The most abundant clone found in ribosomal RNA clone libraries obtained from the world's oceans belongs to the SAR11 phylogenetic group of environmental marine bacteria. Imaging and counting SAR11 bacterial cells in situ has been an important research objective for the past decade. This objective has been especially challenging due to the extremely small size, and hypothetically, the low abundance of ribosomes contained by the cells. To facilitate the imaging of small dim oligotrophic bacterial cells, digital imaging technology featuring very small pixel size, high quantum yield scientific grade CCD chips was integrated with the use of multiple oligonucleotide probes on cells mounted on a non-fluorescing solid substrate. Research into the composition of bacterioplankton populations in natural marine systems follows a two-fold path. Increasing the culturability of microbes found in the natural environment is one research path. Identifying and enumerating the relative fractions of microorganisms in situ by culture-independent methods is another. The accumulation and systematic comparison of ribosomal RNA clones from the marine environment has resulted in a philosophical shift in marine microbiology away from dependence upon cultured strains and toward investigations of in situ molecular signals. The design and use of oligonucleotide DNA probes targeting rRNA targets has matured along with the growth in size and complexity of the public sequence databases. Hybridizing a fluorescently labeled oligonucleotide probe to an rRNA target inside an intact cell provides both phylogenetic and morphological information (a technique called Fluorescence in situ Hybridization (FISH)). To facilitate the imaging of small, dim oligotrophic bacterial cells, digital imaging technology featuring very small pixel size, high quantum yield, scientific grade CCD chips is integrated with the use of multiple oligonucleotide probes on cells mounted on a non-fluorescing solid substrate. This research develops the protocols necessary to acquire and analyze digital images of marine bacterial cells. Experiments were conducted with Bermuda Atlantic Time Series (BATS) environmental samples obtained during cruise BV21 (1998) and B138 (2000). The behavior of the SAR11⁴*Cy3 probe set when hybridized to bacterial cells from these samples was investigated to determine the optimal hybridization reaction conditions. The challenges of bacterial cell counting after cell transfer from PCTE membrane to treated microslides were addressed. Experiments with aged Oregon Coast seawater were performed to investigate the protocol used to transfer cells from membrane to microslides, and examined the distribution of cells and the statistics of counting cells using traditional epifluorescence microscopy and image analysis techniques. / Graduation date: 2002

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.