscopic imaging technique allows simultaneous identification of PSCs and other multiple pancreatic components within3-D tissue environment. It is prospect for intravital observation of dynamic events under natural physiological or pathological conditions, and might help uncover the key mechanisms of exocrine pancreatic diseases, leading to more effective treatments.In the purpose to study exocrine pancreatic diseases in an environment close to natural physiological or pathological conditions, the chamber for intravital imaging of rat pancreatic tissues were designed and used to visualze the intravital dynamic process during acute pancreatitis. In the time-serial nonlinear optical images of the pancreatic tissues in living animals, the leucocytes circulating in the ducts, the leucocytes attached to the endothelium, and the leucocytes moving in the amoeboid migration mode were observed. More importantly, the alterations of multiple components including the morphological changes of PSCs, the activation of PSCs, and the destruction of acinar structure were simultaneously characterized using the label-free nonlinear optical microscopic imaging technique, which makes it possible to observe the interactions between these components. More detailed study of pancreatic tissues in living animals would be of benefit for the observation of the dynamic processes of the interactions between multiple components, and provide a new view for the pathological investigation related with the exocrine pancreatic diseases.