No matter how powerful a microscope is, it cannot distinguish particles which are closer together than approximately λ/2 (250-300nm) due to diffraction. PALM (photo activated localization microscopy) is a technique developed by Betzig et al. in 2006 which can overcome the diffraction limit and locate single molecules to approximately 5nm^[a]. This is accomplished by utilizing photoactivatable fluorescent proteins (PA-FPs) which can be switched on with UV irradiation. Sparse PA-FPs are sequentially switched on, their centers located by fitting them to a Gaussian function, and then are bleached away. This process is repeated until all of the PA-FPs have been found. Their locations are then used to generate a composite image with each spot corresponding to a single molecule with its brightness related to its position uncertainty. We hope to use this technique to study the architecture and stoichiometry of the proteins which form the Ca²⁺-triggered vesicle fusion machine in PC-12 cells in order to study a model of neuronal exocytosis.

PA-FPs will be used to tag syntaxin (Syx), SNAP-25, synaptobrevin (Syb), and synaptotagmin (Syt), the proteins which are thought to make up the vesicle fusion machine. We will then count the number of each protein present in PC-12 cells as well as try to learn about the structure of how they are arranged to ~5nm. This will provide insight into neuronal exocytosis.

This project is in the process of being set up. A Nikon Ti-E microscope has been purchased for performing TIRF microscopy, as well as an Andor EMCCD camera for imaging. Please check back for updates on our progress!