In this dissertation a computer-aided automation design methodology for biotechnology applications is proposed that leads to several design guidelines. Because of the biological nature of the samples that propagate in the automation line, a very specific set of environmental and maximum allowed shelf time conditions have to be followed to obtain good yield. In addition all biotechnology protocols require precise sequence of steps, the samples are scarce and the reagents are costly, so no waste can be afforded. / Includes bibliography. / Dissertation (Ph.D.)--Florida Atlantic University, 2014. / FAU Electronic Theses and Dissertations Collection

Due to the large extent to which important biological mechanisms are conserved evolutionarily, the study of a simple soil nematode, C. elegans, has provided the template for significant advances in biology. Use of this model organism has accelerated in recent years as developments of advanced reagents such as synapse localized fluorescent markers have provided powerful tools to study the complex process of synapse formation and remodeling. Even as much routine biology work, such as sequencing, has become faster and easier, imaging protocols have remained essentially unchanged over the past forty years of research. This, coupled with the ability to visualize small, complex features as a result of new fluorescent reagents, has resulted in genetic screens in C. elegans becoming increasingly labor intensive and slow because microscopy mainly relies on manual mounting of animals and phenotyping is usually visually done by experts. Genetic screens have become the rate limiting factor for much of modern C. elegans research. Furthermore, phenotyping of fluorescent expression has remained a primarily qualitative process which has prevented statistical analysis of subtle features. To address these issues, a comprehensive system to allow autonomous screening for novel mutants was created. This was done by developing novel microfluidic devices to enable high-throughput screening, systems-level components to allow automated operation, and a computer vision framework for identification and quantitative phenotyping of synaptic patterns. The microfluidic platform allows for imaging and sorting of thousands of animals at high-magnification within hours. The computer vision framework employs a two-stage feature extraction to incorporate local and regional features and allows for synapse identification in near real-time with an extremely low error rate. Using this system thousands of mutagenized animals were screened to indentify numerous novel mutants expressing altered synaptic placement and development. Fully automated screening and analysis of subtle fluorescent phenotypes will allow large scale RNAi and drug screens. Combining microfluidics and computer vision approaches will have a significant impact on the biological community by removing a significant bottleneck and allowing large-scale screens that would have previously been too labor intensive to attempt.

The nematode Caenorhabditis elegans has served as one of the primary model organisms in neuroscience. As C. elegans research became more specific, so have the biological tools for manipulating C. elegans improved and matured. Additionally, in some avenues of research, technologies have been developed to manipulate the animals in very efficient and quantitative ways. However, the field of dynamic studies has remained without significant technological support. Dynamic studies focus on processes occurring over time and span a range of time-scales of i) minutes to hours requiring continuous imaging for accurate observation, ii) hours to days requiring periodic imaging of the same animal, and iii) days to weeks requiring daily monitoring. Because of a lack of suitable tools and technologies to perform these studies, researchers have to either apply standard biological methods with limited ability to observe processes dynamically or simply cannot perform such studies with the desired set of experimental conditions. To address this problem, a comprehensive microfluidic toolkit for dynamic studies has been created. The first element is a novel method for reversible and repeatable immobilization at benign conditions in tandem with a microfluidic system for isolated culture of C. elegans with integrated temperature control. The second element is a system for efficient handling of C. elegans embryos in a high-throughput and scalable fashion for chemical and thermal embryonic stimulation with subsequent study of development. The third component is a system capable of selective immobilization of animals’ bodies, while simultaneously facilitating feeding and normal physiological function for live imaging. The last component is capable of culturing animals over their life-span with efficient animal handling, environmental control (temperature and dietary conditions), and high data content experimentation. As a whole, the work in this thesis enables dynamic studies over the whole range of time scales applicable to C. elegans research. These types of studies were previously very difficult or near impossible to perform practically. Now, instead of building population composites to understand the dynamics of a process, risking affecting physiology via the experiment itself, or dealing with extremely labor intensive physical handling of animals, a toolkit for efficient handling of C. elegans facilitating dynamic and direct observation of individual animals is available. The biological applications range from dynamically studying lipid droplet morphology or studying synaptic vesicle transport, through observing the dynamics of synaptic re-arrangement during development or the effect of cancer drugs on development, to performing high-content life-span experiments able to ascertain the relationship between aging and behavior. Additionally, many of the principles in these designs can be expanded to accommodate research on other model organisms, such as other nematode species, zebra fish embryos, or cells and embryoid bodies.