Vaa3d

All imaging experiments were carried out using a dual scan head raster scanning confocal microscope and control software developed by Prairie Systems (Middleton, WI) and incorporated into a BX51 upright microscope (Olympus America, Inc., Center Valley, PA). A x-y translation stage with integrated RIVETS system (Figure 4c) was used to position the tissue for imaging. Two-photon excitation was used with a Mai Tai pulsed Ti:Sapphire laser (Spectraphysics). Custom linescan patterns (Figure 5) were implemented using software generously provided by Bill Karsh (Applied Physics and Instrumentation Group, Janelia Farm Research Campus). All image analysis and presentation was performed using ImageJ (rsbweb.nih.gov/ij/), Matlab 2013a (www.mathworks.com) and plotted using IgorPro with the exception of Figure 6d which was produced using Vaa3D (penglab.janelia.org/proj/Vaa3D/Vaa3D/About_Vaa3D.html).