Shemilt, LA; (2014) Coherent Diffraction Imaging and Ptychography of Human Metaphase Chromosomes. Doctoral thesis , UCL (University College London).

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Abstract

Mitotic chromosome structure is highly important in the packing and safe separation of DNA during cell division. The chromatin fibre, the first packaging of DNA around histone proteins, is coiled into a highly condensed state during the metaphase stage of mitosis where the chromosomes form their characteristic ’X’ shape in order to be split into two daughter cells on cell division. Accessing the 3D structure of the condensed chromatin could provide high resolution information on the location of the genes inside the chromosome during metaphase and the placement of the proteins used in the packaging mechanism. We apply lensless X-ray imaging techniques to chromosomes in an attempt to access their structure. Coherent Diffraction Imaging and Ptychography have potential to achieve wavelength limited resolution in 3D. The techniques directly measure phase that can be used to map the electron density in the sample. Sample preparation is highly important to obtain good quality images from any microscopy technique. We start with studying sample preparations with Fluorescence and Scanning Electron Microscopy to observe the suitability and effects of the protocols. We perform lensless imaging on nuclei and chromosomes prepared with newly designed protocols for this type of X-ray imaging. Images of nuclei were retrieved from the ptychography experiments at 34-ID-C, I-13 and cSAXS. Ptychography was performed with a visible light source on a set of 46 human chromosomes. The images provide a map of electron density that can be used to calculate the mass of chromosomes and nuclei. Mass is a macroscopic structural quantity that can be used to observe the changes to the protein and DNA content in the nucleus during the cell cycle given a high enough resolution. The nuclei measured in this work were in an early mitotic state and gave consistent mass values calculated from the phase measured by ptychography. In the case of chromosomes mass calculated from the ptychography images was used to sort and identify them in a form of karyotyping. This method of identification is novel because it takes into account the whole of the chromosome contents, both protein and DNA, whereas standard methods of karyotyping look at positions of bandsin the chromosomes.

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