Investigation of DNA in Cell Nuclei by Combined Imaging Techniques

Abstract

Biological cells are the basic structural and functional units of all organisms. There are approximately 60 trillion eukaryotic cells within a human body alone. A common feature among eukaryotic cells is the nucleus, a membrane-bound organelle which, in addition to directing the synthesis of proteins and ribosomes, houses deoxyribonucleic acid (DNA). DNA is considered the blueprint of all life as it contains the genetic information necessary to develop and operate living organisms. Genetic mutations can lead to a wide variety of diseases, ranging from Huntington’s and Osteoporosis to Sickle-cell disease. The investigation of DNA allows for the identification of genes which trigger such diseases as well as the ability to diagnose them. Despite the critical importance of DNA, a complete understanding of its hierarchical structure is lacking due to its remarkable complexity. The hierarchical combination of different length scales fundamentally defines the function of a cell. The length scales of DNA structures range from 2 nanometers to 1 micrometer. On intermediate length scales hierarchical structures of several hundred nanometers in size are formed. This thesis uses a combination of scanning small-angle X-ray scattering, X-ray in-line holography, X-ray phase contrast tomography and visible-light fluorescence microscopy to quantitatively probe hierarchical and structural parameters of DNA within whole cell nuclei of lyophilized NIH-3T3 fibroblasts. When correlating visible-light micrographs with X-ray micrographs, information obtained from a specifically-labeled structure can be correlated with structural information obtained using X-rays. Here, analysis of X-ray dark field images, power-law fits of radial intensity profiles and quantitative phase contrast imaging yields access to the length scales of the scatterers, aggregation and morphology and the projected electron and mass densities of the nuclear material. Separate dark field representations for different momentum transfer ranges reveal nuclear regions containing nucleoli, heterochromatin and euchromatin. As revealed by the analysis of radial intensity profiles, the scatterers within these three regions all share similar morphology, but differ in aggregation. In particular, distinct aggregation regions dominated by heterochromatin and some, but not all, nucleoli are observed. X-ray holography is able to clearly distinguish all nucleoli by quantitative electron and mass density analysis. Furthermore, X-ray phase contrast tomography and visible-light fluorescence are able to distinguish the nucleoli. The nucleoli, heterochromatin and euchromatin are not all simultaneously observed in all representation schemes: euchromatin is only pronounced in the dark field, whereas heterochromatin is observed in both the dark field and aggregation representations. Nucleoli are observed in these two representations, as well as the holography, tomography and visible-light fluorescence. Distinct regions of DNA territories are only observed in the visible-light fluorescence data. From a biological point of view, the data show that nucleoli are the densest structures in the nucleus and scatter mostly on length scales up to 60 nm, indicating the existence of sub-structures in this size range, possibly proteins. While the density of heterochromatin and euchromatin is similar and lower than for nucleoli, as revealed by X-ray holography, heterochromatin mostly scatters on length scales above 35 nm and euchromatin scatters on all probed length scales. The aggregation maps reveal that compared to euchromatin, heterochromatin and nucleoli are more aggregated. This thesis demonstrates that only by combining information obtained throughout the various imaging modalities can important nuclear structure be identified and characterized according to size, density and aggregation state. The combined imaging approaches demonstrated in this thesis can be applied to investigate and characterize a large number of biologically complex systems, ranging from the sub-cellular to tissue levels.