Principal setup for intravital 2-photon microscopy of neutrophils in the tibial BM. (A) (1) Schematic overview of the position of imaging in the experimental animal. The animal is anesthetized during the entire procedure. (2) Schematic drawing of the tibial bone in the respective orientation within the animal. (3) Virtual side view of the tibia to demonstrate the thinning approach using an electric drill (silver star). (B) Sample images taken from intravital experiments. Bone (second harmonic generation signal, brown) demonstrating the fine structure of the calcified tibial bone from the outside and inside. Note the appearance of numerous pits on the endosteal surface. Neutrophils (green) and blood vessels (red) of the same stack are shown in a top view as well as in a 60° rotation to demonstrate the thickness of a typical image stack. The respective video is provided as supplemental Video 1. The images are representative of > 20 animals.