Fluorescence lifetime microscopy: a stimulated emission approach

The stimulated emission process has been rarely exploited in spectroscopy and microscopy. Instead, a spectroscopy method based on the pump-probe principle has been frequently used to observe picosecond and femtosecond processes. This common approach has not been applied to microscopy due to the relatively slow acquisition time and the lack of 3D information. We have exploited an idea originally proposed by F. Lytle group in which two pulsed lasers are simultaneously focused on the sample. One laser is used to excite a population of molecules and the second laser to induce stimulated emission. The stimulated radiation is carried away in the same direction of the stimulating laser beam. By collecting the fluorescence emission in other directions, we observe a modulation of the fluorescence signal as a function of the delay between the two laser pulses. The repetition rate of the two lasers is slightly different producing a frequency beating at the laser overlapping volume. We have extended this method to achieve very high spatial and temporal resolution in the microscope environment. By recording only the beating frequency, we obtain a 3D sectional effect similar to two-photon excitation. The harmonic content of the beating signal is limited by the laser pulse width and by the sample frequency response. Information of picosecond processes are extracted by standard frequency-domain methods. Using this principle, we built a stimulated emission microscope that has 3D and fluorescence lifetime capabilities