DS0407 - Exploration du système nerveux dans son fonctionnement normal et pathologique

Acousto-optic Light Patterning for the Imaging of Neurons and Stimulation – ALPINS

Submission summary

In the last decade, cellular and systems neuroscience have experienced a thorough paradigmatic shift following the optogenetic revolution. Optogenetics provides the capability to record and modify the activity of genetically defined cell types in the brain, through light-activated protein-encoded reporters and actuators. Optogenetics thus offers the promise to unravel brain microcircuits dynamics in the behaving animal. Harnessing the full potential of optogenetic tools, however, requires light to be targeted at the right time, to the right cell, and deep inside scattering brain. New light steering techniques, with better dynamics, are therefore required to match the millisecond timescale of neuronal encoding. Illumination of complex patterns in the sample, through spatial shaping of the laser beam, has been proposed as a solution to fit the shape of the recorded or stimulated cell. Deep imaging beyond the standard imaging depths requires using fast phase modulation techniques to focus scattered light, based on the emerging methods developed in particular by the LKB group. However, most of the beam-shaping techniques are limited to a refresh rate of a few Hz, up to a few kHz. A fast beam shaper with sufficient spatial dynamics is currently lacking. Acousto-optic deflectors (AODs) are mainly used as fast 2D pointing devices. In this standard scanning mode, an ultrasonic wave at a constant frequency is used to steer the laser beam at a fixed angle. By using a pair of two orthogonal AODs, light can be scanned in 2D at a rate of about 100kHz. The IBENS groups have pioneered the use of AODs in two-photon fluorescence microscopy in the last years. To spatially shape the laser beam with an AOD, an ultrasonic wave with a frequency varying across the AOD aperture has to be used. But as the ultrasonic wave propagates through the aperture, the beam shape is changing over time, thus preventing the straightforward use of AODs as beam shaper. We have recently proposed a revolutionary method to design an ultra-fast spatial beam shaper based on AOD. By synchronizing the ultrasonic wave in the AOD with the pulses emitted by low repetition rate laser, each pulse interact with a single “frozen” transient ultrasonic wave in the AOD. The pulse can therefore be shaped in any arbitrary manner at a rate of the order of 100kHz. In non-linear microscopy applications, this refresh rate can be obtained with a regenerative amplifier, which provides lower repetition rate (~100 kHz) but hundred times higher energy per pulse as compared to standard fs lasers (100MHz). This proposal consists in the following tasks. 1) We will implement this new technology in a two photon microscope, including beam shaping ability. 2) We will demonstrate its performance by recording and / or stimulating hundreds of neurons in 3D across an entire cortical column with single spike and millisecond resolution. 3) We will take advantage of the high speed of this beam shaper to focus scattered photons and obtain ultra-deep (without the skull) and trans-cranial functional imaging. 4) We will study by this method how barrel cortex neurons may build up representation of expected sensory signals in a head-fixed sensory-motor task. 5) We will record in the cerebellum the population of mossy fibers and Golgi interneurons in a head-fixed animal at rest and during walking episodes. The outcome of this proposal will be the design of a new 3D point access two-photon microscope that will surpass all existing systems in terms of sampling rate, number of recorded / stimulated cells and depth penetration and will provide a technological breakthrough in neurosciences. These new developments will also benefit to the medical research by allowing the longitudinal observation through the skull of the pathological brain in model systems and to medical diagnosis by optical imaging through the skin or the skull.

Project coordination

Laurent BOURDIEU (Institut de Biologie de l’Ecole Normale Supérieure)

The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.

Partner

IBENS Institut de Biologie de l’Ecole Normale Supérieure
LKB Laboratoire Kastler Brossel
IBENS Institut de Biologie de l’Ecole Normale Supérieure

Help of the ANR 467,753 euros
Beginning and duration of the scientific project: - 36 Months

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