Three-week-old mice were anaesthetized with intraperitoneal injection of ketamine and xylazine (this is a cocktail), and the skin around the head region was shaved using a mechanical trimmer and depilatory cream. The mouse was placed on a heated stage, and the head and the ear were supported by a custom-made stage. A glass coverslip was placed against the skin in the junction region between the head and the ear. Image stacks of the skin were acquired with a LaVision TriM Scope II (LaVision Biotec) microscope equipped with a Chameleon Vision II (Coherent) two-photon laser. A laser beam (at 940 nm for GFP and 1040 nm for RFP, respectively) was focused through a ×20 water immersion lens (N.A. 1.0; Olympus) and scanned with a field of view of 0.25 to 0.5 mm2 at 600 Hz. Serial optical sections were acquired in 2–3-µm steps to image a total depth of ~100 µm of tissue in 5-min intervals. Several phases covering the transition from quiescent to growth stages were analysed (telogen to anagen phases). Distinctive inherent landmarks in the skin were used to navigate back to the original field of view and visualize the same follicles in separate experiments. Anaesthesia was maintained throughout the course of the experiment with vaporized isofluorane delivered by a nose cone.