CAREER: Fluorescence Cumulant Analysis: A New Tool for Probing Protein-protein Interactions in Intact Cells

该项目的科学部分侧重于开发和利用先进的荧光技术来研究完整细胞中的蛋白质关联。在这项工作中，荧光涨落光谱(FFS)将与一种基于荧光强度累积量的新统计分析技术相结合。双光子荧光光谱为探测细胞中的生物系统提供了一种独特而强大的方法。该技术是非侵入性的，提供单分子检测灵敏度，提供亚微米空间分辨率，并通过利用平衡涨落来确定动力学。累积量与矩有关，从光子计数的高阶统计量中捕捉信息。该项目的教育部分包括三个部分，每个部分都加强了本科生和研究生对高等生物物理主题的培训。(1)拟议的研究为研究生和研究生在物理学、统计学和生物学之间的交叉学科领域提供了扎实和先进的教育，这一领域在不久的将来将需要更多训练有素的科学家。(2)还将开设一门结合了最新生物物理技术的分子生物物理学课程，如荧光相关光谱、单分子检测和光学镊子。(3)教育部分的重点是为生物学学生举办跨学科的荧光技术实践讲习班。工作坊将有一个强大的实验室部分，学生在那里练习他们所学的东西。这些实验的范围从标准的基于试管的测量到在完整细胞中的先进的双光子显微镜。理论概念、实验室实验和应用的结合将为每个学生提供坚实的荧光光谱学基础，以及对他们未来职业生涯有用的实践知识。

The scientific component of this project focuses on the development and utilization of advanced fluorescence techniques to study protein association in intact cells. In this work fluorescence fluctuation spectroscopy (FFS) will be combined with a new statistical analysis technique based on fluorescence intensity cumulants. Two-photon FFS offers a unique and powerful approach for probing biological systems in cells. The technique is noninvasive, provides single molecule detection sensitivity, delivers submicron spatial resolution, and determines kinetics by exploiting equilibrium fluctuations. Cumulants are related to moments and capture information from higher-order statistics of the photon counts.The educational component of the project contains three parts, each strengthening the training of undergraduate and graduate students in advanced biophysical topics. (1) The proposed research provides graduate and postgraduate students with a solid and advanced education in the interdisciplinary area between physics, statistics and biology, an area where more trained scientists will be needed in the near future. (2) A course on molecular biophysics that incorporates the latest biophysical techniques, such as fluorescence correlation spectroscopy, single molecule detection, and optical tweezers, will be developed as well. (3) The focus of the educational component is an interdisciplinary, hands-on workshop on fluorescence techniques for biology students. The workshop will have a strong laboratory component, where students practice what they have learned. The experiments will range from standard cuvette-based measurements to advanced two-photon microscopy in intact cells. The combination of theoretical concepts, laboratory experiments, and applications will provide each student with a solid foundation in fluorescence spectroscopy together with practical knowledge useful for their further career.