Intravital Imaging of CNS Vasculature

A Leica SP5 confocal microscope equipped with a ~20 Å water immersion lens (Leica HCX-APO-L, N.A. 1.0) and a tunable 16 W Ti/sapphire IR laser tuned to 800 nm (Chameleon Coherent, Inc.) was used for 2PLSM intravital imaging. Four-dimensional XYZT images with an XY dimension of 760 Å × 760 μm and a Z dimension of 100–130 μm (collected at 5 μm Z intervals) were obtained at 20 s intervals. In a parallel study, an independent cohort of nude mice (nu/nu, n = 3) was used to image the CNS vasculature. These mice were maintained on a Teklad 2018 s alfalfa free diet (Harlan Lab) starting 2 weeks before imaging to reduce autofluorescence. Open cranial windows were prepared, as previously described.^{51 (link),67 (link)} Each mouse was administered 200 μg of A647-PhMV-mPEG (~2.56 × 10¹³ particles). Intravital imaging was carried out from preinjection to 2 h postinjection. The data sets were analyzed using Imaris (BitPlane, Inc.) to generate images and to determine clearance.