USING NANOGOLD AS A SPECIFIC MARKER IN TOMOGRAPHY

使用纳米金作为断层扫描中的特定标记

• 批准号: 7597299
• 负责人: MARY MORPHEW
• 金额: $ 1.15万
• 依托单位: UNIVERSITY OF COLORADO
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-08-01 至 2008-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7597299
• 关键词: Acetone; Adherent Culture; Apical; Area; Biological; Cells; Cereals; Chemicals; Computer Retrieval of Information on Scientific Projects Database; Condition; Coupled; Data; Electrons; Epithelium; Freeze Substitution; Freezing; Funding; Gold; Grant; Heavy Metals; Image; Immunoglobulin G; Institution; Intestines; Label; Life; Lipids; MDCK cell; Methods; Microscope; Newborn Infant; Oligonucleotides; Optical Methods; Organelles; Organic solvent product; Peptides; Physiological; Plant Resins; Preparation; Proteins; Protocols documentation; Rattus; Reagent; Research; Research Personnel; Resources; Sampling; Series; Side; Silver; Source; Staining method; Stains; Surface; Suspension substance; Suspensions; System; Technology; United States National Institutes of Health; Work; cold temperature; human SILV protein; in vivo; nanoGold; particle; reconstruction; research study; size; stem; tomography

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. We have been investigating the utility of electron dense markers, particularly gold particles, which can be internalized into live cells under physiological conditions. It is important that these markers are as small as possible, so as not to perturb the system or organelle under study. Nanogold clusters can be covalently coupled to any suitable reactive group such as oligonucleotides, lipids, peptides, proteins and others. They are extremely uniform in size (1.4nm) and provide close to stoichiometric labeling. Once internalized, these tiny particles are usually then enhanced with silver to produce highly visible grains 2-20 nm in size. However, we would like to visualize nanogold particles in tomographic reconstructions without the addition of silver enhancement. For these experiments we either applied nanogold to the surface of epoxy sections containing biological material which had not been previously post stained with heavy metals, or attempted to internalize the nanogold into samples prior to embedding them in resin. Sections (150nm) were imaged at 59,000X in the Technai F30 microscope and tilt series were taken at 1 degree increments over 120 degrees with a pixel size corresponding to 0.39 nm. In the resulting tomographic reconstructions, we were able to clearly see nanogold lying at the section surface. However, nanogold internalized into live cells was not visible, likely because we had not located areas of internalization. We have attempted to use the dark field mode as well as the Gatan Energy Filter to locate nanogold within sections of biological material prior to collecting tilt series data, but these trials were not successful. Our efforts suggest that we must modify our methods of imaging to locate nanogold, either an electron optical method, like STEM dark-field imaging, or a chemical method, like silver enhancement carried out during freeze-substitution. We have, therefore, worked on methods to enlarge nanogold with silver in the presence of organic solvents at low temperature, during freeze substitution. Experiments using nanogold suspensions applied to EM grids, followed by combinations of silver enhancement chemicals dissolved in acetone have been successful in producing particles of 3-5 nm. We have now begun to apply this technology to an in vivo system. The Bjorkman lab has conjugated nanogold particles to IgG and have applied the conjugates at low pH to cultured monolayers of MDCK cells that have been grown under conditions in which the cells form sheets that resemble polarized epithelia. The same reagent has been administered to the intestine of newborn rats Following preparation by rapid freezing we have begun to add silver enhancement reagents to the freeze substitution protocol at -50oC. Enhanced nanogold particles may now be tracked through the vesicular compartments on the apical side of the cells.

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 我们一直在研究电子致密标记物的效用，特别是金颗粒，它可以在生理条件下内化到活细胞中。 重要的是，这些标记物尽可能小，以免干扰所研究的系统或细胞器。纳米金簇可以共价偶联到任何合适的反应性基团，例如寡核苷酸、脂质、肽、蛋白质等。它们的尺寸非常均匀（1.4nm），并提供接近化学计量的标记。一旦内化，这些微小颗粒通常随后用银增强，以产生尺寸为2-20 nm的高度可见的颗粒。然而，我们希望在不添加银增强的情况下在断层重建中可视化纳米金颗粒。对于这些实验，我们要么将纳米金应用于含有先前未用重金属后染色的生物材料的环氧树脂切片的表面，要么试图在将它们嵌入树脂之前将纳米金内化到样品中。 切片（150 nm）在Technai F30显微镜中以59，000 X成像，并且倾斜系列以1度增量在120度上拍摄，像素大小对应于0.39nm。 在由此产生的断层重建中，我们能够清楚地看到纳米金位于截面表面。 然而，内化到活细胞中的纳米金是不可见的，可能是因为我们没有定位内化的区域。 我们尝试使用暗场模式以及Gatan能量过滤器在收集倾斜系列数据之前在生物材料的部分中定位纳米金，但这些试验并不成功。 我们的努力表明，我们必须修改我们的成像方法来定位纳米金，无论是电子光学方法，如STEM暗场成像，还是化学方法，如在冷冻置换过程中进行的银增强。 因此，我们研究了在低温下在有机溶剂存在下用银扩大纳米金的方法。使用纳米金悬浮液施加到EM网格，然后是溶解在丙酮中的银增强化学品的组合的实验已经成功地产生3-5 nm的颗粒。我们现在已经开始将这项技术应用于体内系统。Bjorkman实验室将纳米金颗粒与IgG结合，并在低pH值下将结合物应用于培养的MDCK细胞单层，这些细胞在细胞形成类似极化上皮细胞的条件下生长。 同样的试剂已经被用于新生大鼠的肠道 通过快速冷冻制备后，我们开始在-50 ℃下向冷冻置换方案中添加银增强试剂。增强的纳米金颗粒现在可以通过细胞顶侧的囊泡隔室进行跟踪。