Automated Axon Tracking of 3D Confocal Laser Scanning ...

points are lying on the axons or in the background. This minimizes the chances of seed pointsdetected outside the axons, either due to noise or other structures in the image. Figure 3 showsthe automatic detected seed points in the MIP image and their alignment towards the center.(a)(b)Figure 3 – Automatic detection and alignment of seed points: (a) seed points detected on a grid with a resolution of20 pixels, and (b) Center-aligned seed points. The scale bars in the figures correspond to 6μm.The two-dimensional MIP tracking method begins by defining directional templates for eachof sixteen quantized directions for detecting the edges, angle of orientation, and the center pointsof axons present in the MIP image. As mentioned earlier, the seed points detected automaticallyin the MIP image are aligned towards the center of the axon on which they lie. With the firstunaligned seed point as center, the edges of the corresponding axon are detected by correlatingthe templates with the image at varying distances from this seed point. The angle at which thetemplate response is greatest is considered as the angle of orientation of the axon at thatparticular location. The distances from either side of the center at which the template response isgreatest are considered as the edges. This edge information is then used to align the seed points11