Advances In optical projection tomography

Abstract

Optical projection tomography (OPT) is a 3D imaging technique that can be applied to non- or weakly scattering samples and is often described as the optical equivalent of x-ray computed tomography (CT). Analogous to x-ray CT, OPT acquires wide-field images of a sample from many angles and uses this projection data to reconstruct the 3D distribution, applicable to both absorption and fluorescence contrast. This thesis describes how to implement OPT on a standard wide-field microscope, derives rigorous models for image formation and reconstruction in OPT, and discusses how performance can be improved in terms of spatial resolution and acquisition time through the use of focal scanning, particularly for samples <1 mm in diameter. After a brief overview of 3D optical imaging techniques, a mathematical framework is developed for the standard experimental approaches to OPT based on telecentric imaging, which allows a rigorous comparison with x-ray CT. It is shown that reconstruction of the optical projections using filtered back projection introduces anisotropy to the spatial resolution in the reconstructed images. The OPTiM adapter plate is then described. This open hardware component, together with openly shared software, allows an existing microscope to be adapted for OPT at low cost, thereby increasing the accessibility of OPT for a wide range of researchers. To improve the performance of OPT in terms of spatial resolution and acquisition time, an alternative data acquisition model for OPT is developed that is based on telecentric remote focal scanning. Detailed analysis quantifies the expected improvement in the spatial resolution of the 3D image reconstructions and the reduction in the acquisition time. The derived mathematical framework is also used to identify factors for further optimisation. The focal-scanning concept is extended to “region-of-interest OPT”, where it is shown that the dynamic control of focal plane position can lead to improved signal to background ratios as well as reducing the impact of streak artefacts. The final section of the thesis addresses the equivalence between a non-telecentric optical system and cone-beam CT, which removes the telecentricity requirement of the traditional approach to OPT. Derivation of the associated optical transform leads to a modified form of reconstruction based on the FDK algorithm. It is shown that axial and lateral tracking enables this new OPT approach to acquire 3D images of a sub-volume within a larger body. The optical setup and associated optical transforms for both telecentric and non-telecentric systems are described.