## 1 Introduction  
Microscopy has contributed significantly to our understanding of chromatin/chromosome biology, providing insights into their structure, compaction, and organization. [PERSON], in 1882, observed and illustrated chromosomes using a light microscope and coined the term mitosis [1]. Throughout the cell cycle, chromatin condenses and undergoes conformational changes from interphase to metaphase [2; 3] occupying non-random chromosomal territories (CTs) [4] (reviewed in [5; 6; 7; 8; 9]). These CTs are essential for maintaining overall genome stability and function; however, their spatial positioning in different cells and disease types is unclear and needs further investigation [10; 11; 12; 13]. Another aspect that requires interpretation is how nucleosomes, the 'building blocks' of the chromosome, fold into high order structures such as the controversial 30 nm structure (reviewed in [14; 15; 16; 17; 18]). It is clear that 3D high-resolution imaging is a pre-requisite in addressing unresolved questions related to the spatial organization and structure of chromosomes. Several fluorescence imaging modalities, including Confocal Laser Scanning Microscopy (CLSM) and, more recently, light-sheet fluorescence microscopy (LSFM) [19; 20; 21], have been used to study the live dynamic processes of chromatin condensation and segregation. Fluorescence 3D volume imaging, although useful, has limitations. Its light diffraction is limited to \(\sim\)200 nm, making it challenging to resolve the smaller chromatin structures [22]. Super-resolution microscopy techniques are under development and can attain a resolution far beyond the diffraction limit of light; however, z resolution is still less than that can be achieved by a factor of 2 laterally [23]. Hard X-ray microscopy has been used to image intact chromosomes [24] and nuclei with nuclear substructures including condensed chromatin [25] in 3D using coherent diffraction imaging (CDI) after tilting the sample at different angles. In comparison,electron microscopy (EM) provides resolutions ranging from nanometers down to below an angstrom [26] and has been extensively used to examine the chromosome high order structure and compaction [27]. Transmission electron microscopy (TEM) observations have shown that the metaphase chromosomes have a multilayered structure, whereby each individual layer has the width corresponding to a mononucleosome sheet and has been reviewed [28]. TEM requires the sample thickness to be below 100 nm [29]; therefore, intact chromosomes (over 1 micron thick) would require manual sectioning before imaging. TEM tomography has been performed on chemically treated chromosomes [30] and after serial sections have been collected and aligned in sequence to study the organization of mitotic chromosomes [31]. Furthermore, 3D image reconstructions have been made after sectioning chromosomes and taking micrographs of serial tilted images (generally tilted by \(\pm\)70\({}^{\circ}\)) [32; 33]. Recently, ChromEMT (ChromEM tomography) with improved labelling has been applied to study the 3D ultrastructure of chromatin and mitotic chromosomes after sectioning samples embedded in resin [34]. The serial section TEM (ssTEM) method is useful but poses challenges as it is susceptible to artefacts such as shrinking, distortion and section loss. It also requires manual image alignment of sections and is restricted to minimal volumes [35]. Robust automated volume SEM techniques have been developed that have revolutionized EM (reviewed in [36; 37]). The focused ion beam SEM (FIB-SEM) approach uses an ion source (Gallium (G+) ions) that removes thin layers (down to 5 nm) of the sample, followed by automatic imaging of the exposed surface by the SEM [36; 37; 38], and has been applied to chromosomes [39; 40; 41; 42]. This method, although practical, has a narrow field of view (\(\sim\)20 \(\upmu\)m\({}^{2}\)) and a slow sputtering speed.