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