12 × 3D-ExM enables isotopic expansion of 3D organoid cultures. (A) Representative 3D images of human organoids acquired before and after 12× 3D-ExM. DNA was stained with DRAQ5. The depth of objects in the image are color-coded. Note that the organoids shown in pre- and post-3D ExM were distinct and not identical organoids. 20× water objective (NA = 0.95) was used for imaging. (B) Quantification of the expansion factor of the volume and surface area of individual nuclei within the organoids. Expansion factors of the volume and surface area of the nucleus after 12× 3D-ExM were calculated by the fold change in the cube root of the volume and the square root of the surface area, respectively. Results are shown as the mean ± s.d. Each dot represents a single nucleus. n = 20 cells pooled from two color-coded biological replicates.