## 4 Structural Examination of Chromosomes Using SBFSEM  
SBFSEM has been used to provide information into the structure of chromatin and chromosomes. [PERSON] et al. 2014 published a sample preparation protocol for preparing isolated chromosomes, as a previously published protocol used tissue samples for SBFSEM [53]. This study optimized the sample preparation by cleaning the nuclei and debris from the sample, the concentration of the heavy metal platinum blue stain and the sample density for SBFSEM. The study showed that variation in sample preparation could affect the overall structural details of chromosomes. Chromosomes prepared from polyamine preparations were imaged using SBFSEM after platinum blue staining (Figure 3A(i)). The 3D reconstruction (13 \(\times\) 13 \(\times\) 100 nm) provided an X-shaped mitotic chromosome displaying visible chromatids on both the p and q arms but having no inner structural details (Figure 3A(ii)) [55]. In comparison, methanol acetic acid prepared chromosomes showed internal structural details or cavities along the length of the two sister chromatids (Figure 3B(i)) that were then rendered and measured (Figure 3B(ii)). Some of these cavities were connected to the external space surrounding the chromosome, whereas other cavities were sealed inside the chromosome, forming an average of 6% of the total chromosome volume (Figure 3B(ii)) [49]. Human polyamine chromosomes retain both their  
Figure 2: SBFSEM examination of chromosomes at different mitotic stages. (**A(i)**) A 2D orthoslice of a propane nucleus from the acquired SBFSEM stack showing chromosomes in contrast (white); (**A(ii)**) 3D reconstruction of all 46 chromosomes from a propane nucleus (red); (**A(iii)**) 3D cluster of all 46 modelled and identified chromosomes. Each of the 23 pairs are labelled in different colors. Scale bar—2 μm [44]. (**B(i)**) A 2D orthoslice of a metaphase cell from the acquired SBFSEM images; (**B(ii)**) 3D segmentation of a chromosome complement consisting of 46 metaphase chromosomes (red); (**B(iii)**) 3D reconstructions of 46 metaphase chromosomes labelled in different colors. Scale bar—10 μm [54]. Average diameters during different stages of mitosis were examined by measuring chromosomes imaged using SBFSEM. At early profphase, they measured 0.64 μm [54], 1.53 μm (765 nm for each chromatid) [49], and 1.06 μm [44] at late profphase and 1.15 μm in metaphase [54]. These studies performed on different cell lines provide preliminary data on the 3D gradual chromosome condensation process during mitosis but requires further examination from the same cell line. proteins and nucleic acid after preparation, compared to methanol acetic acid prepared chromosomes that have part of the histones removed, making them less condensed and potentially prone to structural degradation [55]. The role of structural proteins on chromosome stability has been explored using Correlative Light and Electron Microscopy (CLEM) [58]. Using CLEM, specific proteins are immunabeled (fluorescence), giving positional information that can be investigated further for the ultrastructural features using SBFSEM at that same region. Depletion of Ki-67 (Figure 3C) [54] and SMC2 (sc scaffold protein) (Figure 3D) [56], essential proteins for maintaining the mitotic chromosome structure, led to disorganized chromosomal structures having a larger surface area [54,56] (Figure 3C,D). A targeted approach for visualizing chromatin at specific genomic regions combined SBFSEM with in situ hybridization known as 3D electron microscopic in situ hybridization (3D-EMISH). This approach enabled the visualization of 3D chromatin folding at targeted genomic regions using BAC probes (1.7 Mb segment) with ultra-resolution (5 \(\times\) 5 \(\times\) 30 nm in x, y, z dimensions, respectively) after hybridizing biotinylated DNA probes. Many chromatin structures from multiple human lymphoblastoid cells were analyzed simultaneously.