Fluorescence measurements: importance of G-factor correction, magic angle, and observation wavelengths

Excitation and emission (observation) conditions heavily impact fluorescence measurements. Both observed spectra and intensity decays (fluorescence lifetimes), when incorrectly measured, may lead to incorrect data interpretations. In this report, we discuss the role of observation conditions in steady-state and time-resolved (lifetime) fluorescence measurements. We demonstrate the importance of the correction for uneven transmissions of vertical and horizontal polarizations of emission light through the detection system. The necessity of using so-called total fluorescence intensity or intensity measured under magic angle (MA) conditions has been demonstrated for both steady-state and time-resolved fluorescence measurements. The dependence of lifetime measurements on observation (emission) wavelengths is also discussed. Two fluorophores, rhodamine 6G (R6G) and 4,4 Dimethylamino-cyano stilbene (DCS) in two solvents - ethanol and glycerol have been used in order to cover a broad range of dye polarities and solvent viscosities.