Mr. Antonio Lorca Camara Profile

Antonio Lorca Camara

at Institut de la Vision

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Publications (1)

Proceedings Article | 30 May 2022 Presentation

A flexible two-photon endoscope for fast functional imaging and cell-precise optogenetic photo-stimulation of neurons in freely moving animals

Nicolò Accanto, François Blot, Valeria Zampini, Florence Bui, Antonio Lorca Camara, Christophe Tourain, Noam Badt, Ori Katz, Valentina Emiliani

Proceedings Volume PC12144, PC1214404 (2022) https://doi.org/10.1117/12.2624381

KEYWORDS: Neurons, Functional imaging, Optogenetics, Endoscopes, Photostimulation, Brain, Visualization, Reverse engineering, Neuroscience, Neuroimaging

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Understanding the causal relations between neuronal activity and behavior is one of the grand challenges of modern neuroscience. From a theoretical point of view, the requirements for reverse engineering the brain architecture are clear: we need to (1) measure neuronal activity from several individual cells, from which we can make hypothesis on their function; (2) manipulate (activate or inhibit) the same cells to test the accuracy of the hypothesis; (3) doing all this in animals during natural behavior. Two-photon (2P) micro-endoscopy could be the technological answer to these urgent needs and is today a thriving research field. However, 2P micro-endoscopy has so far focused on imaging neuronal activity, rather than manipulating it at will, which limits the ability to directly test possible causal links with behavior. To overcome this limitation, we have developed a new two-photon fiber bundle-based micro-endoscope (2P-FENDO) for the simultaneous functional imaging and optogenetic photostimulation of neurons in freely moving mice. By using computer generated holography, 2P-FENDO is capable of 2P optogenetic photostimulation of several neurons at once with cellular resolution. By optimising excitation and collection efficiencies, 2P-FENDO performs 2P functional imaging at one of the highest speeds so far demonstrated through an endoscope. Key novelty behind these results is the discovery that the fiber bundle, composed of ~15000 individual fiber cores, acts as a temporal multiplexing device, separating the laser pulses from each core in time of the right amount to avoid out of focus 2P fluorescence. This property results in a good axial resolution (< 15 µm) independently of the laser spot size. Proof-of-principle experiments were performed in head restrained and freely moving mice co-expressing jGcaMP7s and the opsin ChRmine in the visual or barrel cortex. On a field of view of 250 µm in diameter, we demonstrated functional imaging at a frame rate of up to 100 Hz and precise photostimulation of single and multiple cells (up to ~ 15, limited by the laser power, which could readily be increased with a more powerful photostimulation laser). With the capability to simultaneously image and control neuronal activity at single-cell resolution in freely moving animals, 2P-FENDO will enable to precisely define the functions of neurons in the brain and their interactions during naturalistic behaviours.

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