Development of 3D x-ray phase-contrast Imaging and analysis tools for tubular and branching structures with applications in colorectal cancer research.

Abstract

X-ray micro-computed tomography (μCT) is a non-destructive imaging technique used to capture the internal three-dimensional (3D) structure of small samples at high spatial resolutions. As it requires a certain level of knowledge in engineering, material science, mathematics and physics, it and can quickly become challenging to follow for readers with diverse academic backgrounds. In particular, biological tissues can have irregular internal structures, with convoluted shapes and orientations. In such cases, structural quantification becomes remarkably difficult as it often requires advanced knowledge in mathematics (topology) and engineering (digital signal processing). To facilitate research that employs μCT to study the internal microstructure of soft tissues such as the colon, a suite of tools, methods, and workflows have been developed here. The study was motivated by the problem of morphological quantification of tubular glands in the colon called crypts. These begin to branch during early-stage colorectal cancer (CRC), which makes crypt budding of central interest in the study of the disease. To study crypt budding during early-stage CRC using μCT, a sample-preparation method was developed here that employs a murine model for CRC and preserves the natural tubular shape of the colon. An X-ray phase-contrast μCT imaging protocol-design flowchart was developed to design X-ray imaging protocols for optimal spatial resolution. The flowchart was then employed to non-destructively image the tubular murine colon samples. These were subsequently unrolled digitally, using a tool developed here called ‘3D cyclorama’. The unrolled views of the colonic samples were used to identify the budding crypts, and a detailed 3D structural phenotype was compiled, employing a mathematical modelling approach that quantified the morphology of individual branches within the crypt. The tools, methods and workflows developed here, although motivated by the study of earlystage CRC, are applicable to a wide range of problems. The protocol-design flowchart will help researchers to understand, adapt and apply key elements of phase-contrast X-ray imaging for their own research questions, involving materials and tissues that do not significantly absorb Xrays. Digital unrolling may be applied to structures including, the colon to study crypt budding, ancient scrolls to retrieve inscribed text, or industrial applications to flatten curved parts. Finally, morphological quantification of budding structures may also be applied to blood vessel and airway networks, or plant roots. Taken together, the work presented in this thesis will help researchers from diverse backgrounds to harness the great potential of X-ray μCT imaging. The developed tools, workflows and methods will support various studies by facilitating the extraction of useful information from μCT data of samples with challenging geometries.

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Identifiers

ORCID for Sylvia Pender: orcid.org/0000-0001-6332-0333

ORCID for Philipp Schneider: orcid.org/0000-0001-7499-3576

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