A novel universal algorithm for filament network tracing and cytoskeleton analysis

Flormann, Daniel; Schu, Moritz; Terriac, Emmanuel; Thalla, Divyendu Goud; Kainka, Lucina; Koch, Marcus; Gad, Annica K. B.; Lautenschläger, Franziska

doi:10.1096/fj.202100048r

Cited by 14 publications

(16 citation statements)

References 22 publications

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“…We then focused on three of the parameters that can be exploited to characterize networks: the ‘mesh hole size’ after exclusion of the artifactual mesh holes; the network ‘connectivity’ (also known as the ‘coordination number’); and the ‘fiber length’. The design and use of this vectoral tracing software is described in [ 39 ]. Here, the mesh hole size is defined in the same manner as for the artifactual mesh hole diameter above (but as <100 nm here) ( Fig 4A ).…”

Section: Resultsmentioning

confidence: 99%

“…The connectivity defines the number of fibers that are connected to a single node ( Fig 4B ). Finally, the fiber length defines the length of a tracing line up to when the break-off angle (defined here as 17°; see also [ 39 ]) is reached ( Fig 4C ).…”

Section: Resultsmentioning

confidence: 99%

“…The home-made vectorial tracing algorithm is available as open source at https://github.com/SRaent/FiNTA , and it is described elsewhere [ 39 ].…”

Section: Methodsmentioning

confidence: 99%

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Scanning electron microscopy preparation of the cellular actin cortex: A quantitative comparison between critical point drying and hexamethyldisilazane drying

et al. 2021

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The cellular cortex is an approximately 200-nm-thick actin network that lies just beneath the cell membrane. It is responsible for the mechanical properties of cells, and as such, it is involved in many cellular processes, including cell migration and cellular interactions with the environment. To develop a clear view of this dense structure, high-resolution imaging is essential. As one such technique, electron microscopy, involves complex sample preparation procedures. The final drying of these samples has significant influence on potential artifacts, like cell shrinkage and the formation of artifactual holes in the actin cortex. In this study, we compared the three most used final sample drying procedures: critical-point drying (CPD), CPD with lens tissue (CPD-LT), and hexamethyldisilazane drying. We show that both hexamethyldisilazane and CPD-LT lead to fewer artifactual mesh holes within the actin cortex than CPD. Moreover, CPD-LT leads to significant reduction in cell height compared to hexamethyldisilazane and CPD. We conclude that the final drying procedure should be chosen according to the reduction in cell height, and so CPD-LT, or according to the spatial separation of the single layers of the actin cortex, and so hexamethyldisilazane.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

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Scanning electron microscopy preparation of the cellular actin cortex: A quantitative comparison between critical point drying and hexamethyldisilazane drying

et al. 2021

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“…Very recently, one of the most complete algorithms for analyzing fibrous patterns from pixelated images has been developed. FiNTA (Flormann et al, 2021) is based on the vector tracing of grayscale images. It provides rich descriptors of fibrous patterns such as connectivity between fibers, hold sizes and shapes, filament density, and length and measures of curvature.…”

Section: Quantificationmentioning

confidence: 99%

Super-Resolution Imaging Approaches for Quantifying F-Actin in Immune Cells

Garlick

Thomas

Owen

2021

Front. Cell Dev. Biol.

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Immune cells comprise a diverse set of cells that undergo a complex array of biological processes that must be tightly regulated. A key component of cellular machinery that achieves this is the cytoskeleton. Therefore, imaging and quantitatively describing the architecture and dynamics of the cytoskeleton is an important research goal. Optical microscopy is well suited to this task. Here, we review the latest in the state-of-the-art methodology for labeling the cytoskeleton, fluorescence microscopy hardware suitable for such imaging and quantitative statistical analysis software applicable to describing cytoskeletal structures. We also highlight ongoing challenges and areas for future development.

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“…Further tools have been developed for the segmentation of microtubules ( Faulkner et al, 2017 ) and their prokaryotic homologue FtsZ ( Liu et al, 2019 ; Özdemir and Reski, 2021 ). Recently, a tool was described that is suitable for segmentation of cytoskeletal networks including keratin networks using data obtained by electron microscopy and super-resolution fluorescence microscopy ( Flormann et al, 2021 ). This tool, however, lacks 3D capability and does not capture information on filament thickness.…”

Section: Introductionmentioning

confidence: 99%

Quantitative mapping of keratin networks in 3D

Windoffer

Schwarz

Yoon

et al. 2022

eLife

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Mechanobiology requires precise quantitative information on processes taking place in specific 3D microenvironments. Connecting the abundance of microscopical, molecular, biochemical and cell mechanical data with defined topologies has turned out to be extremely difficult. Establishing such structural and functional 3D maps needed for biophysical modeling is a particular challenge for the cytoskeleton, which consists of long and interwoven filamentous polymers coordinating subcellular processes and interactions of cells with their environment. To date, useful tools are available for the segmentation and modeling of actin filaments and microtubules but comprehensive tools for the mapping of intermediate filament organization are still lacking. In this work, we describe a workflow to model and examine the complete 3D arrangement of the keratin intermediate filament cytoskeleton in canine, murine and human epithelial cells both, in vitro and in vivo. Numerical models are derived from confocal Airyscan high resolution 3D imaging of fluorescence-tagged keratin filaments. They are interrogated and annotated at different length scales using different modes of visualization including immersive virtual reality. In this way, information is provided on network organization at the subcellular level including mesh arrangement, density and isotropic configuration as well as details on filament morphology such as bundling, curvature and orientation. We show that the comparison of these parameters helps to identify, in quantitative terms, similarities and differences of keratin network organization in epithelial cell types defining subcellular domains, notably basal, apical, lateral and perinuclear systems. The described approach and the presented data are pivotal for generating mechanobiological models that can be experimentally tested.

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A novel universal algorithm for filament network tracing and cytoskeleton analysis

Cited by 14 publications

References 22 publications

Scanning electron microscopy preparation of the cellular actin cortex: A quantitative comparison between critical point drying and hexamethyldisilazane drying

Scanning electron microscopy preparation of the cellular actin cortex: A quantitative comparison between critical point drying and hexamethyldisilazane drying

Super-Resolution Imaging Approaches for Quantifying F-Actin in Immune Cells

Quantitative mapping of keratin networks in 3D

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