Time-resolved fluorescence methods (IUPAC technical report)

This IUPAC Technical Report describes and compares the currently applied methods for measuring and analyzing time-resolved fluorescence traces using phase-modulation fluorometry as well as pulse fluorometry (direct emission decay measurements, single-photon timing, streak camera measurements, fluorescence upconversion, and optical Kerr gating). The paper starts with a brief description of the basic principles for time and frequency domain fluorescence spectroscopy. The fundamental equations are given, and recommendations for adequate use are emphasized. The up-to-date, commonly employed excitation sources and photodetectors are described in detail. The analysis of time-resolved fluorescence data is discussed. Attention is paid to possible artifacts, and remedies are presented on how to avoid them or to account for them. Finally, fluorescence lifetime standards for the nanosecond and picosecond timescales are collected.