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 Kb; Lautenschlaeger, Franziska

doi:10.1101/2021.01.04.425230

Cited by 3 publications

(1 citation statement)

References 23 publications

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

Schu

Terriac

Koch

et al. 2020

Preprint

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The cellular cortex is a 200-nm-thick actin network that lies beneath the cell membrane. It is responsible for the mechanical properties of the cell and is involved in many cellular processes, such as cell migration and interactions with the environment. To develop a clear view of the structure of this meshwork, high resolution imaging is essential, such as electron microscopy. This technique requires complex sample preparation that can lead to artifacts like shrinkage or hole formation. We present a preparation method that reduces artifacts significantly. Here, the final drying step that is typically performed by critical point drying is replaced by hexamethyldisilazane drying. We quantitatively investigated sample integrity after both preparation methods, and show that there are significant advantages of hexamethyldisilazane drying compared to critical point drying. Furthermore, automated analysis of a network is classically performed by thresholding-based software programs, which are sensitive to noise and uneven brightness of images. The here presented analysis that we have developed is based on a vectorial node algorithm. It reproduces all kinds of networks sufficiently to allow derivation of quantitative network-specific parameters, such as mesh hole size. We use this analysis to compare the network structure of cells prepared by these two drying methods, and show that hexamethyldisilazane drying leads to fewer artificial mesh holes compared to critical point drying. We thus present here a significantly improved method to quantitatively investigate the actin cortex of cells, and show that hexamethyldisilazane drying leads to more accurate imaging compared to critical point drying. Insight BoxThe highest resolution for imaging the cellular actin cortex is provided by electron microscopy. Scanning electron microscopy samples require a drying process, usually 3 achieved by critical point drying, which is critical for the sample integrity. We compare the structural defects in the actin cortex of hTert RPE1 cells after critical point drying and a chemical based method, namely hexamethyldisilazane drying. In order to characterize the actin network, we also developed a new vectorial based tracing software. We bring here new tool, both experimental and analytical, which will help to streamline studies of the actin cortex.

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Simple methods for quantifying super-resolved cortical actin

Garlick

et al. 2021

Preprint

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Cortical actin plays a key role in cell movement and division, but has also been implicated in the organisation of cell surface receptors such as G protein-coupled receptors. The actin mesh proximal to the inner membrane forms small fenced regions, or corrals, in which receptors can be constrained. Quantification of the actin mesh at the nanoscale has largely been attempted in single molecule datasets and electron micrographs. This work describes the development and validation of workflows for analysis of super resolved fixed cortical actin images obtained by both Super Resolved Radial Fluctuations (SRRF) and expansion microscopy (ExM). SRRF analysis was used to show a significant increase in corral area when treating cells with the actin disrupting agent cytochalasin D (increase of 0.31 μm2 ± 0.04 SEM), and ExM analysis allowed for the quantitation of actin filament densities. Thus this work allows complex actin networks to be quantified from super-resolved images and is amenable to both fixed and live cell imaging.

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A novel universal algorithm for filament network tracing and cytoskeleton analysis

Cited by 3 publications

References 23 publications

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

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

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

Simple methods for quantifying super-resolved cortical actin

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