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 three-dimensional (3D) stack of serial images. This is achieved by combining automatic methods, visual validation, and interactive correction. After the computation of an initial automatic matching, 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 labour. The software tool was developed in an interdisciplinary collaboration between computer scientists and cell biologists 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 dealing with imperfect data 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 visualisation of the full image stack is hampered by the size of the data (up to 100 GB). Here, we present a specialised, interactive, semi-automatic solution that considers all requirements for large-scale stitching of filamentous structures in serial-section image stacks. To the best of our knowledge, it is the only currently available tool which is able to process data of the type and size presented here. The key to our solution is a careful design of the visualisation and interaction tools for eachThis is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

Three-dimensional structure of kinetochore-fibers in human mitotic spindles

“…This is true for our analysis of MT length distribution ( Figure 4 ; Figure 4-figure supplement 1 ) and our measurements of minus-end distance to the spindle poles and minus-end positioning ( Figure 5 ; Figure 5-figure supplement 2 ). In combination with a semi-automatic segmentation and stitching of MTs (Lindow et al, 2021; Weber et al, 2012), our approach enabled us to reliably model individual MTs overall section borders, thus allowing a quantitative study of MT length and end-positioning in whole spindles. In the future, we will use this routine approach to quantify MT organization also in other mammalian systems, such as RPE-1 and U2OS cells.…”

“…As previously published (Redemann et al, 2014;Weber et al, 2012) MTs were automatically segmented using the ZIBAmira (Zuse Institute Berlin, Germany) software package (Stalling et al, 2005). After manual correction of MT segmentation, the serial tomograms of each recorded cell were stitched using the segmented MTs as alignment markers (Lindow et al, 2021).…”

“…In combination with a semi-automatic segmentation and stitching of MTs (Lindow et al, 2021;Weber et al, 2012), our approach enabled us to reliably model individual MTs overall section borders, thus allowing a quantitative study of MT length and end-positioning in whole spindles. In the future, we will use this routine approach to quantify MT organization also in other mammalian systems, such as RPE-1 and U2OS cells.…”

The Caenorhabditis elegans centrosome is surrounded by a membrane reticulum, the centriculum, that affects centrosome size and function

“…The Amira 6.5.0 (release 2018-03-07; Thermo Fisher Scientific) software with XIMagePAQ, XMesh, and XSkeleton extension packages was used for microtubule segmentation. Microtubules were segmented in individual sections prior to serial stitching based on a previously published template matching and stitching algorithms (Lindow et al, 2021; Weber et al, 2011). Segmented vector datapoints (in tomogram sections #6 and #7) for MTs from the same tomogram dataset (Redemann et al, 2017).…”

A Survey of Visualization and Analysis in High‐Resolution Connectomics

“…Lindow et al [LBD * 21] have developed a software‐assisted algorithm for the alignment and matching of filamentous structures in serial‐section EM tomography. It provides visualization and interaction tools for each registration and reconstruction process to guarantee a real‐time response.…”