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High content cell-based screens are rapidly gaining popularity in the context of neuronal regeneration studies. To analyze neuronal morphology, automatic image analysis pipelines have been conceived, which accurately quantify the shape changes of neurons in cell cultures with non-dense neurite networks. However, most existing methods show poor performance for well-connected and differentiated neuronal networks, which may serve as valuable models for inter alia synaptogenesis. Here, we present a fully automated method for quantifying the morphology of neurons and the density of neurite networks, in dense neuronal cultures, which are grown for more than 10 days. MorphoNeuroNet, written as a script for ImageJ, Java based freeware, automatically determines various morphological parameters of the soma and the neurites (size, shape, starting points, and fractional occupation). The image analysis pipeline consists of a multi-tier approach in which the somas are segmented by adaptive region growing using nuclei as seeds, and the neurites are delineated by a combination of various intensity and edge detection algorithms. Quantitative comparison showed a superior performance of MorphoNeuroNet to existing analysis tools, especially for revealing subtle changes in thin neurites, which have weak fluorescence intensity compared to the rest of the network. The proposed method will help determining the effects of compounds on cultures with dense neurite networks, thereby boosting physiological relevance of cell-based assays in the context of neuronal diseases. |
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