Development of cost-effective Optical Projection Tomography instrumentation for static and dynamic samples

Abstract

Optical projection tomography (OPT) is a technique used to volumetrically image transparent mesoscopic (mm to cm scale) samples, making it well suited to the imaging of both fixed, chemically cleared tissues and live transparent animal models, such as zebrafish (D. rerio). As the optical analogue of x-ray computed tomography, 2D projection images through a sample are sequentially recorded as it is rotated, with the 3D volumetric data reconstructed from the set of projections. OPT may be implemented on an existing widefield imaging system simply by adding the means to rotate the sample. However, as the numerical aperture is typically limited to set the depth of field to equal the sample radius, resolution scales inversely with object size, and the sequential acquisition of projection images precludes the imaging of samples in motion. The work presented in this thesis aimed to increase the accessibility and applicability of OPT, through cost-effective hardware implementation and development of a novel single-shot OPT instrument to enable the imaging of moving samples. I present an evaluation of cost-effective hardware for use in a practical OPT instrument, including the use of CMOS cameras and LED light sources. I also characterised a cost-effective focal scanning OPT implementation, using a commercial scanning lens featuring a liquid tunable element to scan a narrow depth of field through a sample to record high-resolution projection measurements. To enable volumetric imaging of dynamic samples, I developed a single-shot OPT (ss-OPT) instrument, capable of tomography of a ∼1mm^3 volume, at a camera-limited frame rate of 62.5 volumes/s, achieved by simultaneously recording 8 projection images onto 4 cost-effective CMOS cameras, and leveraging compressive sensing reconstruction to retrieve volumetric images from this undersampled data set. The ss-OPT instrument was applied to 3D imaging of ink flowing in water, and freely swimming zebrafish embryos.