Conductive Metallography for Serial Section Electron Microscopy at Nanometer Resolution

纳米分辨率连续切片电子显微镜的导电金相学

• 批准号: 8834483
• 负责人: RICHARD DENIS POWELL
• 金额: $ 22.45万
• 依托单位: NANOPROBES, INC.
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-01-01 至 2016-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8834483
• 关键词: BRAIN initiative; Biochemical; Brain; Brain Mapping; Buffers; Charge; Chromium; Cognition Disorders; Connexins; Conscious; Data; Data Analyses; Deposition; Detection; Development; Dimensions; Drug Formulations; Electron Microscopy; Electrons; Enzymes; Evaluation; Face; Gambusias; Gap Junctions; Goals; Gold; Heavy Metals; Human; Image; Imagery; Imaging technology; Immunoglobulin G; Investigation; Ions; Label; Maps; Mediating; Metals; Methodology; Methods; Microscopic; Molecular; Neurons; Neurosciences; Optics; Osmium; Penetration; Peptides; Preparation; Procedures; Process; Proteins; Protocols documentation; Puerto Rico; Reaction; Reagent; Research; Resolution; Rosa; Ruthenium; Sampling; Scanning; Scanning Electron Microscopy; Series; Specimen; Spinal Cord; Staining and Labeling; Staining method; Stains; Structure; Synapses; System; Techniques; Technology; Temperature; Time; Tissue Embedding; Tissues; Transmission Electron Microscopy; Universities; Variant; antibody conjugate; body system; computerized data processing; connexin 36; data acquisition; density; developmental disease; imaging Segmentation; improved; instrumentation; method development; nanoGold; nanometer; nanoparticle; nanoprobe; nanoscale; neural circuit; neuronal circuitry; novel; particle; public health relevance; reconstruction; screening; therapy development; tool; undecagold

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.

描述（由适用提供）：我们发现了一种新的方法，可以使用一种赋予嵌入式组织的大量电导率的金相染色技术来消除组织样品的扫描电子显微镜检查。这允许增加光束的暴露和停留时间，当与反向散射电子检测和金纳米颗粒标记结合使用时，可能会从当前限制到10 nm到1-2 nm的电流限制增加分辨率。这些试剂构成了重金属染色和靶向酶介导的金属沉积（酶金相或enzmet）的新型组合。这些试剂和程序将逐步研究，以建立形成电导率的机制，确定需要哪些试剂，并简化并优化与其他污渍和标签一起使用的试剂和程序。系统省略处理步骤将用于确定关键反应；然后，将系统省略试剂，以确定哪些试剂必不可少。然后，将对每种试剂进行反应条件（时间，温度，缓冲液和浓度）的控制变化，以推断其机理和作用方式。一旦优化，新的染色方法将与金标记结合使用，使用逐渐较小的金纳米颗粒问题从5到0.8 nm的大小。这些研究将用于确定（a）使用FIB-SEM在大容量样品中可视化的最小金纳米颗粒大小，以及（b）在所有维度和截止尺寸中，问题渗透到最高200°M或更多的样品中，用于金纳米粒子的结合物，从而可以完全渗透。此外，将使用不同尺寸的金纳米颗粒标签进行多个标记，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分别在西部蚊子（gambusia affinis）的脊髓中，同时使用优化的导电性金理染料同时轮廓和分割神经元边界。