Figure 5.
Validation of 3D-ExM resolution using cellular rulers. (A) Representative images of PtK2 cells stained with tubulin (green) and DNA (magenta) acquired before expansion (pre-3D-ExM) or after expansion (4× and 12× 3D-ExM). 60× water objective (NA = 1.20) was used for imaging. Multiple full FOV 12× 3D-ExM images were combined to create a composite image of a single cell. (B) A zoom-in representative 12× 3D-ExM confocal image of microtubule filaments in PtK2 cells. (C) Quantification of full width at half maximum (FWHM) of microtubules in images acquired before or after 4× or 12× 3D-ExM. Both the conventional confocal microscope and STED microscope were used in this experiment and quantification. n = 251, 140, 221, 155, 150, 36 cells pooled from two to three color-coded biological replicates (from left to right). The P value was calculated using Welch’s t-test (two-tailed). **** indicates P < 0.0001. (D) Schematic diagram of the structure of NPC created by BioRender. NP proteins Nup107 and Elys are labeled with yellow and red, respectively. (E) The EM images (upper row) and 12× 3D-ExM images (lower row) of the nucleus and NPs at the edge of the nucleus are shown in the right rows. DNA was stained with DRAQ5. (F) The representative 12× 3D-ExM images of Nup107 and Elys at the edge of a nucleus. (G). Left: an example Nup107 intensity profile at a single NP. Right: the average measured and corrected distance between two Nup107 intensity peaks in the line scan. n = 117 pooled from two biological replicates. (H) Representative image of the surface of RPE1 nuclei stained with Nup107 and DRAQ5. The black regions shown in the DNA channel are nuclear pores that colocalize with the Nup107 punctate signals. (I) Left: example images of the measurement of diameter and circumference of NPs. Right: quantification of the measured or corrected values of diameter and circumference of the NPs. n = 70 NPs pooled from two biological replicates. The calculated circumferences were derived from the diameters of the NPs, while the measured circumferences were obtained independently of their diameters. (J) Quantification of the number of NPs per unit area (1,000 µm2, equivalent to ∼7 µm2 after correction by the expansion factor). n = 30 cells pooled from two biological replicates. (K) Quantification of the estimated total number of NPs per cell. Each dot represents a single cell. n = 15 cells pooled from two biological replicates. All data are shown as the mean ± s.d.

Validation of 3D-ExM resolution using cellular rulers. (A) Representative images of PtK2 cells stained with tubulin (green) and DNA (magenta) acquired before expansion (pre-3D-ExM) or after expansion (4× and 12× 3D-ExM). 60× water objective (NA = 1.20) was used for imaging. Multiple full FOV 12× 3D-ExM images were combined to create a composite image of a single cell. (B) A zoom-in representative 12× 3D-ExM confocal image of microtubule filaments in PtK2 cells. (C) Quantification of full width at half maximum (FWHM) of microtubules in images acquired before or after 4× or 12× 3D-ExM. Both the conventional confocal microscope and STED microscope were used in this experiment and quantification. n = 251, 140, 221, 155, 150, 36 cells pooled from two to three color-coded biological replicates (from left to right). The P value was calculated using Welch’s t-test (two-tailed). **** indicates P < 0.0001. (D) Schematic diagram of the structure of NPC created by BioRender. NP proteins Nup107 and Elys are labeled with yellow and red, respectively. (E) The EM images (upper row) and 12× 3D-ExM images (lower row) of the nucleus and NPs at the edge of the nucleus are shown in the right rows. DNA was stained with DRAQ5. (F) The representative 12× 3D-ExM images of Nup107 and Elys at the edge of a nucleus. (G). Left: an example Nup107 intensity profile at a single NP. Right: the average measured and corrected distance between two Nup107 intensity peaks in the line scan. n = 117 pooled from two biological replicates. (H) Representative image of the surface of RPE1 nuclei stained with Nup107 and DRAQ5. The black regions shown in the DNA channel are nuclear pores that colocalize with the Nup107 punctate signals. (I) Left: example images of the measurement of diameter and circumference of NPs. Right: quantification of the measured or corrected values of diameter and circumference of the NPs. n = 70 NPs pooled from two biological replicates. The calculated circumferences were derived from the diameters of the NPs, while the measured circumferences were obtained independently of their diameters. (J) Quantification of the number of NPs per unit area (1,000 µm2, equivalent to ∼7 µm2 after correction by the expansion factor). n = 30 cells pooled from two biological replicates. (K) Quantification of the estimated total number of NPs per cell. Each dot represents a single cell. n = 15 cells pooled from two biological replicates. All data are shown as the mean ± s.d.

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