Figure 2. Outline of the serial section array scanning electron microscopy (SSA-SEM) method.

SSA-SEM enables the three-dimensional reconstruction of cell nuclei and PML domains combined with the visualization and quantification of VZV capsids with ultrastructural precision. (A) Ribbons of ultrathin serial sections are placed on gelatin-coated glass slides and then carbon-coated to prevent charging effects during SEM imaging. The indicated area (red square) contains about 60 consecutive sections. A standard TEM slot-grid (arrow) commonly used in serial section TEM and a ten-cent coin are shown for size comparison. (B) Low magnification view of a ribbon of serial sections imaged by SEM using a back-scattered electron detector (BSE). (C) Using BSE-SEM, regions of interest (ROI), such as whole cells, nuclei or PML-domains can be identified and then repeatedly imaged in consecutive sections, yielding a stack of unaligned digital images of the ROI. (D) The stack of digital images must be aligned, either manually or automatically, for later 3D reconstruction. (E) Structures of interest, such as electron dense heterochromatin (blue), PML domains (green) and VZV capsids (yellow) are manually or automatically (threshold) traced in each serial section for quantification of numbers, areas or volumes and for the visualization of size, shape and distribution of segmented structures in the final 3D model (F).