A versatile multiple time-gate fluorescence lifetime acquisition module is presented. The module is used as an add-on to a two-photon scanning laser microscope equipped with pulsed excitation for fluorescence lifetime imaging. It enables the recording of fluorescence lifetimes by capturing the intensity decay in four or eight time gates. The module allows fluorescence lifetime imaging at high count rates and with a high efficiency. The module is equipped with computer-controlled variable-delay lines for setting the gate widths. Decay times can be accurately measured in the 0.5 ns–1 μs range. Fluorescence lifetime imaging experiments on a number of single dye solutions with different (mono-exponential) fluorescence lifetimes are in excellent agreement with the reference values obtained with time-correlated single photon counting. Measurements on a test specimen exhibiting a bi-exponential decay demonstrate that the two decay components can be quantified with high accuracy. In a specimen containing six different lifetimes, in the range 2.8 to 12 ns, all lifetimes could be well distinguished. As an example, the results of a pH imaging experiment on oral biofilm using carboxyfluorescein are presented. Here, fluorescence lifetime imaging is used both to separate the short-lifetime autofluorescence from the carboxy-fluorescein fluorescence and to determine the pH in the biofilm.
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