Figure 2. Visualization of cellular cross-sections in dividing cells.

Schematic explaining visualization of cells in different planes in three-dimension (3D). (A) Initially, the rod-shaped Bacillus lies flat on an electron microscopy (EM) grid. (B) Representation of 3D view of a cellular section obtained by cryo-FIB-ET in the xyz coordinate axis. x axis represents the length along the short axis of the cell, y axis represents the length along the long axis of the cell and z axis represents the height of the cellular specimen. (C) Top panel: projection image of the cell in the xy coordinate plane (top view). Bottom panel: the corresponding projection image in the xz coordinate plane when the cell is rotated about its short axis by 90° (side/rotated view). The lateral and septal peptidoglycan (PG) are also indicated. See also Figure 2—figure supplement 1.

Figure 2—figure supplement 1. Cryo-FIB milling workflow and analysis.

(A) Schematic of a rod-shaped cell (gray) subjected to cryo-FIB milling. Two parallel beams of gallium ions (brown) ablate the cellular material from the top and the bottom, leaving a thin slice (~100–300 nm thick) for imaging using cryo-ET. (B) Schematic depicting different sections of the septal disc that can be captured when a rod-shaped cell is FIB-milled depending on the milling angle, orientation of the cell on the electron microscopy grid and orientation of the cell with respect to the tilt axis. (C) Schematic illustrating the orientation of bacterial cells (gray) on a grid square (orange). Since all cells lie flat on the grid with their xy axis parallel to the grid (as shown in Figure 2), the missing wedge is always in a direction that is perpendicular to the xy plane. (D) Schematic depicting the missing wedge issue in cryo-ET workflow. Since we are only able to image from ~ ± 60^o, the areas from −60^o to −90^o and from +60^o to +90^o remain unsampled, leading to missing information for these areas.