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

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Intravital Imaging

NOTE: The time interval between the tumor cell injection and the first intravital imaging session is dependent on the type of tumor cell line used. For the experiments shown in this protocol, 1x 10⁵ GL261 cells were injected and imaged 10 days later.

1. Imaging preparation
   1. Sedate the mouse using isoflurane inhalation anesthesia through a face mask (1.5%–2% isoflurane/O₂ mixture).
   2. Inject the mouse subcutaneously with 100 µL of saline buffer to prevent dehydration.
      NOTE: For long-term imaging, the mouse can be hydrated through a subcutaneous infusion pump.
   3. Place the mouse face-up in an imaging box. Use a metal plate with a 1 mm-diameter hole and small magnets embedded around the aperture to provide fixation of the CIW to the imaging box. Introduce isoflurane through a facemask and ventilate by an outlet on the other side of the box (0.8%–1.5% isoflurane/O₂ mixture). Optionally, use tape to fix the body of the mouse.
      NOTE: Fluorescently labeled dextran for blood vessel visualization or other dyes may be injected intravenously at this point.
   4. Optionally, use a pulse oximeter and a heating probe to monitor the mous's vitals.
      NOTE: In this experiment, it was not necessary to use a pulse oximeter and a heating probe since the mouse was stable throughout the imaging time period (2–3 h). However, for longer imaging times, more thorough monitoring may be needed.
   5. Set the 25x (e.g., HCX IRAPO NA0.95 WD 2.5 mm) water objective to the lowest z-position and add a large drop of water.
      NOTE: The use of a water immersion microdispenser is highly advised for long term experiments since it allows scientists to add water during the experiment. Alternatively, a dry objective can be used.
   6. Transfer the imaging box onto the microscope equipped with a dark climate chamber kept at 37 °C. Bring the objective to the CIW coverslip until the water drop touches it.
   7. Using the epifluorescence mode, observe the tumor through the eyepiece and bring the cells into focus.

2. Time-lapse image acquisition

1. Select several positions of interest to image and record their coordinates in the software. Ensure that the selected positions are representative positions from different sites of the tumor (each tumor can be different, but to ensure consistency, select the same amount of positions that are central to the tumor core and to the edges across all mice).
   NOTE: A tile scan of a part of the tumor or of the whole visible tumor may be performed; however, if the tumor is large, this method will increase the time-lapse between images. In addition, if the tumor cells move fast, it can be challenging to track the same cells over time.
2. Switch to multiphoton mode and tune the laser to the correct wavelength. Note that to avoid photodamage, higher wavelengths are desired.
   NOTE: For Dendra2 imaging, a 960 nm wavelength was used. With the objective used in these experiments, a zoom of 1.3 was sufficient to get a good resolution of the tumor nuclei and scan a representative area of the tumor.
3. Go to the live mode and define a z-stack for each position in order to acquire the maximal volume of tumor cells without compromising the tumor cell resolution. Define the step size between the images as 3 µm.
4. Use the bidirectional mode to increase the scanning speed. The image resolution should be at least 512 x 512 pixels.
5. Acquire images of the tumor volume at different positions every 20 min for 2 h. Add water to the objective before each image acquisition.
   NOTE: Time-lapse images can be automatically acquired by setting the right time-lapse in the software. However, the mouse can move, and the position or z-stack can shift over time, causing data loss. Therefore, it is recommended to perform the time-lapse manually and to check and adjust for xyz shifts in between acquisitions.
6. At this step, optionally, photo-switch the Dendra2 fluorescent marker.
   NOTE: As opposed to time-lapse imaging (which allows studying individual tumor cells properties and how they change over time), this will allow studying the area of tumor cell infiltration in the brain over several days.