Serial Blockface SEM Labels for Assessing Nervous System Plasticity

DESCRIPTION (provided by applicant): Serial blockface scanning electron microscopy (SBFSEM) is a recently developed electron microscopy serial- reconstruction technique which automates the process of sectioning and imaging blocks of tissue by incorporating a microtome into the vacuum chamber of a scanning electron microscope (SEM). SBFSEM provides resolution that is sufficient to trace even the thinnest axons and to identify synapses. It also allows the user to automatically obtain several hundred sections (50-70 nm thick) needed to completely reconstruct the connectivity of neuronal circuits, while potentially providing nanometer-scale resolution for immunogold labeling. SBFSEM will be combined with pre-embedding immunogold labeling to develop a method for the three-dimensional mapping of targets at the molecular level in very large specimens. Conducted in parallel with neuronal mapping, this will allow correlation of localization of specific molecules of interest in the context of high-resolution mapping of neuronal interconnectivity. Reagents will be developed for multiple labeling using different sized gold particles optimized for penetration, minimal background, high solubility, and maximum compatibility with specimen characteristics. Our goal is to make immunogold-SBRSEM a routine method that can be used to map targets within spatially large specimens. Three particle sizes in the range 0.8 to 5 nm will be identified that can be visualized, identified and differentiated using SBFSEM in multiple labeling studies, and used to demonstrate concomitant SBFSEM neuronal tract tracing and pre-embedding labeling of phos- phorylated and non- phosphorylated connexin 35 (Cx35) containing mixed synapses: (a) single and (b) double immunolabeling of neuronal and synaptic targets will be evaluated in the Western Mosquitofish, Gambusia affinis, using the new probes, and best protocols established for labeling and microscopy. PUBLIC HEALTH RELEVANCE: This research will provide new tools with which cell and structural biologists can correlate the observation of structures and processes in large specimens from biological systems, particularly the nervous system but also many other applications in cell biology, at the cellular and macromolecular level. These will enable new insights into how such processes work, and how molecular and macroscopic structure and function are related in normal and disease processes. This knowledge will lead to improved diagnostics and therapeutics in addition to providing improved immunogold probes and contributing to the development of nanobiotechnology.