This article discusses intravital imaging techniques for visualizing live cells in transgenic mice. It highlights the use of imaging windows to observe cellular dynamics in the pancreas without disrupting normal functions.
Intravital imaging involves visualizing cells of a live animal through an imaging window implanted into the animal. It enables long-term observation of cellular dynamics without compromising their functions.
To begin, take an anesthetized transgenic mouse previously implanted with a pancreatic imaging window supported on a metal plate. The base plate spatially separates the pancreas from the bowel, thereby reducing motion artifacts from physiological movements such as peristalsis.
The transgenic mouse expresses green fluorescent protein or GFP under insulin gene promoter expressed in beta cells of islets in the pancreas.
Next, hold the mouse tail firmly and spray disinfectant to remove any surface contaminants. Insert a catheter into the lateral tail vein. Inject a solution containing antibodies tagged with a red fluorescent probe into the tail vein. These antibodies bind to the cells lining the blood vessels, staining them red.
Now, shift the mouse to an intravital microscopy setup. Insert the pancreatic imaging window into the window holder. Start imaging under low magnification to locate green fluorescing islet clusters surrounded by red fluorescing blood vessels. Increase the magnification to obtain high-quality cellular level imaging of individual beta cells.
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 the homeothermic heating pad system. Then, insert the pancreatic imaging window prepared during 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.
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