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.

Statistical analysis of ENDOR spectra.

Electron-nuclear double resonance (ENDOR) measures the hyperfine interaction of magnetic nuclei with paramagnetic centers and is hence a powerful tool for spectroscopic investigations extending from biophysics to material science. Progress in microwave technology and the recent availability of commercial electron paramagnetic resonance (EPR) spectrometers up to an electron Larmor frequency of 263 GHz now open the opportunity for a more quantitative spectral analysis. Using representative spectra of a prototype amino acid radical in a biologically relevant enzyme, the [Formula: see text] in Escherichia coli ribonucleotide reductase, we developed a statistical model for ENDOR data and conducted statistical inference on the spectra including uncertainty estimation and hypothesis testing. Our approach in conjunction with 1H/2H isotopic labeling of [Formula: see text] in the protein unambiguously established new unexpected spectral contributions. Density functional theory (DFT) calculations and ENDOR spectral simulations indicated that these features result from the beta-methylene hyperfine coupling and are caused by a distribution of molecular conformations, likely important for the biological function of this essential radical. The results demonstrate that model-based statistical analysis in combination with state-of-the-art spectroscopy accesses information hitherto beyond standard approaches.

Analysis of patchclamp recordings: model-free multiscale methods and software.

Analysis of patchclamp recordings is often a challenging issue. We give practical guidance how such recordings can be analyzed using the model-free multiscale idealization methodology JSMURF, JULES, and HILDE. We provide an operational manual how to use the accompanying software available as an R-package and as a graphical user interface. This includes selection of the right approach and tuning of parameters. We also discuss advantages and disadvantages of model-free approaches in comparison to hidden Markov model approaches and explain how they complement each other.

Direct characterization of cytoskeletal reorganization during blood platelet spreading.

Blood platelets are the key cellular players in blood clotting and thus of great biomedical importance. While spreading at the site of injury, they reorganize their cytoskeleton within minutes and assume a flat appearance. As platelets possess no nucleus, many standard methods for visualizing cytoskeletal components by means of fluorescence tags fail. Here we employ silicon-rhodamine actin and tubulin probes for imaging these important proteins in a time-resolved manner. We find two distinct timescales for platelet spread area development and for cytoskeletal reorganization, indicating that although cell spreading is most likely associated with actin polymerization at the cell edges, distinct, stress-fiber-like actin structures within the cell, which may be involved in the generation of contractile forces, form on their own timescale. Following microtubule dynamics allows us to distinguish the role of myosin, microtubules and actin during early spreading.

The filament sensor for near real-time detection of cytoskeletal fiber structures.

A reliable extraction of filament data from microscopic images is of high interest in the analysis of acto-myosin structures as early morphological markers in mechanically guided differentiation of human mesenchymal stem cells and the understanding of the underlying fiber arrangement processes. In this paper, we propose the filament sensor (FS), a fast and robust processing sequence which detects and records location, orientation, length, and width for each single filament of an image, and thus allows for the above described analysis. The extraction of these features has previously not been possible with existing methods. We evaluate the performance of the proposed FS in terms of accuracy and speed in comparison to three existing methods with respect to their limited output. Further, we provide a benchmark dataset of real cell images along with filaments manually marked by a human expert as well as simulated benchmark images. The FS clearly outperforms existing methods in terms of computational runtime and filament extraction accuracy. The implementation of the FS and the benchmark database are available as open source.