Quantifying Neuronal Axon Outgrowth Angles

For fluorescence imaging the live cortical samples were rinsed once with phosphate buffered saline (PBS) and then incubated for 30 minutes at 37°C with 50 nM Tubulin Tracker Green (Oregon Green 488 Taxol, bis-Acetate, Life Technologies, Grand Island, NY) in PBS. The samples were then rinsed twice with PBS and immersed in fresh PBS for imaging. Fluorescence images were taken using a standard Fluorescein isothiocyanate -FITC filter: excitation/emission of 495 nm/521 nm. Axon outgrowth was tracked using the NeuronJ plugin for ImageJ (http://rsbweb.nih.gov/ij). For analysis all axons were divided into segments of ∼20 µm per segment. The angle of each segment with respect to the surface direction was measured (see Fig. 1b; nanorods point in the π radians direction for all surfaces, as shown in Fig. 1a), and plotted in histograms that quantify angular axonal outgrowth for each type of surface (see below). All surfaces were imaged using an MFP3D Atomic Force Microscope (AFM), using AC mode operation and AC 160TS cantilevers (Asylum Research, Santa Barbara, CA). Surfaces were imaged both before and after neuronal culture, and no significant change in topography was observed.