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

University Of Minnesota-Twin Cities, Minneapolis MN

Investigators

Abstract

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.