Effects of Photon Losses on Fluorescence Lifetime Imaging Microscopy (FLIM) System Optimization

Several approaches for optimization of fluorescence lifetime imaging microscopy (FLIM) system have been recently suggested. This paper discusses the influences of photons losses on the optimization of FLIM systems based time-correlated single photon counting (TCSPC) technique, considering the limitations associated with detecting the required amount of photons by the system. The fluorescence intensity (FI) and fluorescence lifetime (FLT) were measured in different operating regimes of the imaging system. The relation between parameters such as excitation power, detector gain, laser repetition rate, is also analyzed. Based on data acquisition limitations of typical TCSPC systems, we discuss the considerations for choosing the correct system parameters, which would most influence the accuracy of FLIM experiments. A simple scheme for patent optimization of FLIM systems for different types of fluorescent samples is finally suggested.