Intravital multiphoton microscopy

Depth profile images of the micelle brain distribution were collected on a multiphoton microscope system equipped with a pulse laser (InSight DeepSee, Spectra Physics, Santa Clara, CA) and an Apochromat ×25 water immersion objective lens (2.0 mm WD, 1.10 NA) (Nikon A1R MP, Nikon Corp.). Imaging was performed using an excitation wavelength tuned at 1100 nm. The mice were anaesthetized with 1.5–2% isoflurane, and the head was stabilized with a head holder (SGM-4, Narishige Co., Ltd., Tokyo, Japan). The scalp and dura were removed to create a 3-mm diameter cranial window over the right hemisphere (2 mm lateral from the midline, 2 mm posterior to the bregma). After removing the skull bone, a glass bottom dish (diameter 40 mm, thickness 0.17 mm, WillCo-dish^®, WillCo Wells B.V., Amsterdam, the Netherlands) was attached to the exposed brain tissue.