Kilohertz frame-rate two-photon tomography

Abstract

Point-scanning two-photon microscopy enables high-resolution imaging within scattering specimens such as the mammalian brain, but sequential acquisition of voxels fundamentally limits its speed. We developed a two-photon imaging technique that scans lines of excitation across a focal plane at multiple angles and computationally recovers high-resolution images, attaining voxel rates of over 1 billion Hz in structured samples. Using a static image as a prior for recording neural activity, we imaged visually-evoked and spontaneous glutamate release across hundreds of dendritic spines in mice at depths over 250 μm and frame-rates over 1 kHz. Dendritic glutamate transients in anaesthetized mice are synchronized within spatially-contiguous domains spanning tens of microns at frequencies ranging from 1-100 Hz. We demonstrate millisecond-resolved recordings of acetylcholine and voltage indicators, 3D single-particle tracking, and imaging in Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:

A fast genetically encoded fluorescent sensor for faithfulin vivoacetylcholine detection in mice, fish, worms and flies

“…7a). Previous work showed that 300 µm below the pia, brief stimulation (1 ms, 500 µA) of NB elicits punctate (<2 µm) and rapid (~10 ms) iAChSnFR transients, consistent with synaptic ACh release events 38 . Here we compared dynamics of stimulationevoked ACh transients recorded from neuropil at different cortical depths in response to pulse trains of different lengths (1, 3, or 10 pulses at 20 Hz).…”

“…The sensor is based on a bacterial choline-binding protein, and it shows no apparent effects on neurons following stable expression in rodent brain slice and in living worms, fish, flies, and mice -both in the brain and at the neuromuscular junction (NMJ). We further created both green and yellow variants, the latter enabling kilohertz frame-rate imaging in mouse cortex in vivo with SLAP microscopy 38 . The sensor also allows detection of both increases and decreases of acetylcholine signaling from tonic levels.…”

“…Cholinergic neurons from the basal forebrain (NB) also project to visual cortex (V1), where ACh signaling regulates attention and visual learning 73 . To visualize dynamics of NBderived acetylcholine signals with micron spatial and millisecond temporal resolution, we used two-photon Scanned Line Angular Projection (SLAP) imaging 38 . Yellow-shifted y-iAChSnFR was expressed in L2/3 cortical neurons with AAV2/1.hSynapsin1.y-iAChSnFR, and an electrode implanted in NB ( Fig.…”

Correction of z-motion artefacts to allow population imaging of synaptic activity in awake behaving mice

“…Investigating the operation of these circuits has been greatly advanced by using multiphoton microscopy to image fluorescent reporter proteins that signal events such as changes in cytoplasmic calcium, reflecting spiking activity (Tian et al, 2012), or the release of glutamate at excitatory synapses (Marvin et al, 2013). At present, the large majority of in vivo imaging studies quantify activity as calcium signals in the soma of neurons but we also need to image activity across populations of synapses if the operations of neural circuits are to be unravelled (Lu et al, 2017;Kazemipour et al, 2019;Meng et al, 2019). These connections are key components of all the computations within neural circuits and sites at which neuromodulators act to reconfigure signal flow according to changes in the internal state of the animal.…”

jYCaMP: An optimized calcium indicator for two-photon imaging at fiber laser wavelengths

“…High-power industrial light sources such as Ytterbium-doped fiber lasers (YbFLs) and modelocked semiconductor lasers show great promise to overcome these limitations, as they have increasingly shown feasibility for in-vivo applications [2][3][4][5][6][7] and are becoming widely available at costs orders of magnitude lower and/or power outputs orders of magnitude higher than conventional tunable lasers ( Supplementary Fig. 1).…”