The objective of this research is to develop automated and integrated microsystems for high-resolution imaging and high-throughput phenotyping / laser ablation of C. elegans. These microsystems take advantage of microfluidic technology for precisely handling animals and computer-aid automation for high-throughput processing. We demonstrated automated and high-throughput imaging / sorting and laser ablation of C. elegans. This thesis work is divided into four parts: development of a microsystem for imaging and sorting, development of a microsystem for laser cell ablation, development of a novel temperature measurement method, and development of pressure measurement method in microchannels.
First, a microsystem was developed for high-throughput microscopy at high resolution and sorting. The microfluidic chip integrates novel microfluidic components to trap, position, immobilize, and sort/release animals. To characterize device operation and aid design of the device numerical models were developed. The experimental results demonstrate that the device operates robustly in a completely automatable manner. Additionally, a sophisticated control algorithm developed by Matthew Crane (Dr. Hang Lu¡¯s lab) automates the entire process of image acquisition, analysis, and sorting, which allows the system to operate without human intervention. This microsystem sorted worms based on their fluorescent expression pattern with over 95% accuracy per round at a rate of several hundred worms per hour.
Secondly, the technologies developed for the imaging/sorting system were adapted and further improved to develop a microsystem for high-throughput cell laser ablation of C. elegans. The multiplex ablation module combined with the embryo trap module enables robust manipulation of embryos/L1-stage C. elegans. In addition, software for image processing and automation was developed to allow high-throughput cell ablations. This system performed ablation of a large number of animals and demonstrated accurate ablation by showing behavioral defects of the ablated worms in a chemotaxis avoidance assay.
Thirdly, to aid future development of the microdevices, a novel in situ method for three-dimensionally resolved temperature measurement in microchannels was developed. This method uses video-microscopy in combination with image analysis software (developed by Jaekyu Cho in Dr. Victor Breedveld¡¯s group) to measure Brownian diffusion of nanoparticles that is correlated to temperature. This method offers superior reproducibility and reduced systematic errors. In addition, we demonstrated that this method can be used to measure spatial temperature variations in three dimensions in situ.
Lastly, a method for pressure measurement in microdevices was also developed through collaboration with Hyewon Lee (Dr. Hang Lu¡¯s lab) to aid further device optimization. These micro pressure-sensors are composed of two flow layers with a polydimethylsiloxane (PDMS) membrane in between. The membrane deforms as a function of pressure and its deformation is quantified by a simple image-based method. These sensors offer high-precision pressure measurement in broad sensing ranges. In addition, a pressure transduction scheme combined with imaging-based method enables multiplex pressure measurement for simultaneously detecting pressures in multiple locations in a microsystem.
Overall, the technologies developed in this thesis will establish a solid basis for continuous improvement of the microsystems for multi-cellular model organisms. This high-throughput technology will facilitate a broad range of biological and medical research.
| Identifer | oai:union.ndltd.org:GATECH/oai:smartech.gatech.edu:1853/37163 |
| Date | 05 August 2009 |
| Creators | Chung, Kwanghun |
| Publisher | Georgia Institute of Technology |
| Source Sets | Georgia Tech Electronic Thesis and Dissertation Archive |
| Detected Language | English |
| Type | Dissertation |
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