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结合。在这项工作中，荧光涨落光谱（FFS）将结合一种新的统计分析技术的基础上荧光强度累积。双光子FFS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，并通过利用平衡波动来确定动力学。累积量与矩有关，并从光子计数的高阶统计中捕获信息，该项目的教育部分包括三个部分，每个部分都加强对本科生和研究生的高级生物物理专题培训。(1)拟议的研究为研究生和研究生提供了物理学，统计学和生物学之间的跨学科领域的坚实和先进的教育，在不久的将来将需要更多训练有素的科学家。(2)还将开设一门分子生物物理学课程，其中包括最新的生物物理学技术，如荧光相关光谱学、单分子检测和光镊。(3)教育部分的重点是为生物学学生举办一个跨学科的荧光技术实践讲习班。 该研讨会将有一个强大的实验室组成部分，在那里学生实践他们所学到的。这些实验将从标准的基于比色皿的测量到完整细胞中的先进双光子显微镜。理论概念，实验室实验和应用的结合将为每个学生提供荧光光谱学的坚实基础以及对他们未来职业有用的实践知识。

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.