Conductive Metallography for Serial Section Electron Microscopy at Nanometer Resolution -
DESCRIPTION (provided by applicant): 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.
