Fluorescence Quantum Yields: Methods of Determination and Standards

The fluorescence quantum yield Φ f is a key property that characterizes the ability of a fluorophore to convert absorbed photons into emitted photons under various environmental conditions. Knowledge of it is important for the successful development of fluorometric indication and visualization methods and for the understanding of light-driven processes in the natural sciences. Φ f is a molecule- or material-related parameter and can thus significantly differ from the fraction of absorbed photons that are actually measured as the fluorescence signal of a certain sample, e.g., when the signal is modulated by reabsorption, self-quenching or polarization effects. The determination of Φ f can be performed in absolute measurements or relative to a fluorescent standard material with a known Φ f by optical or calorimetric methods. Here the different procedures and techniques are described and compared, and the suitability of a representative number of dyes that have been proposed as fluorescence reference materials in the past 25 years is discussed. Besides addressing specific issues such as low-temperature measurements and the appropriate choice of standards and measurement conditions, we conclude with recommendations for better standardization and quality management in this area.