Optical microscopy has been an essential technique in cell biology that has permitted the visualization of a variety of sub-cellular processes. When combined with selective labeling methods and fluorescent probes, optical microscopy has allowed the identification of the distribution of specific macromolecules and measurement of ion fluxes as well as the ability to monitor vesicle cycling in nerve terminals. However, because objective-based microscopy is limited by diffraction, the resolution of fluorescence microscopy is about 250 nm limiting studies to the behavior of populations of channels and vesicles. In this study we will apply biological near-field microscopy, with resolution of 20-50nm, to the study of the nerve terminal. Using this approach we will use high spatial resolution fluorescence microscopy to study the activity of single calcium channels and the behavior of individual vesicles in the nerve terminal. In order to achieve our overall objective we have two specific aims of study:
Specific Aim I : We will use near-field optical fibers to map the microdomains of cytosolic calcium elevation during neuronal depolarization.
Specific Aim II : We will determine whether second messengers that modulate synaptic transmission control the """"""""mobility"""""""" of individual synaptic vesicles within the nerve terminal. Success in this project will have significant outcomes by allowing us to demonstrate the feasibility of a method that permits the application of sub-diffraction optics to living samples. In addition we will be able to perform the first investigation of the microdomains of the calcium ion, and will be able to address fundamental questions about synaptic transmission. With the data obtained from this study we will then have the preliminary data that are necessary to successfully compete for RO I style research grant applications.
