Spatially and temporally resolved delivery of stimuli to single cells using nanocapsules and laser manipulation

Thesis (Ph. D.)-- University of Washington, 2005.Cell signaling is at the core of most biological processes. The spatially and temporally resolved stimulation will help elucidate cell signaling, and lead us to a better understanding of signal transduction. We have designed and developed a technique to photolyze individual optically trapped nanocontainers for delivering spatially and temporally resolved stimuli to single cells. A submicrometer-sized nanocontainer, which encapsulates the stimuli of interest, is precisely positioned with respect to a select cell by optical trapping. Application of a single focused UV laser pulse photolyzes the trapped capsule and releases the stimuli onto a localized region of the cell. The spatial resolution of this technique is on the order of several hundreds of nanometers, while the temporal resolution lies in the sub-microsecond range. To achieve and optimize the above strategy, we have explored the synthesis/formation of a series of capsules including liposomes, inorganic and polymeric capsules with tunable size and tailored surface properties. We have created an efficient method to load the polymeric capsules with molecules of interest and developed a polyelectrolyte and silica combined coating approach to eliminate non-controllable leakage after loading. We also initiated a novel technique to quantify the encapsulation efficiency of lipid vesicles on a single-vesicle level, which gives us a unique way to evaluate the encapsulation efficiency more meaningful than the traditional bulk analysis. Besides lipid vesicles, we believe this method can be broadly applied to the study of loading efficiency of other micro/nano-scale containers. We have also characterized the photo-releasing efficiency of liposomes and micro/nano capsules quantitively. Using our delivery technique, we have successfully studied carbachol-evoked calcium signaling in Chinese hamster ovary cells transfected with type-1 muscarinic acetylcholine receptors and explored the mapping of these receptors on the cell membrane