Figure 3.
Validation of isotropic nuclear expansion factors through correlative pre- and post-3D-ExM. (A) Representative images of DAPI-stained RPE1 nuclei before and after 4× 3D-ExM of the same cells. Left: a pre-3D-ExM image. Three example regions of cells are highlighted by bounding boxes of different colors. Right: a direct comparison of the digitally enlarged images (pre-3D-ExM) with the corresponding 4× 3D-ExM images of the same cells. Note that both pre- and post-3D-ExM images were captured under identical imaging conditions (20× WI, NA = 0.95). (B) Quantification of the expansion factor of the same RPE1 nuclei based on their major axis length, minor axis length, and the 2D area before and after 4× 3D-ExM. The expansion factor of the area was determined by taking the square root of the ratio of the post-3D-ExM nuclear area to the pre-3D-ExM nuclear area. Each dot represents a single nucleus. n = 40 cells pooled from two color-coded biological replicates. The horizontal dotted line indicates the gel expansion factor in each experiment. The mean expansion factor is shown above each dot plot. (C) Representative images of DAPI-stained RPE1 nuclei before and after 12× 3D-ExM. Left: a pre-3D-ExM image. Three example regions of cells are highlighted by bounding boxes of different colors. Right: a direct comparison of the digitally enlarged images (pre-3D-ExM) with the corresponding 12× 3D-ExM images of the same cells. Note that both pre- and post-3D-ExM images were captured under identical imaging conditions (20× WI, NA = 0.95). (D) Quantification of the expansion factor of the same RPE1 nuclei based on their major axis length, minor axis length, and the 2D area before and after 12× 3D-ExM. Each dot represents a single nucleus. n = 30 cells pooled from three color-coded biological replicates. The horizontal dotted line indicates the gel expansion factor in each experiment. The mean expansion factor is shown above each dot plot. All data are shown as the mean ± s.d.

Validation of isotropic nuclear expansion factors through correlative pre- and post-3D-ExM. (A) Representative images of DAPI-stained RPE1 nuclei before and after 4× 3D-ExM of the same cells. Left: a pre-3D-ExM image. Three example regions of cells are highlighted by bounding boxes of different colors. Right: a direct comparison of the digitally enlarged images (pre-3D-ExM) with the corresponding 4× 3D-ExM images of the same cells. Note that both pre- and post-3D-ExM images were captured under identical imaging conditions (20× WI, NA = 0.95). (B) Quantification of the expansion factor of the same RPE1 nuclei based on their major axis length, minor axis length, and the 2D area before and after 4× 3D-ExM. The expansion factor of the area was determined by taking the square root of the ratio of the post-3D-ExM nuclear area to the pre-3D-ExM nuclear area. Each dot represents a single nucleus. n = 40 cells pooled from two color-coded biological replicates. The horizontal dotted line indicates the gel expansion factor in each experiment. The mean expansion factor is shown above each dot plot. (C) Representative images of DAPI-stained RPE1 nuclei before and after 12× 3D-ExM. Left: a pre-3D-ExM image. Three example regions of cells are highlighted by bounding boxes of different colors. Right: a direct comparison of the digitally enlarged images (pre-3D-ExM) with the corresponding 12× 3D-ExM images of the same cells. Note that both pre- and post-3D-ExM images were captured under identical imaging conditions (20× WI, NA = 0.95). (D) Quantification of the expansion factor of the same RPE1 nuclei based on their major axis length, minor axis length, and the 2D area before and after 12× 3D-ExM. Each dot represents a single nucleus. n = 30 cells pooled from three color-coded biological replicates. The horizontal dotted line indicates the gel expansion factor in each experiment. The mean expansion factor is shown above each dot plot. All data are shown as the mean ± s.d.

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