Fig. 4: Ex-SMLM of U-ExM expanded centrioles.

a-c 3D dSTORM image of U-ExM expanded and re-embedded Chlamydomonas centrioles stained post re-embedding with anti α-tubulin primary antibody and Alexa Fluor 647 conjugated secondary antibodies measured in MEA buffer. b Zoom-in on highlighted region in (a) revealing the 9-fold symmetry of the shown procentriole. c Side view of two mature centrioles with clearly separated triplets. The inlet shows the cross-sectional profile along the centriole (white box) showing five distinct peaks of microtubule triples (marked with arrow heads). d Comparison of the diameters determined from expanded centrioles measured using different protocols (re-embedded and labeled with Alexa Fluor 647, and imaged in MEA photoswitching buffer, labeled with HMSiR 647 and imaged in double-deionized water or in pH optimized PBS (1x) buffer with pH 7.4). Mean values are 657 ± 90 nm (mean ± sd) for Alexa Fluor 647 in MEA buffer (n = 12 centrioles), 428 ± 74 nm (mean ± sd) for HMSiR in PBS (n = 7 centrioles), and 750 ± 34 nm (mean ± sd) for HMSiR 647 in water (n = 8 centrioles). Data from n = 2 independent experiments for each condition. Divided by the previously analyzed diameter of α-tubulin labeled centriole expansion factors translates into ~3.4x, ~2.2x, and ~3.9x for expanded centrioles labeled with Alexa Fluor 647 in MEA buffer, HMSiR in PBS (1x), and HMSiR 647 in water, respectively. Statistical significance was assessed by one-way ANOVA: the mean values of the diameters are significantly different with p<0.02 (F = 3.80) (*P ≤ 0.05, **P ≤ 0.01). e–g 2D dSTORM image of U-ExM expanded centrioles labeled with HMSiR 647 imaged in water (e–f) or PBS(1x) (g) f Zoom-in on highlighted region in (e). h 3D dSTORM image of unexpanded isolated Chlamydomonas centrioles immunostained with antibodies against glutamylated tubulin and Alexa Fluor 647 conjugated secondary antibodies. Scale bars, 1 µm (a, e), 500 nm (b, f, g), 1.5 µm (c), 250 nm (h).