CHAPTER SEVEN Global Analysis in Fluorescence Correlation Spectroscopy and Fluorescence Lifetime Microscopy Neil Anthony*, Keith Berland* ,1 *Department of Physics, Emory University, Atlanta, Georgia, USA 1 Corresponding author: e-mail address: kberlan@emory.edu Contents 1. Introduction 146 2. Background 147 2.1 Motivation for global analysis 148 3. Theory 149 3.1 Fluorescence signals on the microscope 149 3.2 Time resolved two-photon fluorescence 150 3.3 Observation volumes and gamma factors 154 3.4 Fluorescence lifetime data 156 3.5 Fluorescence correlation spectroscopy 157 4. Methods 158 5. Results 161 5.1 Resolution of concentration and diffusion coefficients when D 1 = D 2 161 5.2 Accurate concentration recovery with unknown molecular brightness 162 5.3 Additional curve fitting enhancements 165 5.4 Model discrimination and resolution requirements 167 6. Conclusions 170 Acknowledgments 170 References 170 Abstract Fluorescence correlation spectroscopy (FCS) and related fluctuation spectroscopy and microscopy methods have become important research tools that enable detailed inves- tigations of the chemical and physical properties of molecules and molecular systems in a variety of complex environments. Information recovery via curve fitting of fluctuation data can present complicating challenges due to limited resolution and/or problems with fitting model verification. We discuss a new approach to data analysis called tFCS that couples multiple modes of signal acquisition, here specifically FCS and fluorescence lifetimes, with global analysis. We demonstrate enhanced resolution using tFCS, including the capability to recover the concentration of both molecular species in a two-component Methods in Enzymology, Volume 518 # 2013 Elsevier Inc. ISSN 0076-6879 All rights reserved. http://dx.doi.org/10.1016/B978-0-12-388422-0.00007-8 145