Calibration approaches for fluorescence lifetime applications using time-domain measurements

This tutorial focuses on presenting experimental protocols for acquiring instrument response functions (IRF) and for calibrating the instruments using reference dyes with validated lifetime in time-domain fluorescence lifetime measurements. Step-by-step preparation of different samples used for the calibrations (scatter solutions, crystals generating second harmonic signal and reference dyes) and the corresponding instrument settings in one- and two-photon excitation are explained. The expected shape of the IRF curves and reference decays are shown using experimentally acquired examples, followed by troubleshooting of the instruments when the expected results are distorted. The discussions focus on the importance of using IRF and reference dyes for adjusting the acquisition parameters of the time-resolved instrument, for data analysis and for comparison and extrapolation of lifetime values between different biological systems.