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.
Matthias Eibl, Sebastian Karpf, Hubertus Hakert, Daniel Weng, and Robert Huber, "Two-photon-excited fluorescence (TPEF) and fluorescence lifetime imaging (FLIM) with sub-nanosecond pulses and a high analog bandwidth signal detection," Proc. SPIE 10069, Multiphoton Microscopy in the Biomedical Sciences XVII, 100691F (Presented at SPIE BiOS: January 31, 2017; Published: 21 February 2017); https://doi.org/10.1117/12.2250831.
Conference Presentations are recordings of oral presentations given at SPIE conferences and published as part of the conference proceedings. They include the speaker's narration along with a video recording of the presentation slides and animations. Many conference presentations also include full-text papers. Search and browse our growing collection of more than 12,000 conference presentations, including many plenary and keynote presentations.