STED images reveal dominant vinculin binding angle in podosomes.

<p>(<b>A</b>) STED image of vinculin rings of podosomes. The vinculin rings appear distinctly polygonal, with vinculin strands apparently nucleating from corner points. (<b>B</b>) Skeletonisation of image <i>A</i> shown in blue. (<b>C</b>) Result of fitting blurred line segments to the underlying image <i>A</i> at corners shown in <i>B</i>. The intensity of the Gaussian line segment is displayed. The background and line segments wider than 10 pixels are not displayed for clarity. (<b>D</b>) Probability distribution of angles at vinculin corners in STED images of fixed cells: a peak at 116° with a shoulder at 136° is observed above the background. (<b>E</b>) Localisation microscopy on live cells shows that the binding angle in podosomes fluctuates in the range of 120° to 135° (mean peak 130.5°5°), but is not time-dependent. (<b>F</b>) Overlay of results from <i>E</i> & <i>F</i>. Dotted lines indicate dominant binding angles. Grey bars represent the inner angles of flat pentagons (108°), hexagons (120°), heptagons (128.6°) and octagons (135°).