Conductive Metallography for Serial Section Electron Microscopy at Nanometer Resolution

描述(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电荷，使用金相染色技术，使嵌入的组织具有整体导电性。这可以增加光束曝光和停留时间，当与背向散射电子检测和金纳米颗粒标记一起使用时，潜在地提供了从大约10 nm的电流限制到低至1-2 nm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(酶金相术，或EnzMet)的新组合。将逐步研究这些试剂和程序，以建立形成导电性的机制，确定需要哪些试剂，并简化和优化用于其他染色和标签的试剂和程序。系统地省略处理步骤将被用来确定关键反应；然后系统地省略试剂将被用来确定哪些试剂是必需的。然后对每种试剂的反应条件(时间、温度、缓冲液和浓度)进行受控变化，以推断其作用机理和模式。一旦优化，新的染色方法将与金标记相结合，使用尺寸从5纳米到0.8纳米的逐渐变小的金纳米探针。这些研究将被用来确定(A)使用火焰离子交换膜扫描电子显微镜在大体积样品中可以看到的最小金纳米颗粒尺寸，以及(B)探针在所有尺寸上对200�m或更大样品的穿透程度，以及允许完全穿透的金纳米颗粒结合物的截止尺寸。此外，还将使用不同大小的金纳米颗粒标记进行多重标记，以区分突触前和突触后蛋白，连接蛋白 35/36和连接蛋白34.7在西方蚊虫(Gambusia Affinis)的脊髓中分别表达，同时使用优化的导电金相染色来轮廓和分割神经元边界。

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.