We have discovered a novel method to eliminate specimen charging during scanning electron microscopic examination of tissue specimens, using a metallographic staining technique that imparts bulk conductivity to embedded tissue. This allows increased beam exposure and dwell time, and when used in conjunction with backscatter electron detection and gold nanoparticle labeling, potentially affords increased resolution from current limits of around 10 nm to as little as 1 - 2 nm. The reagent comprises a novel combination of heavy metal staining and targeted enzyme mediated metal deposition (enzyme metallography, or EnzMet). These reagents and procedure will be investigated step by step in order to establish a mechanism for the formation of conductivity, establish which reagents are required, and simplify and optimize the reagents and procedure for use with other stains and labels. Systematic omission of processing steps will be used to identify the critical reactions; systematic omission of reagents will then be used to determine which reagents are essential. Controlled variation in reaction conditions (time, temperature, buffers and concentrations) will then be conducted for each reagent in order to infer its mechanism and mode of action. Once optimized, the new staining methodology will be combined with gold labeling using progressively smaller gold nanoparticle probes from 5 to 0.8 nm in size. These studies will be used to determine (a) minimum gold nanoparticle size that may be visualized within large- volume samples using FIB-SEM, and (b) extent of penetration of probes into samples up to 200 ?m or more in all dimensions and cutoff sizes for gold nanoparticle conjugates that allow complete penetration. In addition, multiple labeling will be pursued using different sized gold nanoparticle labels to differentiate pre- and post-synaptic proteins, Connexin 35/36 and Connexin 34.7 respectively, in the spinal cord of the Western Mosquitofish (Gambusia affinis) while simultaneously contouring and segmenting neuronal boundaries using the optimized conductive metallographic staining.
A new specimen preparation method and reagents will be developed that may improve the resolution of the electron microscopic analysis of large volume specimens, such as entire neuronal circuits, by serial section methods such as Serial Block Face scanning electron microscopy (SBFSEM) and Focussed Ion Beam scanning electron microscopy (FIB-SEM) from its current limits of around 10 nm to as little as 1 - 2 nm. This will be co-developed with small gold probes that will enable the macromolecular localization of functional components such as proteins with structures such as gap junctions that are too small to be resolved by current methods. This will provide a breakthrough in understanding the structure, function and distribution of components of systems such as neuronal networks. Deliverables will include research tools to bring a new level of resolution to large-scale projects such as the BRAIN initiative to map the structure, function, connectivity and plasticity of neural circuits, but will also be applicable to many larger organs and systems in general. This poses great challenges to conventional microscopic methods because integrative neuroscience requires information on both the distribution of targets within entire neural circuits, and the precise localization of specific functional components of synapses and gap junctions at nanometer-scale resolution. Our approach is intended to provide an enabling technology that will stimulate the development of other complementary tools for brain mapping and large-scale microscopic investigation, such as novel correlative instrumentation and probes, data acquisition and analysis, and imaging technologies.
