Multi-photon excitation fluorescence lifetime imaging

Summary form only given. Multi-photon excitation microscopy (MPEM) offers several advantages over conventional confocal imaging. One of these advantages is the improved in-depth imaging performance that is due to the comparatively long excitation wavelength and the reduced photo damage to the sample. Using fluorescence lifetime imaging (FLIM) the potential of MPEM can be further extended. This combination turns out to be particularly useful for quantitative imaging in thick specimens. The pulsed light source required for MPEM makes the implementation of FLIM in MPE microscopes simple. We employ a simple time-gating scheme. The FLIM module exists of 8 computer controlled gated photon counters. Each gate is opened sequentially after an excitation pulse. This makes the method very efficient and comparatively insensitive to photobleaching effects. Comparisons with time-correlated single photon counting measurements show that accurate results can be obtained for fluorescence decay times greater than 0.5 ns. FLIM can be employed for the quantitative imaging of ion-concentrations and offers several advantages compared with conventional methods such as emission- and excitation-ratio imaging. We present results of pH imaging in thick dental biofilm.