Automated tracing of microtubules in electron tomograms of plastic embedded samples of Caenorhabditis elegans embryos

Weber, Britta; Greenan, Garrett; Prohaska, Steffen; Baum, Daniel; Hege, Hans-Christian; Müller‐Reichert, Thomas; Hyman, Anthony A.; Verbavatz, Jean‐Marc

doi:10.1016/j.jsb.2011.12.004

Cited by 104 publications

(109 citation statements)

References 29 publications

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“…This proved more challenging, as the templates did not always find the inside of tubes but on occasion aligned with neighboring microtubules (or outside noise), resulting in unstable traces that would require manual intervention. After completion of this work a paper appeared which describes such expert feedback during an interactive tracing of microtubules (Weber et al, 2012). Our approach currently lacks a correction for microtubule-specific ‘missing wedge’ artifacts as proposed by Weber et al (2012), which we plan to add in a future software release.…”

Section: Discussionmentioning

confidence: 99%

Automated tracing of filaments in 3D electron tomography reconstructions using Sculptor and Situs

Rusu

Starosolski

Wahle

et al. 2012

Journal of Structural Biology

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The molecular graphics program Sculptor and the command-line suite Situs are software packages for the integration of biophysical data across spatial resolution scales. Herein, we provide an overview of recently developed tools relevant to cryo-electron tomography (cryo-ET), with an emphasis on functionality supported by Situs 2.7 and Sculptor 2.1. We describe a work flow for automatically segmenting filaments in cryo-ET maps including denoising, local normalization, feature detection, and tracing. Tomograms of cellular actin networks exhibit both cross-linked and bundled filament densities. Such filamentous regions in cryo-ET data sets can then be segmented using a stochastic template-based search, VolTrac. The approach combines a genetic algorithm and a bidirectional expansion with a tabu search strategy to localize and characterize filamentous regions. The automated filament segmentation by VolTrac compares well to a manual one performed by expert users, and it allows an efficient and reproducible analysis of large data sets. The software is free, open source, and can be used on Linux, Macintosh or Windows computers.

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Section: Discussionmentioning

confidence: 99%

Automated tracing of filaments in 3D electron tomography reconstructions using Sculptor and Situs

Rusu

Starosolski

Wahle

et al. 2012

Journal of Structural Biology

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“…We assumed that this distortion primarily compensates the distortion of the imaging process. For the tracing, the error was previously analysed for reconstructions of C. elegans centrosomes63. Although the spindle data is larger, the tomogram content is similar to the centrosome data sets, and thus we assumed that the error lies in the same range of 5–10%.…”

Section: Methodsmentioning

confidence: 99%

C. elegans chromosomes connect to centrosomes by anchoring into the spindle network

et al. 2017

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The mitotic spindle ensures the faithful segregation of chromosomes. Here we combine the first large-scale serial electron tomography of whole mitotic spindles in early C. elegans embryos with live-cell imaging to reconstruct all microtubules in 3D and identify their plus- and minus-ends. We classify them as kinetochore (KMTs), spindle (SMTs) or astral microtubules (AMTs) according to their positions, and quantify distinct properties of each class. While our light microscopy and mutant studies show that microtubules are nucleated from the centrosomes, we find only a few KMTs directly connected to the centrosomes. Indeed, by quantitatively analysing several models of microtubule growth, we conclude that minus-ends of KMTs have selectively detached and depolymerized from the centrosome. In toto, our results show that the connection between centrosomes and chromosomes is mediated by an anchoring into the entire spindle network and that any direct connections through KMTs are few and likely very transient.

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“…Once a fiber was fully traced, all points near its centerline were removed from the buffer, and the process was repeated until the buffer emptied. The template matching and tracing algorithm are described in Weber et al (2012) and Rigort et al (2012). By construction, no branching can occur between the fibers.…”

Section: Fiber Segmentationmentioning

confidence: 99%

X-ray Tomographic Imaging of Tensile Deformation Modes of Electrospun Biodegradable Polyester Fibers

et al. 2017

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Electrospinning allows the production of fibrous networks for tissue engineering, drug delivery, and wound healing in health care. It enables the production of constructs with large surface area and a fibrous morphology that closely resembles the extracellular matrix of many tissues. A fibrous structure not only promotes cell attachment and tissue formation but could also lead to very interesting mechanical properties. Poly(3-hydroxybutyrate-co-4-hydroxybutyrate) (P(3HB-co-4HB)) is a biodegradable polyester that exhibits a large (>400%) elongation before failure. In this study, synchrotron X-ray phase contrast imaging was performed during tensile deformation to failure on a non-woven fiber mat of P(3HB-co-4HB) fibers. Significant reorientation of the fibers in the straining direction was observed, followed by localized necking and eventual failure. From an original average fiber diameter of 4.3 µm, a bimodal distribution of fiber diameter (modal diameters of 1.9 and 3.7 µm) formed after tensile deformation. Extensive localized necking (thinning) of fibers between (thicker) fiber-fiber contacts was found to be the cause for non-uniform thinning of the fibers, a phenomenon that is expected but has not been observed in 3D previously. The data presented here have implications not only in tissue regeneration but for fibrous materials in general.

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Automated tracing of microtubules in electron tomograms of plastic embedded samples of Caenorhabditis elegans embryos

Cited by 104 publications

References 29 publications

Automated tracing of filaments in 3D electron tomography reconstructions using Sculptor and Situs

Automated tracing of filaments in 3D electron tomography reconstructions using Sculptor and Situs

C. elegans chromosomes connect to centrosomes by anchoring into the spindle network

X-ray Tomographic Imaging of Tensile Deformation Modes of Electrospun Biodegradable Polyester Fibers

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