High Performance Fluorescence Microscopy Imaging

说明：MHC-I类相关受体FcRN在孕期母体免疫球蛋白(IgG)传递给后代和维持血清免疫球蛋白水平方面起着重要作用。它在细胞内和细胞间传输IGG。通过改变Ig G-FcRN相互作用的亲和力，改变相互作用部位对血清持久性和母胎转运有显着影响。在需要立即预防病原体或毒素(如呼吸道合胞病毒、生物恐怖主义制剂)的情况下，有效利用免疫球蛋白进行被动免疫非常重要。了解免疫球蛋白在细胞内的转运及其对FcRN的依赖，除了生物学意义外，对于在开发和应用具有所需行为的工程抗体方面取得进一步进展也是至关重要的，如血清持久性、母体胚胎移植、剂量要求等。父母提案的目标之一是改善迄今对免疫球蛋白在细胞内的转运及其对FcRN的依赖的了解，使用荧光显微镜技术。 活细胞样本的荧光显微镜既是一项快速发展的技术，也被证明是一种工具，可以在细胞生物学研究中提供重要的见解。这项提案旨在大幅提升作为母公司提案上的PI的共同PI的实验室的荧光显微镜成像能力。其目标是为研究免疫球蛋白贩运问题提供非常先进的能力。特别是，请拨资金购买更多的成像设备，将新设备与现有成像设施相结合，开发定制的采集和分析软件，并对对上级提案进行调查后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用于全内反射显微镜成像和分析的仪器和软件。第二个特定目标的目标是有能力进行具有单分子检测灵敏度的实验，例如研究免疫球蛋白的胞吐作用。这项建议中的方法是基于成像技术的最新进展及其在细胞生物学问题上的应用，特别是单分子敏感性的成像。

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.