Imaging hematopoietic cell movement in the tibial BM of a live mouse. (A) Setup of the mouse in the imaging chamber. The animal is ventilated through a tracheal tubus by a mechanical small animal respirator receiving a mixture of O2/isoflurane for narcosis at the depicted rates and volumes. The gross area of bone thinning and imaging is boxed and the approximate position of the microscope lens is indicated with a dashed arrow. Imaging is performed anywhere within the red (thinned) area of the tibia. All fluorescently marked cells detectable within this area are recorded by individual Z-stacks. (B) A close-up micrograph of a mouse fixed for imaging with the position of the tibia indicated (white arrow). (C) Drawing of the bones of the hind leg as they are positioned for the animal shown in panel B. (D) Enlargement of the tibial bone, rotated 90° clockwise with the area of preparation indicated by the red color. (E) Virtual side view through the tibial bone in the area of imaging to demonstrate the process of careful bone thinning by an electric drill (silver star), which is constantly monitored with a stereomicroscope until finished. The residual bone surface is kept at 30 to 50 μm thickness, whereas a nondrilled tibia has a wall thickness of approximately 100 μm. Of note: extreme care has to be taken to not totally remove the covering bone. If this happens, the contents of the BM will leak out, rendering imaging impossible in a live mouse with intact blood circulation. The figure on the right shows as an example rendered images of a bone with fluorescently labeled (green) cells below the bone surface. The typical XYZ dimensions of the area of imaging are depicted. (F) To demonstrate the practicability of the transplantation/visualization technique, BM cells were stained with CFSE (green) or CTO (red) and transplanted together in equal numbers in recipient animals. Cells were visualized by 2-photon intravital microscopy in the region close to the endosteum in the tibia (brown = autofluorescence signal of the calcified bone) 16 hours postinjection. The corresponding movie is available as supplemental Video 1.
