Development of a microfluidic flow cytometer for plant protoplast analysis and manipulation by means of laser radiation pressure
Date
2022-12
Authors
Heerschap, Seth
Major Professor
Advisor
Dong, Liang
Shinar, Joseph
Lübberstedt, Thomas
Biswas, Rana
Wang, Xuefeng
Committee Member
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Abstract
Since the early 2000s with the development of soft lithography techniques, there has been a wide expansion of research involving microfluidic systems, particularly, flow cytometry. The advent of these highly capable microfluidic cytometry systems has brought new opportunities for research which in turn has begun to provide new applications in the industry. In the first chapter we review the current state of microfluidic cytometry by first looking at the cell analyzing techniques in the literature and then examining several methods for particle manipulation and sorting.
We develop a PDMS based, all-fiber microfluidic cell cytometer in which we tested with both beads and Arabidopsis mesophyll protoplasts and were successful in distinguishing both RFP and GFP labeled plant cells. We employ several techniques to improve data quality: By adding hydrodynamic focusing we improve data consistency and reduce clogging. To improve signal to noise ratio we fabricated a series of PDMS micro-lenses, improving light capture. We also improve detection by examining two different signal processing algorithms and finding methods to more intelligently detect events. We examine the effects of cell trauma by running several viability tests running the cells through the channels at different speeds and find evidence of a breakdown of viability at protoplast speeds of over 40 mm/s.
The effects of electromagnetic (EM) radiation pressure are studied with both beads and Arabidopsis mesophyll protoplast on a fabricated microfluidic system. We find that geometric scattering theory agrees well with our experimental results for both beads and protoplast cells. We report a calculated force of 2pN onto 2.5µm polystyrene beads from a 1.27W fiber laser. This setup provides feasibility for the sorting of Arabidopsis mesophyll protoplast by means of EM radiation pressure.
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dissertation