Scanless two-photon voltage imaging

Abstract

Parallel light-sculpting methods have been used to perform scanless two-photon photostimulation of multiple neurons simultaneously during all-optical neurophysiology experiments. We demonstrate that scanless two-photon excitation also enables high-resolution, high-contrast, voltage imaging by efficiently exciting fluorescence in a large fraction of the cellular soma. We present a thorough characterisation of scanless two-photon voltage imaging using existing parallel approaches and lasers with different repetition rates. We demonstrate voltage recordings of high frequency spike trains and sub-threshold depolarizations in intact brain tissue from neurons expressing the soma-targeted genetically encoded voltage indicator JEDI-2P-kv. Using a low repetition-rate laser, we perform recordings from up to ten neurons simultaneously. Finally, by co-expressing JEDI-2P-kv and the channelrhodopsin ChroME-ST in neurons of hippocampal organotypic slices, we perform single-beam, simultaneous, two-photon voltage imaging and photostimulation. This enables in-situ validation of the precise number and timing of light evoked action potentials and will pave the way for rapid and scalable identification of functional brain connections in intact neural circuits.

Two-photon voltage imaging with rhodopsin-based sensors

“…We then performed whole-cell current clamp recordings and simultaneous 2P scanless imaging using targeted holographic illumination (Figure 2A). The optical setup to generate a temporally-focused (TF) computer-generated holographic (CGH) spot was as described previously 12 . Here, we used a high-repetition laser tuned to 940 nm to project a disk with a radius and axial extent of ∼ 10 μm, approximately the size of the soma of the neurons in the polymorphic layer of dentate gyrus predominantly patched (Figure 2B).…”

“…If not stated otherwise in the respective method section, imaging datasets were exported as tiff files and analysed using a pipeline written entirely in Python, described previously 12 and derived from 38 .…”

“…Patch-clamp experiments with illumination using GPC (Supplement figure S6) were performed in ND7/23 cells expressing Jarvis, pAce or JEDI as described above. The setup and realisation of the GPC spot are described in Sims and Bendifallah et al 12 . Here, we projected a large (305 ± 5 μm 2 ) temporally-focused spot with an axial resolution of 4.3±0.1 μm using a Coherent Chameleon Discovery laser (1.2 W, 80 MHz repetition rate) tuned to 940 nm.…”

“…To facilitate in-vivo AP detection, especially in depth or densely labelled preparations, the ideal GEVI should not only be bright and have fast kinetics, but should also be compatible with two-photon (2P) excitation. However, to date, only the ASAP-type sensors have been successfully used for AP detection under 2P illumination [7][8][9][10][11][12] . Although rhodopsin-based sensors are intrinsically brighter than the ASAP-type ones 6,13 , they are considered incompatible with 2P illumination and no 2P detection of APs has been reported to date 14,15 .…”

High-throughputin vivosynaptic connectivity mapping of neuronal micro-circuits using two-photon holographic optogenetics and compressive sensing

“…This step can be avoided by integrating in the experimental protocol with optical readout of presynaptic activity using GCaMP imaging 58,69,71 or using voltage indicators. In the latter case, the evoked presynaptic response as well as precise spike times can be verified 104 to correct for any jitter in synaptic inputs, which is key for accurate reconstruction 74 .…”