| This dissertation describes the development of four new techniques which exploit recent advances in optical microscopy, microfluidics, and soft lithography. The first two of these techniques are used to control the temperature within microfluidic devices: One employs thermoelectric coolers to demonstrate cooling localized to select areas of a chip, while the other utilizes a cooling chamber to manipulate the ambient temperature around the chip. With these techniques aqueous droplets can be frozen in a still-flowing immiscible phase during transit through microfluidic channels. Single cells can be encapsulated in these droplets and frozen, and optimal conditions for freezing and thawing cells in droplets can be determined.;The third technique described in this dissertation utilizes soft lithography of poly(dimethylsiloxane) and agarose to generate arrays of microislands suitable for primary neuronal cell culture. These islands encourage the formation of autapses, or self-synapses, by confining single neurons to a small area. Single neurons grown on these microislands displayed three distinct neurite-arbor morphologies, which did not correlate with island shape or number of synapses. This method of island generation yields a greater number of single-neuron islands per culture and permits more rapid visualization of islands than pre-existing techniques.;The fourth and final technique described in this dissertation is a microfluidic device for neuronal growth that permits the creation of bulk samples of cultured axonal material. Utilizing a chip designed to isolate axons from dendrites and cell bodies, neurons are grown for 14--17 days until a dense mat of axonal material is present in the axonal area of the chip. These axons are then lifted off of the culture surface and removed from the device, yielding a sample of pure axonal material. Samples of axonal transport carrier vesicle-motor complexes obtained using this device can be individually analyzed using single-organelle TIRF microscopy. These complexes were labeled with antibodies to the synaptic vesicle proteins p38, SV2A, and VAMP2, and the anterograde axonal transport motor KIF1A, after which antibody overlap was evaluated using single-organelle TIRF microscopy. The results confirm a previously discovered association between KIF1A and p38 and show that KIF1A may also transport SV2A- and VAMP2-containing carrier vesicles. |
