Figure 5: SIM and dSTORM imaging of protein phosphorylation at actin nodules.

(a) SIM images of a human platelet stained with Alexa488-phalloidin (left panel) and Alexa568-anti-pTyr (middle panel) and the merged image (right panel, actin=magenta, pTyr=green). The presence of tyrosine phosphorylated proteins can be seen as punctate staining across the platelet with a greater intensity and larger foci observed at actin nodules (two examples indicated by arrows). (b) TIRF image of Alexa488-phalloidin labelled human platelet (left panel) with arrows indicating the actin nodules and a dSTORM image of the same cell labelled with Alex647-anti-pTyr (right panel). This confirms the results seen with SIM in that tyrosine phosphorylated proteins are more concentrated at actin nodules (arrows). The asterisk indicates an actin nodule that resolves as two separate foci of pTyr labelling in the super-resolution image. (c) Enlargement of the boxed region from (b) of the pTyr signal at a single actin nodule (left panel). Quantitative cluster mapping of the image (right panel) confirms that phosphorylated proteins are highly clustered at the actin nodule as indicated by the high L(r) value (according to the pseudocolour scale shown to the right of the cluster map). Scale bars, a–b: 2 μm, c: 0.5 μm.