To begin, place an intubated mouse with an open thoracotomy onto a base plate of the imaging chamber with the open left chest facing up. With silk tape, secure the mouse over its face, front paws and tail. Apply glue to the bottom side of the cover glass attached to the top plate.
Next, lower the top plate until the cover glass contacts the lung, allowing the glue to touch the lung's surface. Hold the cover glass against the lung. Inflate the lung for one to two seconds, so that the area of glue adheres to the surrounding tissue.
Place a thumb nut over each bolt and secure the top plate. For two photon imaging, place the entire imaging chamber with the mouse onto a microscope stage. Use dichroic filters to separate the signals from the GFP reporter, Q dots, and collagen generate SHG signal.
Next, set a titanium sapphire femtosecond pulse laser to an excitation wavelength of 890 nanometers at the lowest possible power. Apply one milliliter of water over the cover glass on the top plate. Now lower the water immersion objective into the water and focus on the surface of the lung.
Turn on the base plate heater, keeping the temperature between 35 to 37 degrees Celsius. Acquire image stacks with acquisition software of choice. At 24 hours post-transplant, there were more neutrophils in the lung relative to two hours post-transplant.
