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Clement Molinier

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Proceedings Article | 4 March 2019 Presentation

Multiplexed temporally focused light shaping for precise in-depth optogenetic stimulation (Conference Presentation)

Nicolò Accanto, Clement Molinier, Emiliano Ronzitti, I-Wen Chen, Dimitrii Tanese, Daniel Tomer, Eirini Papagiakoumou, Ori Katz, Valentina Emiliani

Proceedings Volume 10886, 108860R (2019) https://doi.org/10.1117/12.2507772

KEYWORDS: Optogenetics, Multiplexing, Brain, Scattering, Wavefronts, Microscopes, Neurons, Light scattering, Laser scattering, Two photon excitation microscopy

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Optical wavefront shaping is a powerful technique to control the distribution of light in the focus of a microscope. Combined with optogenetics, it holds great promise for a precise manipulation of neuronal activity with light. A better understanding of complex brain circuits however, requires advanced and flexible optical methods capable of simultaneously photo-exciting multiple neurons, possibly using dedicated excitation shapes, arbitrarily distributed in the three dimensions, with single-cell resolution. At the same time, the study of deep brain structures with all optical techniques, even in the multi-photon regime, is limited by scattering to a depth of few hundreds µm. Here we first present a new optical scheme, based on the spatio-temporal shaping of a pulsed laser beam, to project several tens of spatially confined two photon excitation patterns in a large volume. Using two spatial light modulators and the temporal focusing technique we are able to produce at least 4 different extended excitation patterns, with single cell axial confinement, that we independently multiplex at the sample volume an arbitrary number of times. We fully characterise the optical response of the system, discuss the possibility of simplifying it at the expenses of flexibility, and subsequently exploit it to perform multi-cell volumetric excitation in both Drosophila and zebrafish larvae. Finally, we summarise our recent efforts towards the extension of such method to a micro endoscope, which could be used for the study of complex neural circuits in deep brain structures, thus overcoming the limitations imposed by scattering.

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