Objective: The liver plays a central role in lipid metabolism. During fasting and feeding, the fatty acid trafficking between adipose tissue and liver induces accumulation and dissociation of dynamic hepatic lipid droplets (LDs). Herein, we established an intravital 2-photon imaging technique to longitudinally visualize the dynamic in vivo alteration of hepatic LD deposition during fasting and refeeding in the liver of live mouse. Methods: Intravital 2-photon imaging of liver was performed to observe hepatic LD alteration induced by fasting for different periods of time, 12, 24, and 48 hours followed by refeeding. Hepatic LDs were fluorescently labelled in vivo by intravenous injection of Seoul-Flour 44 and visualized by custom-built intravital 2-photon microscope. Results: Significant increases of the number and size of hepatic LDs were observed by intravital 2-photon imaging of the liver after 12 hours of fasting. The degree of hepatic LD accumulation continuously increased with fasting up to 48 hours. Remarkably, with refeeding for 24 hours, the hepatic LDs accumulated by fasting were fully dissociated and the LD occupancy in the liver was recovered to the normal state. Conclusion Utilizing intravital 2-photon microscope with in vivo systemic fluorescent labeling of LD in live mice, dynamic alterations of hepatic LDs such as accumulation and dissociation by fasting and refeeding were successfully visualized at a subcellular level in vivo. The established method enabling the in vivo visualization of LDs will be a useful tool to investigate the pathophysiology of various diseases associated with dysregulated lipid metabolism.

Longitudinal imaging of murine pancreas is technically challenging due to the mechanical softness of the tissue influenced by peristalsis. Here, we report a novel pancreatic imaging window for long-term stabilized cellular-level observation of the islets in the pancreas in vivo. By spatially separating the pancreas from the bowel movement and physiologic respiration with a metal plate integrated in the imaging window, we successfully tracked the pancreatic islets up to three weeks and visualized the dumbbell-shape transformation from the single islet. This window can be a useful tool for long-term cellular-level visualization of the microstructure in the pancreas.

Immunoreactive dynamics of tumor-infiltrating lymphocytes (TILs) within the tumor microenvironment in breast cancer are not well understood. This study aimed to investigate the spatiotemporal cellular dynamics of TILs in breast cancer models. Breast cancer cells were implanted into the dorsal skinfold chamber of BALB/c nude mice, and T lymphocytes were adoptively transferred. Longitudinal intravital imaging was performed, and the spatiotemporal dynamics of TILs were assessed. In the 4T1 model, TILs progressively exhibited increased motility, and their motility inside the tumor was significantly higher than that outside the tumor. In the MDA-MB-231 model, the motility of TILs progressively decreased after an initial increase. TIL motility in the MDA-MB-231 and MCF-7 models differed significantly, suggesting an association between programmed death-ligand 1 expression levels and TIL motility, which warrants further investigation. Furthermore, intravital imaging of TILs can be a useful method for addressing dynamic interactions between TILs and breast cancer cells.

Dental pulp is composed of nerves, blood vessels, and various types of cells and surrounded by a thick and hard enamel-dentin matrix. Due to its importance in the maintenance of tooth vitality, there have been intensive efforts to analyze the complex cellular-level organization of the dental pulp in teeth. Although conventional histologic analysis has provided microscopic images of the dental pulp, 3-dimensional (3D) cellular-level visualization of the whole dental pulp in an intact tooth has remained a technically challenging task. This is mainly due to the inevitable disruption and loss of microscopic structural features during the process of mechanical sectioning required for the preparation of the tooth sample for histological observation. To accomplish 3D microscopic observation of thick intact tissue, various optical clearing techniques have been developed mostly for soft tissue, and their application for hard tissues such as bone and teeth has only recently started to be investigated. In this work, we established a simple and rapid optical clearing technique for intact mouse teeth without the time-consuming process of decalcification. We achieved 3D cellular-level visualization of the microvasculature and various immune cell distributions in the whole dental pulp of mouse teeth under normal and pathologic conditions. This technique could be used to enable diverse research methods on tooth development and regeneration by providing 3D visualization of various pulpal cells in intact mouse teeth.