Abstract

Imaging tools that allow for high resolution high-speed network-level brain activity mapping are of high interest in neuroscience and in other biomedical applications studying biological population dynamics in scattering tissues in vivo. Currently nonlinear optical microscopy plays a key role in imaging neural activity in behaving animals. In scattering tissue, two-photon microscopy in combination with raster scanning approaches have been widely applied, but one of the most difficult challenges is to achieve sufficient image contrast deep in scattering samples [1]. Due to limited number of emitted photons available for image reconstruction, accessing neuronal dynamics with sufficient temporal resolution for large-scale volumetric measurements of neural activity in scattering tissue is challenging. For high-speed raster scanning multiphoton imaging, fundamentally the speed limit is set by the pulse repetition rate of the femtosecond laser used, i.e. at least one laser pulse must be used per image pixel. In practice, this limit is also ruled by a compromise of the speed and inertia of the imaging and beam steering hardware, as well as the maximum allowed incident laser power on the sample to avoid damage or excessive heating. On the other hand, the available pulse peak intensity and operation wavelength of the driving laser source play also a significant role for achieving deep tissue imaging. To counteract the power decrease with depth, in order to maintain sufficient intensity at the focus at significant depths in scattering media, it is key to increase the energy of the excitation pulse. Yb-fiber chirped pulse amplifiers providing femtosecond, multi µJ-level pulses at very high repetition rates are very attractive for high-speed functional imaging. In this work, we present a µJ-level, MHz repetition rate all-fiber integrated chirped pulse amplifier delivering clean ~180 fs pulses (Fig. 1 (a–c)), that enabled us to demonstrate a new approach for fast volumetric raster scanning microscopy, using temporal focusing [2] to produce an enlarged point spread function of 5x5x10 µm (matching the typical size of neurons in the mouse cortex) to allow for single laser pulse per vowel excitation [3]. This is important because it allows to sample the imaging volumes with the minimally required number of voxels, which leads to faster volume sampling and higher signal-to-noise ratios for the same average laser power. The large stretching ratio of the pulses in the Yb-fiber amplifier allows us to change the repetition rate and output energy of the system over a large range, without compromising the pulse fidelity. At MHz repetition rate, we have obtained up to 10 W of average power from the system using less than 60% of the available pump power, and at 100 kHz, compression of 1 W of output power (10 µJ pulse energy) from a similar system, using the same stretcher, but less efficient gratings yielded 160 fs pulses [4]. With the system set to a repetition rate of 4 MHz and 600 nJ output energy, we have performed high-speed volumetric functional imaging in the brain of living, awake mice, using a microscope with a sculpted focal volume of 5x5x10 µm³ (xyz) through temporal focusing. We demonstrate high-speed single neuron-resolution three-dimensional imaging in mouse brain in-vivo, on GCamp6 and jRGECKO1 labelled neurons, over large volumetric FOVs (up to 500x500x500 µm) at multi-Hertz (3-6 Hz) update rate, see Fig. 1 (d) and (e).

© 2017 IEEE