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Rosenzweig, Zeev


When developing new techniques to analyze neuro-chemical microenvironments, it is important to realize the incredible variability in the cellular content and the response to stimulation between cells and within a single cell. Conventional analysis techniques yield an average result to describe the content and function of cells. This approach often misses important information since the onset of pathological conditions is always initiated in a small number of cells. New minimally invasive single cell analysis techniques are required for single cell studies in order to gain new insights and understanding of cells' functions. The objective of my Ph.D. study was to fabricate, characterize, and apply submicrometric fluorescence sensors for the analysis of neuron cells. This dissertation will report the fabrication of miniaturized fluorescence sensors for Ca2+, pH and Zn2+ analysis. In the first approach, liposomes (phospholipid vesicles) were used as miniaturized containers for fluorescent sensing reagents. Liposome-based fluorescence sensing technology offers several advantages over commonly used fluorescence sensing techniques including high spatial resolution, protection of the sensing dye from quenchers and high biocompatibility. However, liposome based sensors were found to be unstable in the cellular environment. The second approach was to synthesize submicrometric particle-based fluorescence sensors named lipobeads to replace the fluorescent liposomes in cellular studies. Lipobeads are polystyrene particles that are coated with a phospholipid membrane. One unique advantage of fluorescent sensing lipobeads is the ability to immobilize hydrophobic indicator molecules in the phospholipid membrane. This enables the use of these indicators in aqueous media since the lipobeads are fully water miscible. The lipobeads also proved to be highly biocompatible in cellular studies. This is attributed to their phospholipid bilayer membrane, which is similar in structure to cell membranes. The dissertation will describe the analytical properties of fluorescence sensing lipobeads and their application in studying zinc ion release and pH changes near neuron cells under physiological conditions, conditions of neuronal injury and stress and acidic cortical spreading depression during stroke like conditions.


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