This study demonstrates intravital imaging techniques using optical imaging windows to track cancer cell behavior in a live mouse brain. By employing multiphoton microscopy, researchers can visualize individual cells within their native microenvironment.
Intravital imaging through optical imaging windows facilitates high-resolution tracking of individual cells to study their behavior in the native microenvironment in a live animal.
To begin, take an anesthetized mouse with its brain pre-injected with cancer cells and having an implanted cranial window fit inside a metal ring. The cancer cells express nuclear fluorescent proteins.
Position the mouse, face up, in an imaging box. The magnetic holder in the imaging box helps secure the implanted cranial window.
Now, transfer the imaging box inside the environmentally controlled imaging chamber of an inverted microscope to analyze the behavior of cancer cells in the brain.
Under multiphoton mode, set the laser to a suitable wavelength. Focus on the tumor region to detect the fluorescence from cancer cells.
Define a z-stack for each position to acquire multiple intravital images of cancer cells in different optical planes.
This step enables the capture of a significant proportion of cancer cells in the tumor microenvironment without affecting cell resolution.
Subsequently, acquire images from different positions of the tumor for an extended duration.
Tracking the path of cancer cells in different optical planes helps determine individual tumor cell migration using suitable software.
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.
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