Intravital multiphoton microscopy of murine skull BM. (a) Schematic overview of the imaging region of mouse skull. (Left) Set-up of an anaesthetized mouse on a custom-made intravital imaging stage. (Middle) Removal of skin and exposure of the skull bone. (Right) A snapshot of the skull BM microenvironment in a LysM-GFP mouse, consisting of GFP⁺ neutrophils (green), sinusoids (vasculature labeled with TRITC dextran; red), and bone collagen matrix (second harmonic generation; blue). Bar, 200 µm. (b) Representative time-lapse images showing resting state neutrophil activity in skull BM in a chimeric mouse (LysM-GFP BM transferred into irradiated mT/mG mice; n = 3 mice). The white box specifies the region viewed at high magnification (bottom). The arrow indicates the direction of blood flow, and arrowheads indicate GFP⁺ neutrophils migrating through the sinusoidal endothelium (red), followed by their release into the circulation (white dashed line). Time scale (minutes:seconds) is shown above images. Bars, 40 µm. See also Video 1. (c) Kinetics of circulating neutrophil numbers in WT mice in response to a single s.c. injection of 250 µg/kg G-CSF or 5 mg/kg plerixafor treatment (n = 4 mice per group per time point). All data are shown as mean ± SEM. A representative set of data were shown from three independent experiments. (d) The experimental procedure for multiphoton imaging of the skull BM. This involved preparation of the mouse followed by 2 h of imaging, which involved 10 min of basal imaging and post–G-CSF or -plerixafor s.c. injection.