Conductive Metallography for Serial Section Electron Microscopy at Nanometer Resolution

描述（由申请人提供）：我们发现了一种新的方法，可以在组织样本的扫描电子显微镜检查过程中消除样本充电，使用金相染色技术，为包埋组织提供体电导率。这允许增加射束曝光和停留时间，并且当与反向散射电子检测和金纳米颗粒标记结合使用时，可能提供从约10 nm到小至1 - 2 nm的电流限制的增加的分辨率。该试剂包括重金属染色和靶向酶介导的金属沉积（酶金相学或EnzMet）的新组合。将逐步研究这些试剂和程序，以建立电导率形成的机制，确定所需的试剂，并简化和优化用于其他染色剂和标记的试剂和程序。系统性省略处理步骤将用于识别关键反应;然后系统性省略试剂将用于确定哪些试剂是必需的。然后将对每种试剂进行反应条件（时间、温度、缓冲液和浓度）的受控变化，以推断其作用机制和模式。一旦优化，新的染色方法将与金标记相结合，使用尺寸从5到0.8 nm的逐渐变小的金纳米颗粒探针。这些研究将用于确定（a）使用FIB-SEM在大体积样品中可以可视化的最小金纳米颗粒尺寸，以及（B）探针在所有尺寸和允许完全渗透的金纳米颗粒缀合物的截止尺寸上渗透到样品中高达200 μ m或更大的程度。此外，将使用不同尺寸的金纳米颗粒标记物进行多重标记，以区分突触前和突触后蛋白质，连接蛋白， 35/36和连接蛋白34.7，同时使用优化的导电金相染色对神经元边界进行轮廓和分割。

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.