Quantifying Three-dimensional Chromatin Organization Utilizing Scanning Transmission Electron Microscopy: ChromSTEM

Abstract

Chromatin organization over a wide range of length scales plays a critical role in the regulation of gene expression and deciphering these processes requires high-resolution, three-dimensional, quantitative imaging of chromatin structure in vitro. Herein we introduce ChromSTEM, a method which utilizes high angle annular dark field imaging and tomography in scanning transmission electron microscopy in combination with DNA-specific staining for electron microscopy. We utilized ChromSTEM to quantify chromatin structure in cultured cells and tissue biopsies through local DNA distribution and the scaling behavior of chromatin polymer. We observed that chromatin is densely packed with an average volume concentration of over 30% with heterochromatin having a two-fold higher density compared to euchromatin. Chromatin was arranged into spatially well-defined nanoscale packing domains with fractal internal structure and genomic size between 100 and 400 kb, comparable to that of topologically associated domains. The packing domains varied in DNA concentration and fractal dimension and had one of the distinct states of chromatin packing with differential ratio of DNA content to the chromatin volume concentration. Finally, we observed a significant intercellular heterogeneity of chromatin organization even within a genetically uniform cell population, which demonstrates the imperative for high-throughput characterization of chromatin structure at the single cell level.

Nanoscale Chromatin Imaging and Analysis (nano-ChIA) platform bridges 4-D chromatin organization with molecular function

“…We calculated the average mass scaling from individual mass scaling centered on all the voxels within one packing domain, and quantified the D and R f . Assuming the highest intensity in the tomograms represents 100% unhydrated DNA, we converted the voxel values to DNA density and calculated the average genomic size of PDs using that of a single packing domain with the average packing scaling and average domain radius (29).…”

Nanoscale Chromatin Imaging and Analysis (nano-ChIA) Platform Bridges 4-D Chromatin Organization with Molecular Function

“…Hi-C), neutron scattering, soft x-ray tom ography (SXT), super-resolution microscopy, partial wave spectroscopic (PWS) microscopy, and more recently developed chromEMT, which combines DNA-specific DNA labeling and transmission electron tomography,. These techniques have provided critical insights into the principles of 3D chromatin structure, but they have fundamental limitations rooted the philosophy of experimental designs, characterization resolution, or sample preparation protocols (6). Because no individual technique can fully elucidate the chromatin organization and its relation to molecular function at all length and time scales (7), it is necessary to create an ensemble combining complementary techniques.…”

Investigation of DNA in Cell Nuclei by Combined Imaging Techniques

“…Rather than the classically considered hierarchically folded assembly, the authors concluded that chromatin can best be described as a disordered and flexible granular chain. Motivated by the work of Ou et al [42], Li et al [43] introduced a method to quantitatively image the 3D distribution of chromatin in A549 lung cancer cells. The method, termed "ChromSTEM", exploits high angle annular dark field imaging and tomography in scanning transmission electron microscopy in combination with a DNA-specific fluorescent label compatible with electron microscopy.…”