ABSTRACT
Fluorescence lifetime imaging is the best approach to image molecular and cellular processes. A typical measurement of fluorescence lifetime consists of two independent measurements. One measurement of observed fluorescence intensity decay and the second measurement of Instrument Response Function (IRF). This chapter discusses the importance of obtaining/measuring and considering in calculation an appropriate IRF. It reviews the factors that affect IRF such as speed of light and optimal (maximum signal) wavelength and excitation wavelength. Intensity decay frequently will depend on the setting of polarizer on observation and it is crucial to keep magic angle conditions. Today most time-resolved fluorescence measurements are made using time-domain technology (time-correlated single-photon counting). Occasionally, frequency-domain technology can also be used. In an ideal case, the initial intensity observed with a polarizer oriented parallel to the excitation light polarization will be three times greater than the intensity observed through a polarizer oriented orthogonally.
