Fig. 3: 3D nanoscale imaging of the nucleus through iterative HD-MIBI.
From: Subcellular localization of biomolecules and drug distribution by high-definition ion beam imaging
a Representative single-plane HD-MIBI images at different depths of a HeLa cell nucleolus. HeLa cells were stained with anti-nucleolin-19F/FITC, and 785 individual planes were acquired to obtain HD-MIBI images of a nucleolus from its appearance to its disappearance. Single planes every 100 depths show a distinctive molecular distribution of nucleolin in the 3D space. See Fig. S19 for images of each individual plane. Blue, red, and green arrows indicate x-axis, y-axis, and z-axis, respectively. b (Left) 3D surface reconstruction of images of nucleolin staining of a nucleolus shown in panel (a). (Right) Overviews of the same nucleolus along x-axis (blue arrow), y-axis (red arrow), and z-axis (green arrow) with the origin represented as a black dot. c Representative 3D surface reconstruction of nucleolus (green), centromeres (red), and nuclear speckles (cyan). HeLa cells were stained with anti-nucleolin-19F/FITC, anti-CENP-A-81Br/Cy3, and anti-SC35-biotin (recognized by streptavidin-197Au/FITC). The image consists of the 3D reconstruction of a stack of 40 consecutive planes. d Representative 3D reconstruction of nucleolin (cyan), phosphorus (blue), H3K9me3 (magenta), H3K27Ac (green), and SC35 (red) in a HeLa cell stained with anti-nucleolin-19F/FITC, anti-H3K9me3-81Br/Cy3, anti-H3K27Ac-127I/Cy5, and anti-SC35-biotin (recognized by streptavidin-197Au/FITC). The image consists of the 3D reconstruction of a stack of 400 consecutive planes. Enlarged images from the boxed region show details of marker distribution. e (Left) Workflow of iterative HD-MIBI: (1) Five to ten depths at high current are acquired in a cell of interest at 25 × 25 μm to identify a ROI. (2) Iterative acquisition is performed by focusing the beam into the ROI at lower currents in a smaller area of 5 × 5 μm or 10 × 10 μm. (Right) (Top) Representative region of the IdU signal (127I) in a nucleus of a HeLa cell labeled for 24 h with IdU (127I). (Bottom) A 10 × 10 μm ROI was acquired for super-resolution imaging of chromatin folding. Enlarged image of the boxed region shows fine detail of IdU labeled chromatin. n = 2. f Quantification of the resolution of iterative HD-MIBI imaging of a nucleolus. From left to right: (1) –e image of a HeLa cell reveals subnuclear structures including the nucleoli chosen for iterative HD-MIBI. (2) A 3 × 3 μm ROI was acquired by iterative HD-MIBI for nucleolin (19F). n = 2. (3) An enlarged view from the boxed region in image 2 showing the nanoscale organization of nucleolin (19F). (4) Line scan along the red line in the boxed region in image 3 demonstrates identification of molecules spaced about 30 nm. (5) An enlarged view of the boxed region in image 3. See Fig. S21 for additional examples. g Iterative HD-MIBI 3D reconstruction. HeLa cells were stained with anti-FBL-81Br/Cy3, anti-nucleolin-127Ir/Cy5, and anti-NPM1-biotin (detected with streptavidin-197Au/FITC). Iterative HD-MIBI was performed on a site with three nucleolin. The −e image confirmed that the ROI was acquired. Nucleolin (cyan), phosphorus (blue), FBL (green), and NPM1 (red) were used for 3D reconstruction from 40 consecutive planes. These images identify the granular component (GC, NPM1-positive), dense fibrillar component (FBL- and nucleolin-positive), and perinucleolar heterochromatin (PNC, phosphorus-high).
