Data Analysis Methods in Time‐Resolved Fluorescence Spectroscopy: A Tutorial Review

Fluorescence spectroscopy and related techniques benefit from exceptional sensitivity and have become engrained in a variety of fields from biosciences to materials sciences. Measuring time-domain fluorescence decays is nowadays a routine task in many laboratories across these different fields. Perhaps surprisingly, a correct data analysis of these fluorescence decay curves presents a formidable challenge and requires extensive insight in the problems associated with fitting this type of data. As a result, the reported analysis of these decays is usually limited to a non-linear least squares fit of a sum of a few exponential terms to the data. This review aims to expose the intricate field of data analysis in time-resolved fluorescence spectroscopy to a broader audience, from researchers interested in understanding the photophysics of their system to readers and reviewers trying to understand the merits of specific methods. Challenges associated with this type of kinetic experimental data are outlined and the clever analysis strategies devised by researchers across different disciplines are introduced and discussed in detail. A section on freely available scripts and software facilitating the analysis is included towards the end. We encourage the reader to try their hand at the worked examples.