Figure 3. TG-based iterative expansion.

a, Monomeric cleaving of TG (monomer 2’’’) after re-embedding in a second hydrogel. A reducing agent, tris(2-carboxyethyl)phosphine) (TCEP), was applied to cleave the disulfide bonds in the TG polymer network. b, HeLa cell with beta-tubulin immunostaining, expanded by cleavable TG-based 2-round iterative expansion. Expansion factor, 15.6x. Scale bar, 5 µm (78.2 µm). Results are representative of 10 cells from the same cell culture. c, Top, magnified view of the boxed region in b. Scale bar, 100 nm (1.56 µm). Bottom, transverse line intensity profile of the microtubule in a single xy-plane in the dotted box (circle) and the fitted sum of two Gaussians (red line). The line intensity profile was averaged in the direction parallel to the microtubule axis over a length of 200 nm. d, Histogram of peak-to-peak distance between microtubule sidewalls in HeLa cells (n = 336 segments of a length of 200 nm from 10 cells in one culture). e, Left, HeLa cell with two-colour labelling of clathrin coated pits/vesicles and microtubules, expanded by TG-based 2-round iterative expansion. Expansion factor, 15.6x. Scale bar, 10 µm (156 µm). Right, magnified view of the boxed region for each colour channel. Scale bars, 1 µm (15.6 µm). Results are from 1 cell from the same cell culture preparation as in b.