12Understanding information processing in the brain requires us to monitor neural activity 13 in vivo at high spatiotemporal resolution. Using an ultrafast two-photon fluorescence 14 microscope (2PFM) empowered by all-optical laser scanning, we imaged neural activity in 15 vivo at 1,000 frames per second and submicron spatial resolution. This ultrafast imaging 16 method enabled monitoring of electrical activity down to 300 µm below the brain surface 17 in head fixed awake mice. 18 The ability to monitor neural signaling at synaptic and cellular resolution in vivo holds the key to 19 dissecting the complex mechanisms of neural activity in intact brains of behaving animals. The 20 past decade has witnessed a proliferation of genetically encoded fluorescence indicators that 21 monitor diverse neural signaling events in vivo, including those sensing calcium transients, 22 neurotransmitter and neuromodulator release, and membrane voltage [1]. Most popular are the 23 calcium indicators (e.g., GCaMP6 [2]) and glutamate sensors (e.g., iGluSnFR [3]), with their 24 success partly attributable to their slow temporal dynamics (e.g., rise and decay times of 100 -25 1000's milliseconds for GCaMP6 and tens of milliseconds for iGluSnFR), which can be adequately 26 sampled with conventional 2PFM systems. Indeed, using point scanning and near-infrared 27 wavelength for fluorescence excitation, 2PFM can routinely image calcium activity hundreds of 28 microns deep in opaque brains with submicron spatial resolution [4-6].29Imaging faster events, however, is more challenging. Indicators reporting membrane 30 voltage, arguably the most direct and important measure of neural activity, have rise and decay 31 times measured in milliseconds. Too fast for the frame rate of conventional 2PFM to match, their 32 in vivo imaging demonstrations were mostly carried out by widefield fluorescence microscopy with 33 comparatively poor spatial resolution and limited to superficial depths of the brain [7, 8]. In other 34 words, the capability of state-of-the-art indicators has outstripped our ability to image them at 35 sufficiently high speed, especially at high spatial resolution and in large depths of scattering brains.
36The imaging speed of conventional 2PFM is limited by laser scanners, such as 37 galvanometric mirrors, to tens of frame per second (fps) [5, 6]. Its effective pixel dwell time 38 (typically microseconds) is much longer than the ultimate limit imposed by the fluorescence 39 lifetime (typically nanoseconds), below which substantial crosstalk of fluorescence from 40 neighboring pixels can occur [9]. By leveraging an all-optical, passive laser scanner based on a 41 concept termed free-space angular-chirp-enhanced delay (FACED) [10], here we demonstrated 42 2PFM at 1,000 fps, with the pixel dwell time flexibly configured to reach the fluorescence lifetime. 43 We applied it to ultrafast monitoring of calcium activity, glutamate release, and action potentials 44 Figure 1: Principles and resolution of a 2PFM with a FACED module. ...