Surface structures consisting of chromatin fibers in isolated barley ( Hordeum vulgare ) chromosomes revealed by helium ion microscopy

The chromosome compaction of chromatin fibers results in the formation of the nucleosome, which consists of a DNA unit coiled around a core of histone molecules associated with linker histone. The compaction of chromatin fibers has been a topic of controversy since the discovery of chromosomes in the 19th century. Although chromatin fibers were first identified using electron microscopy, the chromatin fibers on the surface of chromosome structures in plants remain unclear due to shrinking and breaking caused by prior chromosome isolation or preparation with alcohol and acid fixation, and critical point drying occurred into dehydration and denatured chromosomal proteins. This study aimed to develop a high-quality procedure for the isolation and preparation of plant chromosomes, maintaining the native chromosome structure, to elucidate the organization of chromatin fibers on the surface of plant chromosomes by electron microscopy. A simple technique to isolate intact barley ( Hordeum vulgare ) chromosomes with a high yield was developed, allowing chromosomes to be observed with a high-resolution scanning ion microscopy and helium ion microscopy (HIM) imaging technology, based on a scanning helium ion beam. HIM images from the surface chromatin fibers were analyzed to determine the size and alignment of the chromatin fibers. The unit size of the chromatin fibers was 11.6 ± 3.5 nm and was closely aligned to the chromatin network model. Our findings indicate that compacting the surface structure of barley via a chromatin network and observation via HIM are powerful tools for investigating the structure of chromatin.