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

Abstract

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.