## 2 SBFSEM Method  
Nanoscale 3D imaging of large biological sample volumes can be achieved using SBFSEM [43]. The concept was first proposed in 1981 [45] and initially commercialized as 3 view(tm) (Gatan, Abingdon, UK) [46], a fully automated sectioning and image collection system. This approach gives a high cutting speed and a wide area cutting [47]. The spatial resolution is determined by the lateral (x-y) and slice thickness in the z-direction [48]. In the x-y plane, a pixel size of 3-4 nm is possible [47], and in the z-direction, the voxel size is approximately 20-25 nm [49; 50]. A schematic of the SBFSEM workflow is shown in Figure 1.  
An extensive sample preparation procedure similar to preparation procedures for TEM is crucial for obtaining good quality images [43]. First, the sample is fixed using aldehydes followed by heavy metal staining to enhance contrast for high electron density. The sample is then dehydrated and infiltrated with resin, followed by embedding and polymerization before imaging [51]. Choices of fixatives (including glutaraldehyde and formaldehyde), heavy metal stains (e.g., osmium, uranium, lead, platinum, etc.), and resins (e.g., Epon812, Hard Plus, Spurr's, Durcupan, etc.) are used dependent on the sample type being imaged. Overall, the sample must be able to (i) withstand the vacuum, the high energy, including the current of the electron beam, (ii) give suitable image contrast and (iii) be structurally rigid [36].  
Before the sample is mounted in an SBFSEM chamber, the resin block face must be trimmed to a suitable size and pyramidal shape. It is coated with a metallic layer (e.g., gold) to prevent charging. The sample is mounted in the SEM chamber from which air is pumped out to generate a vacuum. Thereafter electron beam energies of 1-3 keV are typically applied [52]. The block surface of the specimen is imaged using SEM, and a backscattered electron (BSE) detector is used to collect the signal from heavy atoms [50]. The procedure involves sectioning using a diamond knife in the built-in ultramicrotome that resides in the chamber of the SEM. After imaging the block face, the specimen height is raised (generally between 25-100 nm) and the newly exposed surface is imaged, and the removed slice is discarded. This cut-and-image cycle is left to run continuously, acquiring thousands of aligned serial images, making it a fully automated process (Figure 1). Normal practice is to leave the SBFSEM running overnight, which acquires approximately 1000 images. A total of 345 images (11 \(\times\) 11 \(\times\) 25 nm) [44] took approximately 6.5 h.  
Acquired data stacks are then reconstructed and analyzed using different software packages such as DigitalMicrograph(r) 3.5 (Gatan 3 View(r) system software), Fiji, Imaris, Amira, Avizo and Ilasktik, providing unique and vital 3D information. This includes quantification by measuring various parameters of the sample, i.e., shape, volume, distribution [35]. Figure 1B(i-iii) shows an example of three sections from a data stack with segmented boundaries of a chromosome. Figure 1B(iv) shows a single chromosome after reconstruction [44].  
Below we highlight the studies that have been carried out to date in applying SBFSEM for analyzing chromatin and chromosomes. The experimental conditions and findings of these studies are summarized in Table 1.  
Figure 1: Principle of SBFSEM and a 3D model of a single chromosome. (**A**) Showing basic workflow of SBFSEM volume imaging; (**B**) different slices from an SBFSEM data stack showing segmentation (i–iii) and (iv) a 3D modelled X-shaped chromosome; scale bar—0.5 \(\upmu\)M [44].