This protocol describes stabilized and repetitive cellular-level in vivo imaging of the pancreas with a novel pancreatic intravital imaging window. The main advantage of this technique is that it is possible to perform motionless, three-dimensional imaging with resolution up to the cellular level over three weeks in the pancreas of a live mouse. Begin by preparing the surgical platform and disinfecting the surfaces with 70%ethanol.
After anesthetization, use a rectal probe with a homeothermic-controlled heating pad to monitor the body temperature. Remove hair from the left flank of the mouse and apply alcohol and iodine alternatively, rotating from the incision site to the outer surface, never going back to the incision site, and ending with iodine. Then, make a 1.5-centimeter incision on the left flank of the mouse, dissecting the skin and the muscle.
Use a black or nylon 4-0 suture to perform a purse string suture in the incision margin. Pull the spleen carefully with ring forceps and identify the pancreas. Placing the window at the flank of the mouse, pass the spleen and the pancreas through the open space of the window.
Then, gently place the pancreas on the plate of the imaging window while placing the spleen on the open space of the window. Inject PANC-1 nuclite red directly into the pancreas. Use a 31-gauge catheter needle to apply drops of n-Butyl cyanoacrylate glue to the margin of the imaging window, ensuring a minimal amount of glue is applied.
Gently apply a 12-millimeter round cover glass to the margin of the imaging window. Then, hold the suture loop to fit into the lateral groove of the window and tie it three times. Finally, to prevent the interruption of these tight stitches when the mice are awake, cut the maximum proximal site of the tie.
Begin by switching on the intravital microscope, including the laser power. To insert a vascular catheter, apply pressure on the proximal side of the tail with the index and third finger. If necessary heat the tail with a lamp.
Disinfect the tail vein with a 70%ethanol spray, then insert a 30-gauge catheter into the lateral tail vein and visualize the regurgitation of blood in the PE-10 tube. Apply silk tape on the catheter to stabilize it. Inject FITC dextran and TMR dextran, or other fluorescent probes as appropriate, according to the combination of fluorescent probes.
Insert a rectal probe to automatically control the body temperature with a homeothermic heating pad system. Then, insert the pancreatic imaging window prepared during the intravital microscopy setup into the window holder. Transfer the mouse from the surgical platform to the imaging stage.
To perform intravital imaging, start with imaging the pancreas at a low magnification, such as four times, to scan the whole view of the pancreas in the pancreatic imaging window. Once the region of interest has been determined, switch to a higher magnification objective lens, like 20 times or 40 times, to perform cellular-level imaging with lateral and axial resolution approximately 0.5 micrometers and 3 micrometers respectively. Perform Z-stack or time-lapse imaging to observe the 3D structure or cellular-level dynamics such as cell migration.
Intravital microscopy combined with the pancreatic intravital imaging window provides long-term tissue stability that enables the acquisition of high-resolution imaging to track individual islets for up to three weeks. The window implanted in a C57 black 6 N mouse with intravenously-injected anti-CD31 antibody, conjugated with an Alexa 647 fluorophore, facilitated wide-area imaging and magnified 3D-imaging of the pancreas. Acinar cells were identified in the averaged images of pancreatic tissue in the adjacent vasculature, visualized using autofluorescence and anti-CD31 antibody respectively.
Using the mosaic imaging method, which combines a wide-field view with high-resolution imaging, approximately 40 to 50 islets with the adjacent vasculature were visualized in an MIP-GFP mouse. A previous study showed that islets can be tracked for up to three weeks using this stable imaging method. To visualize cancer cells, PANC-1 nuclite red cells were directly implanted into the mouse pancreas during surgery and nearby vessels were stained with anti-CD31 conjugated with Alexa 647.
Using this protocol, wide-field images of pancreatic cancer were generated. This helped delineate the margin of the tumor, as well as achieve high-resolution 3D images at the single cell level. The most critical step in this method is the skillful implantation of the pancreatic imaging window in the mouse.
Using other combinations of the fluorescent mouse cells, and antibody probes, dynamic interaction of nearby cells with either islets or cancel cells could be identified. This method can be widely applied by those exploring the change of the islet in various pathophysiological conditions and micro-environments of the pancreatic cancer in situ.
