(A) Confocal images of microtubules in HeLa cells, stained with a rat α-tubulin antibody (YL1/2) which recognises tyrosinated tubulin, and an Affimer for polymerised tubulin, conjugated to Alexa Fluor 647. Images of an interphase and metaphase cell, together with an image of the cytokinetic furrow are shown. Arrows in the metaphase cell point to astral microtubules that are predominantly labelled with the antibody. Arrows in the cyokinetic furrow indicate the central region (Fleming body). Scale bar is 10 μm. (B) 3D dSTORM images of microtubules in a HeLa cell, labelled with Alexa Fluor 647 conjugated to a primary antibody to rat α-tubulin (left) and an Affimer for polymerised tubulin (right). These images are from separate cells. Localisations were aggregated into 10 nm bins and projected onto a single plane, with Gaussian smoothing. Scale bar 1 µm. (C) Intensity profile across the microtubule image labelled in (B) (yellow box), averaged along 510 nm of its length. The central decrease in intensity reflects the hollow structure of the microtubule. (D) Comparison of the average microtubule image intensity profile with antibody staining (dashed, mean of 6 microtubule sections), Affimer staining (solid, mean of 8 microtubule sections) and actual microtubule size (black circle). The FWHM of each average profile (as in (C)) was found for a Gaussian fit and a Gaussian distribution is plotted here using the mean FWHM for each staining method.