Fig 1.
Schematic overview of the morphometric features of the cell, nucleus, and focal adhesions (FAs), providing information about the effects of cell type, physical environment, and pharmacological drugs on cell response.
The cell type, physical properties of the environment, and pharmacological drugs are known to affect cellular, nuclear and FA morphology. With the developed algorithm we were able to detect these changes and translate them into quantifiable parameters.
Fig 2.
Overview of the steps of the automated image analysis pipeline.
Representative immunofluorescent image of Human Vena Saphena Cells (HVSCs), (A) stained for the actin cytoskeleton (green), nucleus (blue), and focal adhesions (magenta). To automatically detect and analyze cells (B), nuclei (C) and focal adhesions (D), corresponding grey-scale images were processed using the automated image analysis pipeline. Scale bars: 50 μm.
Fig 3.
Automatic cell and nucleus segmentation.
Representative immunofluorescence images of the actin cytosketeleton and nuclei and the corresponding segmentation results, with each identified cell and nucleus shown in a different color. Scale bar: 50 μm.
Fig 4.
Detection of a single cell, nucleus and focal adhesions (FAs).
Representative grey-scale images of the actin cytoskeleton, nucleus, and FAs of HVSCs on a substrate homogeneously coated with fibronectin. The detected outlines are shown in green, blue, and magenta, respectively, and the orange rectangles marked areas show zoom-in images of the cell, nucleus and FAs. The white arrows indicate some small actin-rich membrane protrusions that were not detected.
Fig 5.
Quantitative analysis of the morphological features of a single cell, nucleus and individual focal adhesions (FAs) obtained under different experimental conditions, e.g. cell type, pharmacological, and substrate manipulation.
A: Representative immunofluorescence images of the actin cytoskeleton (green), nucleus (blue), and FAs (magenta) for a HVSC (control) and a MEF (cell type) on a substrate homogeneously coated with fibronectin, HVSC in the presence of 10 μM of Y-27632 (drugs), and HVSC on 5x5 μm lines of fibronectin (red, anisotropy). The detected outlines are shown in green, blue, and magenta respectively. Scale bar: 50 μm. B: Analyzed morphological features of a single cell, and corresponding nucleus and FAs for the control, cell type (MEF), drug (Y-27632) and environment (anisotropy) situation. C: Boxplot of the orientation of the FAs comparing cells cultured on isotropic and anisotropic substrates. The box and whisker plot indicate the median (black line in the box), 25th percentile (bottom line of the box), 75th percentile (top line of the box), and 5th and 95th percentiles (whiskers). Next to this, the orientation of the cell and nucleus are represented by the green and blue lines, respectively. 90° represents the direction of the lines.
Fig 6.
The effect of the Rho-associated kinase (ROCK) inhibitor Y-27632 on the morphological features of focal adhesions (FAs) in Human Vena Saphena Cells (HVSCs).
A: Representative immunofluorescence images of the actin cytoskeleton (green), nucleus (blue), FAs (magenta) and zoom-in images of FAs of HVSCs treated with different doses (0-20 μM) of ROCK inhibitor or DMSO (control). Scale bar: 50 μm. Quantitative analysis of FA area (B), FA aspect ratio (C), and fraction of FAs with a defined length (D) reveals that Y-27632 affects FA morphology. At least 20 cells were analyzed per each condition and the results are expressed as the mean ± standard error of the mean (SEM). To assess differences between the different concentrations of ROCK inhibitor on the morphological features of the FAs, the One-Way ANOVA with a Bonferroni post-hoc test was used. ***: p < 0.001.