Microfabricated Fluorescence-Activated Cell Sorters (μFACS) for Screening Bacterial Cells

Microfabricated Fluorescence-Activated Cell Sorters (μFACS) for Screening Bacterial Cells

2002 (Submitted May 13, 2002) | Anne Yen-Chen Fu
This thesis by Anne Yen-Chen Fu, submitted in partial fulfillment of the requirements for a Doctor of Philosophy degree at the California Institute of Technology, focuses on the development of microfabricated fluorescence-activated cell sorters (μFACS) for screening bacterial cells. The primary goal is to create an inexpensive, robust, and effective device that can perform high-throughput cell sorting with minimal cross-contamination and higher sensitivity compared to conventional flow cytometry (FACS). The thesis outlines the use of soft lithography, a micromachining technology, to fabricate elastomeric microfluidic devices. These devices are designed to replace flow chambers in conventional FACS, offering advantages such as novel sorting algorithms, immediate reagent dispensing, and the ability to handle biohazardous/infectious cells in a safer environment. Key contributions include: 1. **Development of an Elastomeric Microfabricated Electrokinetic Sorter**: This device uses electrokinetic flow to sort cells and particles, demonstrating its effectiveness in sorting micron-sized latex beads and bacterial cells. 2. **Integration with Microvalves and Micropumps**: The second generation of the μFACS is integrated with microvalves and micropumps for pressure-driven sorting, improving flow control, cell trapping, and sample dispensing. 3. **High Throughput Screening of GFP Variants**: The μFACS is used for high-throughput screening of green fluorescent protein (GFP) variants, achieving rapid and efficient sorting for directed evolution. 4. **Digital Genetic Circuits**: The μFACS is applied to analyze digital genetic circuits in *E. coli* cells, demonstrating its versatility in cellular computation. 5. **Flow Cytometry of Magnetotactic Bacteria**: The μFACS is integrated with a superconducting quantum interference device (SQUID) microscope to detect the magnetic field strengths of magnetotactic bacteria, providing new insights into biomagnetism. The thesis concludes by discussing the future applications and potential of μFACS in various fields, including high-throughput screening, directed evolution, digital genetic circuits, and biomagnetism. The μFACS is presented as a versatile and cost-effective tool for cell sorting and analysis, with potential for integration into other technologies for advanced biological research.This thesis by Anne Yen-Chen Fu, submitted in partial fulfillment of the requirements for a Doctor of Philosophy degree at the California Institute of Technology, focuses on the development of microfabricated fluorescence-activated cell sorters (μFACS) for screening bacterial cells. The primary goal is to create an inexpensive, robust, and effective device that can perform high-throughput cell sorting with minimal cross-contamination and higher sensitivity compared to conventional flow cytometry (FACS). The thesis outlines the use of soft lithography, a micromachining technology, to fabricate elastomeric microfluidic devices. These devices are designed to replace flow chambers in conventional FACS, offering advantages such as novel sorting algorithms, immediate reagent dispensing, and the ability to handle biohazardous/infectious cells in a safer environment. Key contributions include: 1. **Development of an Elastomeric Microfabricated Electrokinetic Sorter**: This device uses electrokinetic flow to sort cells and particles, demonstrating its effectiveness in sorting micron-sized latex beads and bacterial cells. 2. **Integration with Microvalves and Micropumps**: The second generation of the μFACS is integrated with microvalves and micropumps for pressure-driven sorting, improving flow control, cell trapping, and sample dispensing. 3. **High Throughput Screening of GFP Variants**: The μFACS is used for high-throughput screening of green fluorescent protein (GFP) variants, achieving rapid and efficient sorting for directed evolution. 4. **Digital Genetic Circuits**: The μFACS is applied to analyze digital genetic circuits in *E. coli* cells, demonstrating its versatility in cellular computation. 5. **Flow Cytometry of Magnetotactic Bacteria**: The μFACS is integrated with a superconducting quantum interference device (SQUID) microscope to detect the magnetic field strengths of magnetotactic bacteria, providing new insights into biomagnetism. The thesis concludes by discussing the future applications and potential of μFACS in various fields, including high-throughput screening, directed evolution, digital genetic circuits, and biomagnetism. The μFACS is presented as a versatile and cost-effective tool for cell sorting and analysis, with potential for integration into other technologies for advanced biological research.
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