Cranial Window-based Intravital Imaging in a Mouse Model: An Imaging Technique to Study the Behavior of Pre-injected Cancer Cells in the Brain of a Murine Model

At the appropriate experimental time point, place the mouse face up in an imaging box and set the 25x water objective to the lowest z-position. Add a large drop of water to the objective and transfer the imaging box into the 37 degrees Celsius dark climate chamber of the microscope.

Bring the objective to the cranial imaging window coverslip until the water drop touches the coverslip, and using the epifluorescence mode, observe the tumor through the eyepiece to bring the cells into focus.

Tune the laser to the correct wavelength and select live mode. After selecting several positions of interest for imaging, record their coordinates in the software. Define a z-stack for each position to acquire the maximal volume of tumor cells without compromising the tumor cell resolution, with the step size between images set to 3 micrometers.

Then, acquire images of the tumor volume at different positions every 20 minutes for 2 hours, adding water to the objective before each image acquisition. After the last image is acquired, transfer the mouse to a heating pad with monitoring until full recovery.