A comparison between hollow cone illumination and wavelet transform methods for chromatin fiber orientation measurement

Nucleosomes are proven to be the fundamental unit of chromosome structure. The stacking and folding of the nucleosome fibers within a chromosome is not fully understood. One of the reasons for the incomplete understanding of chromosome internal structure is that a nucleosome, about 11 nm in diameter, can not be resolved within the large chromatids (∼ 700 nm diameter) of a chromosome. In a transmission electron microscope (TEM), the large difference in size between the small diameter nucleosomes and a chromosome results in an extremely low contrast arising from individual nucleosomes. Consequently, the nucleosome fiber can not be detected within an intact chromosome. In this study, we compared two different methods in TEM, namely the hollow cone illumination (HCI) TEM and wavelet transform (WT) analysis on bright-field TEM (BFTEM) images, to analyze internal structure of chromosomes at length scales ranging from 10 to 30 nm. Isolated chromosomes were expanded and the orientation of the chromatin fibers was measured by HCI TEM and by WT applied to BFTEM. We demonstrated that the results obtained by the two methods are in an agreement.