AbstractsBiology & Animal Science

"In accordance with the requirements for completion of a Doctorate of Philosophy at Macquarie University, February 2014". Thesis by publication Bibliography: pages 153-172. Ch. 1. Introduction  – Ch. 2. Paper 1. Automated detection of rare-event pathogens through time-gated luminescence scanning microscopy  – Ch. 3. Paper 2. Time-gated orthogonal scanning automated microscopy (OSAM) for high-speed cell detection and analysis ; Paper 3. Resolving low-expression cell surface antigens by time-gated orthogonal scanning automated microscopy  – Ch. 4. Paper 4. Time-resolved scanning cytometry in the μs region: rapid discrimination of lifetime-tunable microspheres  – Ch. 5. Paper 5. Tunable lifetime multiplexing using luminescent nanocrystals  – Ch. 6. Conclusions and perspectives. "Detection, quantification, or localisation of particular cells or molecules quickly, sensitively and accurately, is fundamental to many areas of modern biomedical research and industry: from understanding sub-cellular processes and biochemical pathways to the early detection of diseases in clinical settings. However, the targets-of-interest are often in a tiny minority within a surrounding abundance of biological and biochemical substances, which presents significant problems for rapid detection of trace of biological analytes in large-volume samples. Current fluorescence detection based on fluorescent biolabels is seriously limited by autofluorescence from non-target detritus and organisms and spectral overlapping associated with multichannel detection in the spectral domain. These limits can be addressed using time-gated luminescence detection techniques based on molecular probes with luminescence lifetimes in a μs-to-ms region rather than the nanosecond lifetimes of standard fluorescent labels, so that autofluorescence background can be effectively suppressed. This thesis explores novel time-gated luminescence strategies to develop a new generation of analytical systems - time-domain scanning cytometry - for high-throughput, ultrasensitive detection, quantification and localization of trace biomolecules and rare-event cells in biological samples. The first part of this thesis presents two practical scanning strategies for fully automated time-gated luminescence detection of rare cell types in 2-dimensional samples. We first report a "step-by-step" scanning scheme incorporating the time-gated detection with wide-field scanning followed by imaging confirmation. This minimises the requirements for image acquisition, storage and processing to specific areas-of-interest identified as containing target analytes. We then present a more sophisticated "on-the-fly" scanning scheme employing continuous-motion scanning which involves X-axis scanning for rapid identification of target cells and orthogonal Y-axis scanning for accurate localisation and quantitative measurement of cells. This accelerates the scanning speed of a microscopy slide by a factor of 20, reducing whole-sample scanning time from 1 hour to ~ 3 minutes, and enables…