Excitation-resolved area-normalized emission spectroscopy: a rapid and simple steady-state technique for the analysis of heterogeneous fluorescence

Excitation-resolved area-normalized emission spectroscopy (ERANES) is proposed as a new steady-state fluorescence technique for the investigation of heterogeneous fluorescence (HGF) from a mixture of fluorophores and fluorophores present in various environments and proteins. The presence of a single isoemissive point was used to confirm the presence of two absorbing and emitting species in the system. The isoemissive point was found to occur at the wavelength where the ratio of wavelength dependent fluorescence quantum yield of the emissive species equals to the ratio of their total fluorescence quantum yield. The application of the ERANES method for resolving HGF from a mixture of fluorophores having similar or different fluorescence lifetimes with a relatively high degree of fluorescence spectral overlap was demonstrated. When compared to excitation fluorescence (EF) matrix and time-resolved methods, ERANES was found to be a simple analytical method for analyzing HGF from a mixture of fluorophores, and from fluorophores present in heterogeneous media, such as cells, membranes, etc., and for analyzing protein fluorescence, without the requirement for sophisticated instrumentation and data analysis.