Tutorial: measurement of fluorescence spectra and determination of relative fluorescence quantum yields of transparent samples.

ABSTRACT

The measurement of fluorescence spectra and the determination of fluorescence quantum yields in transparent samples are conceptually simple tasks, but these procedures are subject to several pitfalls that can lead to significant errors. Available technical reports and protocols often assume that the reader possesses a solid theoretical background in spectroscopy and has ample experience with fluorescence instrumentation, but this is often not the case given the many applications of fluorescence in diverse fields of science. The goal of this tutorial is to provide a didactic treatment of the topic that will hopefully be accessible to readers without extensive expertise in the field of fluorescence. The article covers the theoretical background needed to understand the origins of the most common artifacts researchers can expect. Possible artifacts are illustrated with examples to help readers avoid them or identify them if present. A step-by-step example of a fluorescence quantum yield determination in solution is provided with detailed experimental information to help readers understand how to design and analyze experiments.