A novel algorithm to generate kymographs from dynamic axons for the quantitative analysis of axonal transport

The biological and clinical relevance of axonal transport has driven the development of a variety of new approaches to its study, including the generation of fluorescence or brightfield movies of moving cargoes within axons. Kymograph analysis is a simple and effective tool used to analyze axonal transport in neurons. Typically, kymographs are built by having a user trace the path of the axon in one frame of a time-lapse movie and extracting intensity profiles from subsequent frames along that path. This method cannot accommodate movies in which translation of the axon, or changes in axonal orientation or geometry, occur. Both are frequently observed in long-term movies of neurons, both in vitro and in vivo. To solve this problem and automate the creation of kymographs from these movies, we developed a two step algorithm. The first step implemented a simple image registration algorithm that aligned axons based on identification of a reference point on the axon in each image. The second step used a Hough transformation (HT) to automatically detect the axonal contour in each frame. Intensity profiles along this contour were then used to construct a kymograph. This algorithm was able to build an accurate kymograph of mitochondrial and actin transport in dynamic cultured sensory neurons, which were not amenable to previously used analytical methods. Although developed as a tool for analyzing transport, this algorithm is easily modified to analyze movies for the directionality and speed of axonal outgrowth, another metric of interest to neuroscientists.