We propose a new FRET-based fluorescent molecule that can be used to collect instrument response function. We used 5-carboxyfluorescein as a donor and BHQ2 as an acceptor. The decrease of lifetime was from 4.00 ns to 18 ps for new compound. The brightness of Fl-BHQ2 is two to three orders of magnitude stronger than Raman signal. The solution of Fl-BHQ2 can be used to characterize temporal responses of photodetectors used in spectroscopy/microscopy. We used Fl-BHQ2 fluorescence as a reference in time-domain measurements and we got the same or even better results as those with light scattering. (C) 2010 Elsevier B. V. All rights reserved.