DETERMINATION OF NANOPARTICLE LOCALIZATION IN PC12 NEURONS BY TEM

该子项目是利用NIH/NCRR资助的中心赠款提供的资源的许多研究子项目之一。子项目和研究者（PI）可能从另一个NIH来源获得主要资金，因此可以在其他CRISP条目中表示。所列机构为中心，不一定是研究者所在机构。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（PC 12）作为我工作的模型。在一个典型的实验中，我将细胞与纳米颗粒悬浮液一起孵育特定的时间，清洗多余的，并通过相差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TEM样品制备和显微镜技术，这样我就能够确定颗粒是否确实被内吞或简单地沉积在表面上。由于纳米颗粒的尺寸小（10-30 nm），它们的位置不可能通过相位显微镜精确定位，尽管观察到一些可见的变色。此外，我想估计与细胞相关的颗粒的浓度。我曾试图通过电感耦合等离子体发射光谱法（ICP-OES）分析计算颗粒浓度，但该技术很容易因水溶液中的微小差异而发生偏差。我有一些准备ESEM生物样品的经验，但我没有执行TEM样品准备的某些方面，如Epon包埋。我将非常感谢专家的培训和指导，学习如何准备样品和操作TEM在NCMIR

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