An integrated enhancement and reconstruction strategy for the quantitative extraction of actin stress fibers from fluorescence micrographs

Abstract

BackgroundThe stress fibers are prominent organization of actin filaments that perform important functions in cellular processes such as migration, polarization, and traction force generation, and whose collective organization reflects the physiological and mechanical activities of the cells. Easily visualized by fluorescence microscopy, the stress fibers are widely used as qualitative descriptors of cell phenotypes. However, due to the complexity of the stress fibers and the presence of other actin-containing cellular features, images of stress fibers are relatively challenging to quantitatively analyze using previously developed approaches, requiring significant user intervention. This poses a challenge for the automation of their detection, segmentation, and quantitative analysis.ResultHere we describe an open-source software package, SFEX (Stress Fiber Extractor), which is geared for efficient enhancement, segmentation, and analysis of actin stress fibers in adherent tissue culture cells. Our method made use of a carefully chosen image filtering technique to enhance filamentous structures, effectively facilitating the detection and segmentation of stress fibers by binary thresholding. We subdivided the skeletons of stress fiber traces into piecewise-linear fragments, and used a set of geometric criteria to reconstruct the stress fiber networks by pairing appropriate fiber fragments. Our strategy enables the trajectory of a majority of stress fibers within the cells to be comprehensively extracted. We also present a method for quantifying the dimensions of the stress fibers using an image gradient-based approach. We determine the optimal parameter space using sensitivity analysis, and demonstrate the utility of our approach by analyzing actin stress fibers in cells cultured on various micropattern substrates.ConclusionWe present an open-source graphically-interfaced computational tool for the extraction and quantification of stress fibers in adherent cells with minimal user input. This facilitates the automated extraction of actin stress fibers from fluorescence images. We highlight their potential uses by analyzing images of cells with shapes constrained by fibronectin micropatterns. The method we reported here could serve as the first step in the detection and characterization of the spatial properties of actin stress fibers to enable further detailed morphological analysis.Electronic supplementary materialThe online version of this article (doi:10.1186/s12859-017-1684-y) contains supplementary material, which is available to authorized users.

Nanoscale Architecture of the Cortical Actin Cytoskeleton in Embryonic Stem Cells

“…The images were rendered at a pixel size of 20 nm, yielding a typical image size of 4096 3 4096 pixels. Curvilinear features in the reconstructed images were then enhanced using line and orientation filter transforms (Sandberg and Brega, 2007;Zhang et al, 2017aZhang et al, , 2017b to accentuate filamentous structures and suppress non-filamentous features. The enhancement calculation was performed using a neighborhood radius parameter of 200 nm.…”

Simple methods for quantifying super-resolved cortical actin

“…As such, normal membrane associations should be severely disrupted, as indicated by our data. The accurate representation of cytochalasin D effects by our analysis workflow therefore strengthens justification for broader application and to assess subtle changes in cortical actin network organisation in response to inhibitors, stimulation, etc.. Skeletonisation analysis methods work well for continuous, well separated filaments, such as microtubules, intermediate filaments, or even thicker actin stress fibers (see Zhang et al (2017) for implementation of their technique SFEX to quantify actin stress fibers in TIRF images). When considering super resolved fine actin meshworks, however, discontinuity and reduced fluorescence intensity is an inherent issue.…”

Comprehensive profiling of the STE20 kinase family defines features essential for selective substrate targeting and signaling output

“…(A) NIH-3T3 cells stably expressing the indicated PAK4 mutants were imaged following staining with phalloidin (cyan), α-vinculin (magenta), and DAPI (blue). (B) The number of actin fibers per cell were identified using SFEX software [54] to analyze blinded images. More than 30 cell images were analyzed for each condition.…”

“…Images were randomly selected from each condition and image set, and blindly scored for actin disassembly using a 7-point scale, with lower values indicating a less extensive stress fiber network. The number of fibers per cell was also quantified in blinded images using SFEX for automated fiber identification [54].…”