FilamentSensor 2.0: An open-source modular toolbox for 2D/3D cytoskeletal filament tracking.

Cytoskeletal pattern formation and structural dynamics are key to a variety of biological functions and a detailed and quantitative analysis yields insight into finely tuned and well-balanced homeostasis and potential pathological alterations. High content life cell imaging of fluorescently labeled cytoskeletal elements under physiological conditions is nowadays state-of-the-art and can record time lapse data for detailed experimental studies. However, systematic quantification of structures and in particular the dynamics (i.e. frame-to-frame tracking) are essential. Here, an unbiased, quantitative, and robust analysis workflow that can be highly automatized is needed. For this purpose we upgraded and expanded our fiber detection algorithm FilamentSensor (FS) to the FilamentSensor 2.0 (FS2.0) toolbox, allowing for automatic detection and segmentation of fibrous structures and the extraction of relevant data (center of mass, length, width, orientation, curvature) in real-time as well as tracking of these objects over time and cell event monitoring.

A Focal Adhesion Filament Cross-correlation Kit for fast, automated segmentation and correlation of focal adhesions and actin stress fibers in cells.

Focal adhesions (FAs) and associated actin stress fibers (SFs) form a complex mechanical system that mediates bidirectional interactions between cells and their environment. This linked network is essential for mechanosensing, force production and force transduction, thus directly governing cellular processes like polarization, migration and extracellular matrix remodeling. We introduce a tool for fast and robust coupled analysis of both FAs and SFs named the Focal Adhesion Filament Cross-correlation Kit (FAFCK). Our software can detect and record location, axes lengths, area, orientation, and aspect ratio of focal adhesion structures as well as the location, length, width and orientation of actin stress fibers. This enables users to automate analysis of the correlation of FAs and SFs and study the stress fiber system in a higher degree, pivotal to accurately evaluate transmission of mechanocellular forces between a cell and its surroundings. The FAFCK is particularly suited for unbiased and systematic quantitative analysis of FAs and SFs necessary for novel approaches of traction force microscopy that uses the additional data from the cellular side to calculate the stress distribution in the substrate. For validation and comparison with other tools, we provide datasets of cells of varying quality that are labelled by a human expert. Datasets and FAFCK are freely available as open source under the GNU General Public License.