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Two-photon excited fluorescence (TPEF) microscopy and fluorescence lifetimeimaging (FLIM) are powerful imaging techniques in bio-molecular science. Theneed for elaborate light sources for TPEF and speed limitations for FLIM,however, hinder an even wider application. We present a way to overcome thislimitations by combining a robust and inexpensive fiber laser for nonlinearexcitation with a fast analog digitization method for rapid FLIM imaging. Theapplied sub nanosecond pulsed laser source is synchronized to a high analogbandwidth signal detection for single shot TPEF- and single shot FLIM imaging.The actively modulated pulses at 1064nm from the fiber laser are adjustablefrom 50ps to 5ns with kW of peak power. At a typically applied pulse lengthsand repetition rates, the duty cycle is comparable to typically usedfemtosecond pulses and thus the peak power is also comparable at same cw-power.Hence, both types of excitation should yield the same number of fluorescencephotons per time on average when used for TPEF imaging. However, in the 100psconfiguration, a thousand times more fluorescence photons are generated perpulse. In this paper, we now show that the higher number of fluorescencephotons per pulse combined with a high analog bandwidth detection makes itpossible to not only use a single pulse per pixel for TPEF imaging but also toresolve the exponential time decay for FLIM. To evaluate the performance of oursystem, we acquired FLIM images of a Convallaria sample with pixel rates of 1MHz where the lifetime information is directly measured with a fast real timedigitizer. With the presented results, we show that longer pulses in themany-10ps to nanosecond regime can be readily applied for TPEF imaging andenable new imaging modalities like single pulse FLIM.

proceedings of spie, the international society for optical engineering/proceedings of spie

Two-photon-excited fluorescence (TPEF) and fluorescence lifetime imaging  (FLIM) with sub-nanosecond pulses and a high analog bandwidth signal  detection