High Performance Fluorescence Microscopy Imaging

产品说明：MHC I类相关受体FcRn在妊娠期间将母体γ球蛋白（IgG）递送给后代和维持血清IgG水平中起重要作用。它在细胞内和细胞间转运IgG。 通过改造相互作用位点改变IgG-FcRn相互作用的亲和力对IgG的血清持久性和母胎转运具有显著影响。IgG在被动免疫中的有效使用在需要针对病原体或毒素的立即保护的情况下是非常重要的（例如，RSV，生物恐怖主义制剂）。 除了生物学兴趣之外，了解IgG的细胞内运输及其对FcRn的依赖性对于在开发和施用具有关于血清持久性、母体胎儿转移、剂量要求等的期望行为的工程化抗体方面取得进一步进展也是至关重要的。母公司提案的目标之一是改善对IgG的细胞内运输的迄今非常有限的理解，及其对FcRn的依赖性。 活细胞样品的荧光显微术既是一种快速发展的技术，也已被证明是一种可以在细胞生物学研究中提供重要见解的工具。 本提案旨在显著提升共同主要研究者（即母提案的主要研究者）实验室的荧光显微镜成像能力。 目标是为研究IgG贩运问题提供非常先进的能力。特别是，需要资金购买更多的成像设备，将新设备与现有成像设施结合起来，开发定制的采集和分析软件，并对母提案调查中产生的关键实验进行测试。 第一个具体目标是实现高速双色荧光显微镜的仪器和软件。第二个具体目标是实现全内反射显微镜成像和分析的仪器和软件。 第二个具体目标的目的是能够进行具有单分子检测灵敏度的实验，例如IgG胞吐作用的研究。 该提案中的方法基于成像技术及其在细胞生物学问题中的应用的最新进展，特别是具有单分子灵敏度的成像。

DESCRIPTION: The MHC class I related receptor FcRn plays an important role in the delivery of maternal gamma globulins (IgGs) to offspring during pregnancy and in maintaining serum IgG levels. It transports IgGs within and across cells. Changing the affinity of the IgG-FcRn interaction by engineering the interaction site has a significant influence on serum persistence and maternal-fetal transport of IgGs. The effective use of IgGs in passive-immunization is of major importance in situations where immediate protection against a pathogen or toxin is needed (e.g., RSV, bioterrorism agents). Understanding intracellular trafficking of IgGs and its dependence on FcRn is, aside from the biological interest, also crucial to make further progress in the development and administration of engineered antibodies with desired behavior as regards to serum persistence, maternal fetal transfer, dosage requirements, etc. One of the goals of the parent proposal is to improve on the as yet very limited understanding of intracellular trafficking of IgGs, and its dependence on FcRn using fluorescence microscopy techniques. Fluorescence microscopy of live cell samples is both a rapidly evolving technology and has been shown to be a tool which can provide significant insights in cell biological investigations. This proposal seeks to significantly upgrade the fluorescence microscopy imaging capability of the laboratory of the co-PI who is the PI on the parent proposal. The goal is to make available very advanced capabilities to the study the IgG trafficking problem. In particular, funds are sought for the purchase of additional imaging equipment, the integration of the new equipment with the existing imaging facility, the development of custom acquisition and analysis software, and its testing on key experiments arising from the investigations on the parent proposal. The first specific aim is to implement instrumentation and software for high speed dual color fluorescence microscopy. The second specific aim is to implement instrumentation and software for total internal reflection microscopy imaging and analysis. The objective of the second specific aim is to have the ability to perform experiments with single molecule detection sensitivity, such as the study of IgG exocytosis. The approach in this proposal is based on recent advances in imaging technology and its applications to cellular biology problems, in particular imaging with single molecule sensitivity.