Evaluation of Time-Resolved Fluorescence Data: Typical Methods and Problems

Abstract

The temporal characterisation of the light emission by fluorescing molecules can be used to extract a variety of different parameters, such as intramolecular distances or environmental changes. However, deriving a set of lifetimes from given raw data may prove to be a complex task, depending on influences such as the dynamics of the underlying process, the separation of the lifetime parameters or the statistical properties of the measurement. Whereas nowadays computational power provides the possibility to extract the lifetime parameters with sufficient speed, aspects like parameter accuracy and the interpretation of the extracted values can still be challenging. In this contribution, the underlying mathematical approaches to the analysis of time-resolved fluorescence data are outlined with emphasis on time-correlated single photon counting (TCSPC). Some peculiarities of these approaches are discussed with respect to their impact on recently emerging techniques like fluorescence lifetime imaging (FLIM).