The long term objective of this proposal is to develop instrumentation for molecule-specific imaging of biological cells. Preliminary data is provided demonstrating the feasibility of sputtering intact molecular ions from a complex biological matrix. The method is based on the use of a focused ion beam to desorb molecules from an area which can be as small as 500 A in diameter. To achieve the necessary sensitivity, desorbed molecular ions are detected using a high postionization with a novel high repetition rate, high power, 200 fs Ti:sapphire laser system. There are three parallel strategies we propose for achieving the objective. First, we plan to optimize the use of the fs laser system for multiphoton ionization experiments. This goal is important, since the technology has only become available during the last year. Second, we plan to continue to increase the capability of preparing cell surfaces for mass spectral characterization in vacuo. In addition to existing freeze-fracture capabilities we hope to add jet- freeze spraying and crymicrotoming to expose the inside of biological material. During this phase, issues related sample preparation, matrix effects, quantitation and possible chemical interferences will be sorted out in detail. Finally, we plan to apply this technology to elucidating the molecular aspects of exocytosis. These studies will include imaging the contents of single neurotransmitter vesicles, imaging cells frozen during stimulated release to identify the distribution of release sites and vesicle content, and imaging catecholamine neurotransmitters at synaptic connections after stimulation and freeze sectioning. This work will provide novel information leading to a molecular and single cell understanding of exocytosis from adrenal cells, mast cells, and the cell body of a neuron with fully grown neurites. The fundamental biochemistry and physiology involved in these processes is critically important to our understanding of nervous system disorders (schizophrenia, Parkinson's disease), allergic reactions, cancer and the mechanism of neurocommunication.
