Three-dimensional segmentation of vesicular networks of fungal hyphae in macroscopic microscopy image stacks

Automating the extraction and quantification of features from three-dimensional (3-D) image stacks is a critical task for advancing many applications, such as the analysis of biological structures. The quantification of biological resistance of a plant tissue to fungal infection through the analysis of attributes such as fungal penetration depth, fungal mass, and branching of the fungal network at large scales requires the union of 3D image acquisition and analysis. From an image processing perspective, these tasks reduce to segmentation of vessel-like structures and the extraction of features from their skeletonization. In order to sample multiple infection events for analysis, we have developed an approach we refer to as macroscopic microscopy. However, macroscopic microscopy produces high-resolution image stacks that pose challenges to routine approaches and are difficult for a human to annotate to obtain ground truth data. We present a synthetic hyphal network generator, a comparison of several vessel segmentation methods, and a minimum spanning tree method for connecting small gaps resulting from imperfections in imaging or incomplete skeletonization of hyphal networks. Qualitative results are shown for real microscopic data. We believe the comparison of vessel detectors on macroscopic microscopy data, the synthetic vessel generator, and the gap closing technique are beneficial to the image processing community.