Supplementary Figure 2: Accuracy of bitmap image-based Sholl Analysis.
(a) Maximum intensity projection of an Alexa 594 dye-filled layer-5 pyramidal neuron of juvenile mouse visual cortex1. Arrowheads highlight the apical tuft (top) and the soma (bottom). (b) Manually reconstructed dendrites (blue) and axon (magenta) of the neuron in (a). (c) Linear Sholl plots from bitmap images (dots), following either manual segmentation ("user segm.") or automated segmentation of the image stack. For comparison, results from reconstruction-based analysis are plotted for the axon (dashed magenta line) and dendrites (solid blue line). The plot of the left corresponds to the basal region populated by both axonal and dendritic arbors, while the plot on the right corresponds to apical dendrites. (d) Tukey mean-difference plot to examine the agreement between the bitmap approach and traced reconstruction approach for three different neurons (numbered and colorcoded). Each data point represents the count of intersections at a particular distance from the apical bifurcation: pairwise differences between the two approaches at each distance are plotted against each mean. The 95% limits of agreement for individual cells are shown to the right, as is the average for all three cells and the average bias (dotted lines). 1. Buchanan, K.A. et al. Target-specific expression of presynaptic NMDA receptors in neocortical microcircuits. Neuron 75, 451-466 (2012).
