Near-native state imaging by cryo-soft-X-ray tomography reveals remodelling of multiple cellular organelles during HSV-1 infection

Nahas, Kamal L; Connor, Viv; Harkiolaki, Maria; Crump, Colin M.; Graham, Stephen C.

doi:10.1101/2021.10.11.463900

Cited by 8 publications

(31 citation statements)

References 112 publications

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“…We have used Contour to segment mitochondria in a recent preprint where we studied how mitochondrial morphology changes during HSV-1 infection. We found that mitochondria transitioned from a heterogenous morphology in uninfected U2OS cells to a more consistently elongated and branched formation as the infection progressed (24) . Contour can be used to segment other cellular compartments based on intensity and width, such as lipid droplets (Fig.…”

Section: Applications Of Contour To Analyse Geometry Of Cellular Compartmentsmentioning

confidence: 79%

“…We have applied Contour to one study, where we investigated how HSV-1 infection alters the morphology of cellular compartments, and we were able to segment mitochondria in multiple tomograms (24) . The dependency on low projection intensity and width for the segmentation does pose some limitations.…”

Section: Discussionmentioning

confidence: 99%

“…Cytoplasmic vesicles often have a highly contrasting membrane but a light lumen, making it difficult to segment such features when applying a minimum width restriction. Although we did not use Contour to segment cytoplasmic vesicles in our recent study (24) , we used Contour to calculate the longest widths of each vesicle that we manually segmented using Segmentation Editor in Fiji (2) . We therefore show that Contour can be used in conjunction with other segmentation tools to calculate quantitative data.…”

Section: Discussionmentioning

confidence: 99%

“…cytoplasmic vesicles) other segmentation tools can be used to generate segmented volumes that can be imported into Contour for quantitation. We have used Contour in a recent preprint to study how the morphology of mitochondria and cytoplasmic vesicles change during infection with herpes simplex virus-1 (HSV-1) (24) . We generated multiple segmented volumes with Contour and found that mitochondria became more elongated and vesicles reduced in width as the infection progressed (24) .…”

Section: Introductionmentioning

confidence: 99%

“…We have used Contour in a recent preprint to study how the morphology of mitochondria and cytoplasmic vesicles change during infection with herpes simplex virus-1 (HSV-1) (24) . We generated multiple segmented volumes with Contour and found that mitochondria became more elongated and vesicles reduced in width as the infection progressed (24) . In this paper we discuss the algorithm and applications of this segmentation tool to cryoSXT data.…”

Section: Introductionmentioning

confidence: 99%

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Contour, a semi-automated segmentation and quantitation tool for cryo-soft-X-ray tomography

Nahas

Fernandes

Crump

et al. 2021

Preprint

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Cryo-soft-X-ray tomography is being increasingly used in biological research to study the morphology of cellular compartments and how they change in response to different stimuli, such as viral infections. Segmentation of these compartments is limited by time-consuming manual tools or machine learning algorithms that require extensive time and effort to train. Here we describe Contour, a new, easy-to-use, highly automated segmentation tool that enables accelerated segmentation of tomograms to delineate distinct cellular compartments. Using Contour, cellular structures can be segmented based on their projection intensity and geometrical width by applying a threshold range to the image and excluding noise smaller in width than the cellular compartments of interest. This method is less laborious and less prone to errors from human judgement than current tools that require features to be manually traced, and does not require training datasets as would machine-learning driven segmentation. We show that high-contrast compartments such as mitochondria, lipid droplets, and features at the cell surface can be easily segmented with this technique in the context of investigating herpes simplex virus 1 infection. Contour can extract geometric measurements from 3D segmented volumes, providing a new method to quantitate cryo-soft-X-ray tomography data. Contour can be freely downloaded at github.com/kamallouisnahas/Contour.Impact StatementMore research groups are using cryo-soft-X-ray tomography as a correlative imaging tool to study the ultrastructure of cells and tissues but very few tomograms are segmented with existing segmentation programs. Segmentation is usually a prerequisite for measuring the geometry of features in tomograms but the time- and labour-intensive nature of current segmentation techniques means that such measurements are rarely across a large number of tomograms, as is required for robust statistical analysis. Contour has been designed to facilitate the automation of segmentation and, as a result, reduce manual effort and increase the number of tomograms that can be segmented. Because it requires minimal manual intervention, Contour is not as prone to human error as programs that require the users to trace the edges of cellular features. Geometry measurements of the segmented volumes can be calculated using this program, providing a new platform to quantitate cryoSXT data. Contour also supports quantitation of volumes imported from other segmentation programs. The generation of a large sample of segmented volumes with Contour that can be used as a representative training dataset for machine learning applications is a long-term aspiration of this technique.

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Section: Applications Of Contour To Analyse Geometry Of Cellular Compartmentsmentioning

confidence: 79%

Section: Discussionmentioning

confidence: 99%