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Cryoelectron tomography of the cell nucleus using scanning transmission electron microscopy and deconvolution processing technology has highlighted a large-scale, 100- to 300-nm interphase chromosome structure, which is present throughout the nucleus. This study further documents and analyzes these chromosome structures. The paper is divided into four parts: 1) evidence (preliminary) for a unified interphase chromosome structure; 2) a proposed unified interphase chromosome architecture; 3) organization as chromosome territories (e.g., fitting the 46 human chromosomes into a 10-μm-diameter nucleus); and 4) structure unification into a polytene chromosome architecture and lampbrush chromosomes. Finally, the paper concludes with a living light microscopy cell study showing that the G1 nucleus contains very similar structures throughout. The main finding is that this chromosome structure appears to coil the 11-nm nucleosome fiber into a defined hollow structure, analogous to a Slinky helical spring [https://en.wikipedia.org/wiki/Slinky; motif used in Bowerman et al., eLife 10, e65587 (2021)]. This Slinky architecture can be used to build chromosome territories, extended to the polytene chromosome structure, as well as to the structure of lampbrush chromosomes.

A molecular architecture is proposed for a representative mitotic chromosome, human chromosome 10. This architecture is built on an interphase chromosome structure based on cryo-electron microscopy (cryo-EM) cellular tomography [J. Sedat et al., Proc. Natl. Acad. Sci. U.S.A., in press], thus unifying chromosome structure throughout the complete mitotic cycle. The basic organizational principle for mitotic chromosomes is specific coiling of the 11-nm nucleosome fiber into large scale, ∼200-nm interphase structures, a Slinky [https://en.wikipedia.org/wiki/Slinky; motif cited in S. Bowerman et al., eLife 10, e65587 (2021)], then further modified with subsequent additional coiling for the final mitotic chromosome structure. The final mitotic chromosome architecture accounts for the dimensional values as well as the well-known cytological configurations. In addition, proof is experimentally provided by digital PCR technology that G1 T cell nuclei are diploid with one DNA molecule per chromosome. Many nucleosome linker DNA sequences, the promotors and enhancers, are suggestive of optimal exposure on the surfaces of the large-scale coils.

Cryo-electron tomography (cryo-ET) allows for the high-resolution visualization of biological macromolecules. However, the technique is limited by a low signal-to-noise ratio (SNR) and variance in contrast at different frequencies, as well as reduced Z resolution. Here, we applied entropy-regularized deconvolution (ER-DC) to cryo-ET data generated from transmission electron microscopy (TEM) and reconstructed using weighted back projection (WBP). We applied deconvolution to several in situ cryo-ET datasets and assessed the results by Fourier analysis and subtomogram analysis (STA).

The complex environment of biological cells and tissues has motivated development of three-dimensional (3D) imaging in both light and electron microscopies. To this end, one of the primary tools in fluorescence microscopy is that of computational deconvolution. Wide-field fluorescence images are often corrupted by haze due to out-of-focus light, i.e., to cross-talk between different object planes as represented in the 3D image. Using prior understanding of the image formation mechanism, it is possible to suppress the cross-talk and reassign the unfocused light to its proper source post facto. Electron tomography based on tilted projections also exhibits a cross-talk between distant planes due to the discrete angular sampling and limited tilt range. By use of a suitably synthesized 3D point spread function, we show here that deconvolution leads to similar improvements in volume data reconstructed from cryoscanning transmission electron tomography (CSTET), namely a dramatic in-plane noise reduction and improved representation of features in the axial dimension. Contrast enhancement is demonstrated first with colloidal gold particles and then in representative cryotomograms of intact cells. Deconvolution of CSTET data collected from the periphery of an intact nucleus revealed partially condensed, extended structures in interphase chromatin.

Malaria is a potentially fatal infectious disease caused by the obligate intracellular parasite Plasmodium falciparum. The parasite infects human red blood cells (RBC) and derives nutrition by catabolism of hemoglobin. As amino acids are assimilated from the protein component, the toxic heme is released. Molecular heme is detoxified by rapid sequestration to physiologically insoluble hemozoin crystals within the parasite's digestive vacuole (DV). Common antimalarial drugs interfere with this crystallization process, leaving the parasites vulnerable to the by-product of their own metabolism. A fundamental debate with important implications on drug mechanism regards the chemical environment of crystallization in situ, whether aqueous or lipid. This issue had been addressed previously by cryogenic soft X-ray tomography. We employ cryo-scanning transmission electron tomography (CSTET) to probe parasite cells throughout the life cycle in a fully hydrated, vitrified state at higher resolution. During the acquisition of CSTET data, Bragg diffraction from the hemozoin provides a uniquely clear view of the crystal boundary at nanometer resolution. No intermediate medium, such as a lipid coating or shroud, could be detected surrounding the crystals. The present study describes a unique application of CSTET in the study of malaria. The findings can be extended to evaluate new drug candidates affecting hemozoin crystal growth. [ABSTRACT FROM AUTHOR]