Figure 3.

Confocal imaging techniques to improve depth of imaging. (A) A schematic of typical confocal microscope imaging parameters showing a whole brain slice optically sectioned at 20 µm intervals. Ten z-stack images were used to create a single composite 200 µm image of a LN18-GFP tagged spheroid invading on a brain slice in the X/Y direction. Scale bar = 500 µm. The re-slice function in ImageJ was used to extract an image in the Z-direction along the yellow dotted line. (B) A schematic displaying a whole 300 µm brain slice physically re-sliced on the vibratome into perpendicular 200 µm sections, which are then flipped 90°, rotated clockwise and laid flat onto a microscope slide. A single z-plane image was captured by confocal microscopy revealing a LN18-GFP tagged spheroid invading into a brain slice in the Z-direction. Scale bar = 250 µm. Brain slices were stained for GFAP to mark astrocytes (red).