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Title: Visible light detection in microflow cytometry : advancing miniaturisation and detection methods
Author: Zmijan, Robert
ISNI:       0000 0004 5922 4965
Awarding Body: University of Southampton
Current Institution: University of Southampton
Date of Award: 2016
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The advent of optical flow cytometry in the 1980s created a growing demand for flow cytometers in biomedical research and inspired scientists to build or modify commercial instruments. Oceanographers use flow cytometers to perform phytoplankton analysis in remote locations on board ships. Our current knowledge of the role of algae and phytoplankton and their impact on global climate is limited by the frequency with which it can be analysed. Cytometric analysis of phytoplankton life cycles in their natural environment has the potential to revolutionise our understanding, but requires the use of miniaturised devices to operate at remote locations. To date, many research groups have developed unique flow cytometer systems to enable in-situ phytoplankton analysis. The development and miniaturisation process continues, and there is still a need to improve detection limits, modularity, and maintenance requirements. Furthermore, recent advancements in image processing and the availability of high resolution and high sensitivity cameras enable combining flow cytometry with microscopy. Image flow cytometry offers better morphological information, and unlike traditional cytometry, has the potential to screen multiple particles at a time, increasing the throughput. High throughput is also highly desired in an early detection and monitoring of hematological diseases such as leukemia, and other cancers. The aim of this thesis is to contribute to the development process of the traditional micro flow cytometry and imaging cytometry. Two kinds of devices were described and investigated in this thesis. The first device was a micro-flow cytometer chip based on optical fibre coupled architecture with an integrated micro lens that was fabricated to demonstrate an integrated fluorescence collection method by using a miniature cylindrical lens with a graded refractive index profile. The fluorescence collection efficiency was found comparable to a standard microscope objective. The use of the micro lens represents a significant progress in miniaturisation and assembly without sacrificing of performance. Practical use of the device was demonstrated by optical differentiation of phytoplankton cultures by detection of fluorescence, forward, and side scattered light. In addition, the device was tested with plain polystyrene, and fluorescent particles. The results were compared against two commercial instruments. The second device presented in this thesis is an imaging flow cytometer that uses acoustic levitation to assemble particles into a sheet structure. A high resolution, low noise CMOS camera is then used to capture images of thousands of cells with each frame. While ultrasonic focussing has previously been demonstrated for 1D cytometry systems, extending the technology to a planar, much higher throughput format and integrating imaging is non-trivial, and represents a significant jump forward in capability, leading to diagnostic possibilities not achievable with current systems. A galvo mirror was used to track the images of the moving cells permitting exposure times of 10ms at frame rates of 50Hz with near-single-pixel motion blur. At 80 Hz, a throughput of over 200k particles per second was achieved with particle velocities reaching 104 mm/s. The factors affecting motion blur and throughput were studied, and demonstrated the system with fluorescent beads, leukocytes, chondrocyte cell lines, and algae.
Supervisor: Morgan, Hywel Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available