Figure 2. TG-mediated expansion of cells and tissues.

a, Image of TG (using monomer 2’’’) as synthesized (left, pre-expansion) and after swelling in deionized water (right, post-expansion). The two gels were cast in circular molds with identical dimensions. Grid size, 5 mm. b, TG labelled with fluorescein in pre- (left, 2’; same sizes and shapes when 2’’ and 2’’’ were used) and post-expansion (right, 2’, 2’’ and 2’’’) states. Irregular boundaries on the post-expansion images reflect the meniscus of water used to expand the gels. Scale bar, 5 mm. c, Left, HEK293 cells with alpha-tubulin immunostaining in pre- (inset) and post-expansion states. Expansion factor, 2.85x. Scale bars, 20 µm. Right, magnified views of the boxed regions 1 (top) and 2 (bottom). Scale bars, 1 µm [bottom, 2.85 µm; here and after, unless otherwise noted, scale bar sizes are provided at pre-expansion scale (i.e., biological scale) with the corresponding post-expansion size (i.e., physical size) indicated in parentheses]. Results are representative of 5 cells from the same cell culture. d, Root-mean-square (RMS) error curve for HEK293 cell expansion (blue line, mean; shaded, standard deviation; n = 8 cells from one culture). Inset, non-rigidly registered and overlaid pre- (green) and post-expansion (magenta) images used for the RMS error analysis. Scale bar, 20 µm (57 µm). e, Pre- (left top) and post-expansion (left bottom and right) Thy1-YFP mouse brain slices. Expansion factor, 3.00x. Scale bars, 5 mm (left) and 10 µm (right, 30 µm). The gelled brain slice on the left bottom panel was immunostained against YFP after the proteolysis step to enhance fluorescence. Inset, magnified view of the boxed region. Scale bar, 1 µm (3 µm). Results are representative of 2 brain slices from the same single batch of brain slice preparation for each panel. f, Pre-expansion (left column) and post-expansion (right column) Thy1-YFP mouse brain slices immunostained with Homer1 primary antibody and Alexa Fluor 647 (AF647)-conjugated secondary antibody, using sodium polyacrylate/polyacrylamide gel (PAAG, top row) and TG (bottom row). Expansion factor, 3.93x (PAAG) and 2.72x (TG). Scale bars, 300 µm (top right, 1.18 mm; bottom right, 815 µm). Results are representative of 3 brain slices from the same single batch of brain slice preparation for each gel type. g, Fluorescence retention of AF647, Cy5, and AF680 with TG and PAAG in immunostained mouse brain slices processed as in f (bar height, mean; black dots, individual data points; error bar, standard error of the mean; n = 3 brain slices from one mouse).