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: 7722821
• 负责人: MARY MORPHEW
• 金额: $ 0.92万
• 依托单位: UNIVERSITY OF COLORADO
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-08-01 至 2009-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7722821
• 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利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 我们一直在研究电子致密标记的用途，特别是金粒子，它可以在生理条件下内化到活细胞中。重要的是，这些标记要尽可能小，以免干扰正在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基团，如寡核苷酸、脂类、多肽、蛋白质等。它们的大小极其一致(1.4 nm)，并提供接近化学计量比的标记。一旦内化，这些微小的颗粒通常会被银增强，产生大小为2-20 nm的高度可见的颗粒。然而，我们希望在没有添加银增强的情况下，在断层重建中可视化纳米金颗粒。在这些实验中，我们要么将纳米金应用于含有生物材料的环氧片的表面，这些生物材料以前没有被重金属后染色，或者试图在将样品嵌入树脂之前将纳米金内化到样品中。切片(150 Nm)在Technai F30显微镜上以59,000X成像，倾斜序列以1度递增超过120度，像素大小对应于0.39 nm。在由此产生的断层重建中，我们能够清楚地看到纳米金躺在切片表面。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。在收集倾斜系列数据之前，我们曾尝试使用暗场模式以及Gatan能量过滤器来定位生物材料部分中的纳米金，但这些试验都没有成功。我们的努力表明，我们必须修改成像方法来定位纳米金，要么是电子光学方法，如STEM暗场成像，要么是化学方法，如冷冻替代过程中进行的银增强。 因此，我们致力于研究在冷冻替代过程中，在有机溶剂存在的情况下，在低温下用银扩大纳米金的方法。将纳米金悬浮液应用于EM栅格，然后使用溶解于丙酮的银增强剂的组合，已成功地生产出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。比约克曼实验室已经将纳米金颗粒与免疫球蛋白偶联，并在低pH值下将偶联物应用于培养的单层MDCK细胞，这些单层细胞是在细胞形成类似极化上皮的薄片的条件下生长的。将同样的试剂注射到新生大鼠的肠道中，经快速冷冻制备后，我们开始在-50oC的冷冻替代方案中添加银增强剂。增强的纳米金颗粒现在可以通过细胞顶端的囊泡隔间进行追踪。