4 × and 12 × 3D-ExM enable isotropic 3D nuclear expansion. (A) Representative images of HeLa and PtK2 nuclei before or after 4× or 12× expansion using the original or our 3D-ExM protocol. The corrected yellow scale bar was calculated by dividing the original measured length in 3D-ExM images by the gel expansion factor. The expansion factor (exp factor) displayed on top of each image represents the average lateral nuclear expansion factor plotted in Fig. 2 B. (B) Quantification of the lateral and axial expansion factors in both HeLa and PtK2 nuclei using the original ExM or 3D-ExM method. Each dot represents a single nucleus. L: lateral, A: axial. n = 48, 103, 62, 58, 53, 52, 42, 34, 41, 41, 46, 43, 68, 68, 45, 40 cells (from left to right) pooled from two to three color-coded biological replicates. The P value was calculated using Welch’s t-test (two-tailed). **** indicates P < 0.0001. (C) Left: representative images of nuclei of RPE1 cells before and after 12× 3D-ExM. Right: quantification of the lateral and axial expansion factors in RPE1 nuclei using 12× 3D-ExM. n = 44 and 33 cells (from left to right) pooled from three color-coded biological replicates. (D) The 3D view of a RPE1 nucleus. Both XZ and YZ views are shown. DAPI was used for pre-ExM imaging and DRAQ5 was used for post-ExM imaging. 60× water objective (NA = 1.20) was used for imaging. All data are shown as the mean ± s.d.