Application of 3D-ExM in visualiz ing centriole architecture, kinetochore structure, and karyotype determination. (A) Left: representative images of the centriole structure in RPE1 cells from the top view and side view perspectives before and after 4× 3D-ExM. Three 4× 3D-ExM example images of different centrioles were shown. Both Pre-3D-ExM and 4×-3D-ExM images were acquired using 100x oil objective (NA = 1.45). Right: the schematic diagram of centriole structures and the distribution of target molecules (CEP164 and acetylated-Tubulin) from the top view and side view. Note that both anti-CEP164 and anti-Ac-Tub antibodies were raised in mice, and thus the same anti-mouse secondary antibody was used for both targets in this experiment. (B) Quantification of mother centriole length and the expected length obtained from the previous study. Each dot represents a single centriole. n = 15 centrioles. (C) Quantification of distal appendage diameter from the top view perspective and the expected size obtained from the previous studies. Each dot represents a single centriole. n = 23 centrioles. (D) Left: an example 12× 3D-ExM image labeled by anti-Hec1 and anti-Tubulin antibodies in PtK2 metaphase cells. Right: the zoom-in image of the highlighted region shown in the left image. (E) Representative pre-3D-ExM images of a metaphase PtK2 cell stained with DAPI. Both maximum projection and consecutive single Z plane images (interval: 1.2 μm) are shown. Note that individual chromosomes cannot be identified with this image resolution. (F) The centromeric index of each chromosome in the normal karyotype is shown in Fig. 8 A. (G) The relative chromosome volume of each PtK2 chromosome in the normal karyotype is shown in Fig. 8 A. (H) The centromeric index vs relative chromosome volume of each PtK2 chromosome in the normal karyotype shown in Fig. 8 A.