Kilohertz two-photon SLIDE microscopy using a newly developed 780 nm excitation laser

Abstract

The system presented here is an evolution of the recently introduced spectro-temporal laser imaging by diffractive excitation (SLIDE) (1) microscopy technique. In order to excite endogenous fluorescence, a new flexible and fiberbased laser source at 780nm was developed. The fiber-based FDML-MOPA (2) was amplified to high peak and average powers by rare-earth Erbium fiber amplifiers. Afterwards, broadband quasi-phase-matched frequencydoubling using a Fan-out PPLN crystal was employed. The output is a 10 nm wide swept, pulsed laser around 780 nm with a pulse peak power of 150 W, 44 ps pulse duration and a pulse repetition rate of 82 MHz (250 pulses at 347 kHz sweep rate). The sweep rate is converted to line scans by a diffraction grating and sent to a microscope for two-photon excitation of UV-excited dyes or endogenous autofluorescence. For detection, the signal is captured with a 4 GS/s high speed digitization card leading to 2kHz fluorescence lifetime imaging (FLIM) acquisition.In this work, we present first images of 780nm SLIDE obtained at 2kHz frame-rate. Through the additional use of an piezo objective scanner, we are able to perform 3D imaging at 20Hz volume rate. We have also used this novel system for high-speed LiDAR imaging at a frequency of 2 kHz, using the recently introduced SLIDE-based timestretch LiDAR approach.