DETERMINATION OF NANOPARTICLE LOCALIZATION IN PC12 NEURONS BY TEM

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是针对中心的，而不一定是针对调查员的机构。该研究项目涉及开发用于生物应用的磁性纳米颗粒。我正在使用对神经生长因子有反应的大鼠外周神经元(PC12)作为我工作的模型。在一个典型的实验中，我用纳米颗粒悬浮液孵化细胞一段特定的时间，清洗多余的，并通过相位对比或荧光显微镜观察它们。目前，我们假设细胞从溶液中摄取颗粒，因此观察到的细胞形态差异归因于细胞内颗粒的存在。在NCMIR，我想学习透射电子显微镜样品制备和显微技术，这样我就能够确定颗粒是真的内吞还是仅仅停留在表面。由于纳米粒子的尺寸很小(10-30 nm)，虽然观察到了一些可见的变色，但通过相位显微镜无法精确定位它们的位置。此外，我想估计与细胞相关的颗粒浓度。我曾尝试用电感耦合等离子体光发射光谱分析(ICP-OES)来计算颗粒浓度，但这项技术很容易受到水溶液中微小差异的影响。我有一些为环境扫描电子显微镜准备生物样品的经验，但我还没有进行过透射电子显微镜样品的某些方面的准备，如EPON嵌入。我非常感谢NCMIR在学习如何准备样品和操作瞬变电磁方面的专家培训和指导

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. The research project involves the development of magnetic nanoparticles for biological applications. I am using rat peripheral neurons (PC12) that respond to nerve growth factor, as a model for my work. During a typical experiment, I incubate cells with a suspension of nanoparticles for a specific time, wash the excess, and view them by phase contrast or fluorescence microscopy. Currently, we are assuming that the cells uptake particles from solution, so observed differences in cell morphology are attributed to the presence of particles within the cell. At NCMIR, I would like to learn TEM sample preparation and microscopy techniques, so I will be able to determine whether particles are indeed endocytosed or simply settling on the surface. Because of the small size of the nanoparticles (10-30 nm), their location is not possible to pinpoint by phase microscopy, although some visible discoloration is observed. Furthermore, I would like to estimate the concentration of particles associated with the cell. I have attempted to analytically calculate particle concentration by inductively coupled plasma optical emission spectrometry (ICP-OES), but the technique is easily skewed by minor differences in aqueous solution. I have some experience preparing biological samples for ESEM, but I have not performed certain aspects of TEM sample prep, such as Epon embedding. I would greatly appreciate the expert training and guidance in learning how to prepare samples and operate the TEM at NCMIR