Begin by positioning an anesthetized mouse on its right side on a heated surgical stand to expose its left abdomen. Anchor the mouse's front end and hind limbs with paper tape, ensuring spleen visibility. Next, disinfect the skin at the surgical site by applying antiseptic generously.
Confirm that the mouse is fully anesthetized by administering a toe pinch. Using forceps, lift the skin of the upper left abdomen and make a 10-millimeter circular incision in the skin and muscle layer using castroviejo scissors. Locate the pancreas attached to the spleen to determine the direction in which the cross stitch should be placed.
Use a 5/0 silk suture to place the first stitch in the muscle layer at the identified location. Secure the end with three to five knots. Continue to stitch directly across the incision.
Then cut and leave a tail of approximately five centimeters. Repeat the stitching, this time perpendicular to the original stitch. Next, carefully lift and position the pancreas over the cross stitch.
Place a purse string stitch approximately one millimeter from the hole, interlacing the skin and muscle layer. Position the window frame so that the circular incision edges are located within the window's groove. Now, fasten the implanted window by tightly tying down the 5/0 silk.
Load 100 microliters of liquid cyanoacrylate adhesive into a 10-millimeter syringe. Apply a delicate stream of compressed air toward the tissue for about 10 seconds to dry it. Using forceps, clasp the window frame by its outer edge and lift carefully to ensure separation of the pancreas from the base of the window frame.
Dispense a thin layer of liquid cyanoacrylate adhesive along the recess of the window, avoiding the pancreas tissue. Then use vacuum pickups to lift the five-millimeter cover slip. Gently position the cover slip in the center of the optical window frame and maintain light pressure for approximately 25 seconds to allow the adhesive to set.
Using forceps, detach the cover slip from the vacuum pickups. Next, tighten the cross stitch sutures to secure the pancreas to the cover slip and cut the suture ends. Finally, remove the tape from the mouse.
Switch off the isoflurane vaporizer and relocate the mouse to a clean cage. Increased lateral and axial stability of the pancreatic tissue was observed after the settling period. Imaging with the SWIP showed lower levels of drift compared to the abdominal and pancreas imaging windows.
Serial imaging of the murine pancreas was performed after cerulein treatment. Lobule relocation was observed after two consecutive days. The SWIP can also be used for long-term imaging, allowing visualization and quantification of vacuole motility.
For example, the average speed of vacuoles in the murine pancreas increased by approximately 10%after cerulein treatment, relative to PBS. Cerulein treatment also increased the average turning frequency of subcellular structures by 10%However, there was no significant difference in net speed, directionality, or cumulative distance between treatments. SWIP enables visualization and capture of tumor cell migration.
Collective cluster cell migration of tumor cells was observed over short time periods. Single cell migration of tumor cells and macrophages was also observed.
