Neurient: an algorithm for automatic tracing of confluent neuronal images to determine alignment.

A goal of neural tissue engineering is the development and evaluation of materials that guide neuronal growth and alignment. However, the methods available to quantitatively evaluate the response of neurons to guidance materials are limited and/or expensive, and may require manual tracing to be performed by the researcher. We have developed an open source, automated Matlab-based algorithm, building on previously published methods, to trace and quantify alignment of fluorescent images of neurons in culture. The algorithm is divided into three phases, including computation of a lookup table which contains directional information for each image, location of a set of seed points which may lie along neurite centerlines, and tracing neurites starting with each seed point and indexing into the lookup table. This method was used to obtain quantitative alignment data for complex images of densely cultured neurons. Complete automation of tracing allows for unsupervised processing of large numbers of images. Following image processing with our algorithm, available metrics to quantify neurite alignment include angular histograms, percent of neurite segments in a given direction, and mean neurite angle. The alignment information obtained from traced images can be used to compare the response of neurons to a range of conditions. This tracing algorithm is freely available to the scientific community under the name Neurient, and its implementation in Matlab allows a wide range of researchers to use a standardized, open source method to quantitatively evaluate the alignment of dense neuronal cultures.