Fig. 6: Simultaneous in vivo imaging of the glia limitans and the vascular and leptomeningeal CNS borders.

Aqp4-mRuby3 mice were crossed with VE-cadherin-GFP mice creating CNS border reporter mice (Aqp4-mRuby3; VE-cadherin-GFP). Representative images of 2P-IVM imaging of the brain and spinal cord surface of CNS border reporter mice through cranial window (a), skull thinning (b) and cervical spinal cord window (c) are shown. 2P excitation wavelengths of 920 nm and 1045 nm were used simultaneously. a Representative YZ and XY MIP 2P images of the brain surface through a cranial window of a CNS border reporter mouse. Right panel shows a 2 µm thick optical section from the yellow inset on the merged XY MIP image. The AQP4-mRuby3 signal (red) allows for imaging of the glia limitans (white arrowheads) and the VE-cadherin-GFP signal (green) depicts the adherens junctions from the endothelial cells and the leptomeningeal fibroblasts (white arrows). b, c Representative YZ MIP 2P images of the brain (b) and spinal cord (c) surface through a skull thinning (b) and a spinal cord window (c) of a CNS border reporter mouse are shown in the top panels. Bottom panels show representative XY MIP 2P images excluding the skull and dura mater (b) or the dura mater (c) following the dotted line at the YZ MIP images. Right panel shows a 2 µm thick optical section from the yellow inset on the merged XY MIP image. The AQP4-mRuby3 (red) signal allows for imaging of the glia limitans (white arrowheads) and the VE-cadherin-GFP signal (green) depicts the adherens junctions from the endothelial cells and the leptomeningeal fibroblasts (white arrows). Data are representative of 3 mice per condition. GL glia limitans, BV blood vessel lumen, SAS subarachnoid space, MIP maximum intensity projection, SHG second harmonic generation.