The system presented here is a further development of the recently introduced spectro-temporal laser imaging by diffractive excitation (SLIDE) microscopy technique. To excite endogenous fluorescence, a new flexible and fibre-based laser source at 780 nm has been developed. The fiber-based FDML-MOPA was amplified to high peak and average powers using rare earth erbium fiber amplifiers. Broadband quasi-phase-matched frequency doubling using a fan-out PPLN crystal was then employed. The output is a 10 nm wide swept pulsed laser at 780 nm with a peak pulse power of 150 W, a pulse duration of 44 ps and a pulse repetition rate of 82 MHz (250 pulses at a sweep rate of 347 kHz). The sweep rate is converted into 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 digitisation card, resulting in 2kHz fluorescence lifetime imaging (FLIM) acquisition. In this paper we present the first images of 780nm SLIDE obtained at 2kHz frame rate. Through the additional use of a 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 2kHz using the recently introduced SLIDE-based time-stretch LiDAR approach.
The recently developed SLIDE microscope enables rapid imaging in nonlinear two-photon microscopy, where frame rates of 4 kHz are achieved. Such fast acquisition speeds coupled with the molecular specificity of fluorescence markers and the high optical resolution in the sub-μm range allow volume scan rates at 40 Hz. A commercially available Fourier Domain Mode Locked Laser system (Optores GmbH, Munich, Germany) was used as the light source emitting at 1060 nm (Bandwidth 15 nm). An electro-optical Modulator (EOM) splits the light of a single sweep duration up into 600 pulses with 30 ps pulse duration each. Each of it is then spatial separated by a diffractive grating. Only one scanner is needed for beam steering to excite the slow axis resulting to a frame rate of 4 kHz. Using a piezo driver for the objective of the microscope at a frequency of 20 Hz, a live 4D volume scan of 40 Hz with 600 x 400 x 100 voxel is possible. Until now, SLIDE systems were bulky and bound onto a fixed optical desk. The Medical Laser Centre Lübeck developed a transportable and reliable SLIDE system, so that this new and highly innovative technology can be made available to various biological laboratories in Europe. This work was conducted in the framework of the EU project “Faircharm.”
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