Application of 12 × 3D-ExM in chromosome identification and examination of the detailed architecture of chromosomes. (A) Top: example 12× 3D-ExM images of three mitotic PtK2 cells with one wild-type and two aneuploid karyotypes (gain or loss of Chr. Y2). Bottom: the entire karyotype of individual chromosomes for each cell. 3D rendering of mitotic chromosomes was performed using Imaris (Andor). DNA was stained with DRAQ5. (B) An example 12× 3D-ExM image of an anaphase HeLa cell with the chromosome bridge composed of two twisted chromosome strands. DNA was stained by DAPI. 60× oil objective (NA = 1.40) was used for this imaging.