Semi-automatic stitching of filamentous structures in image stacks from serial-section electron tomography

Abstract

We present a software-assisted workflow for the alignment and matching of filamentous structures across a 3D stack of serial images. This is achieved by combining automatic methods, visual validation, and interactive correction. After an initial alignment, the user can continuously improve the result by interactively correcting landmarks or matches of filaments. Supported by a visual quality assessment of regions that have been already inspected, this allows a trade-off between quality and manual labor. The software tool was developed to investigate cell division by quantitative 3D analysis of microtubules (MTs) in both mitotic and meiotic spindles. For this, each spindle is cut into a series of semi-thick physical sections, of which electron tomograms are acquired. The serial tomograms are then stitched and non-rigidly aligned to allow tracing and connecting of MTs across tomogram boundaries. In practice, automatic stitching alone provides only an incomplete solution, because large physical distortions and a low signal-to-noise ratio often cause experimental difficulties. To derive 3D models of spindles despite the problems related to sample preparation and subsequent data collection, semi-automatic validation and correction is required to remove stitching mistakes. However, due to the large number of MTs in spindles (up to 30k) and their resulting dense spatial arrangement, a naive inspection of each MT is too time consuming. Furthermore, an interactive visualization of the full image stack is hampered by the size of the data (up to 100 GB). Here, we present a specialized, interactive, semi-automatic solution that considers all requirements for large-scale stitching of filamentous structures in serial-section image stacks. The key to our solution is a careful design of the visualization and interaction tools for each processing step to guarantee real-time response, and an optimized workflow that efficiently guides the user through datasets. Author summaryElectron tomography of biological samples is used for a 3D reconstruction of filamentous structures, such as microtubules (MTs) in mitotic and meiotic spindles. Large-scale electron tomography can be applied to increase the reconstructed volume for the visualization of full spindles. For this, each spindle is cut into a series of semi-thick physical sections, of which electron tomograms are acquired. The serial tomograms are then stitched and non-rigidly aligned to allow tracing and connecting of MTs across tomogram boundaries. Previously, we presented fully automatic approaches for this 3D reconstruction pipeline. However, large volumes often suffer from imperfections (i.e. physical distortions) caused during sectioning and imaging, making it difficult to apply fully automatic approaches for matching and stitching of numerous tomograms. Therefore, we developed an interactive, semi-automatic solution that considers all requirements for large-scale stitching of microtubules in serial-section image stacks. We achieved this by combining automatic methods, visual validation and interactive error correction, thus allowing the user to continuously improve the result by interactively correcting landmarks or matches of filaments. We present large-scale reconstructions of spindles in which the automatic workflow failed and where different steps of manual corrections were needed. Our approach is also applicable to other biological samples showing 3D distributions of MTs in a number of different cellular contexts.