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In the past 10 years, the use of light has become irreplaceable for the optogenetic study and control of neurons and neural circuits. Optical techniques are however limited by scattering and can only see through a depth of few hundreds µm in living tissues. GRIN lens based micro-endoscopes represent a powerful solution to reach deeper regions. In this work we demonstrate that cutting edge optical methods for the precise photostimulation of multiple neurons in three dimensions can be performed through a GRIN lens. By spatio-temporally shaping a laser beam in the two-photon regime we project several tens of spatially confined targets in a volume of at least 100 × 150 × 300 µm 3 . We then apply such approach to the optogenetic stimulation of multiple neurons simultaneously  in vivo  in mice. Our work paves the way for an all-optical investigation of neural circuits in previously inaccessible brain areas.

Multiplexed temporally focused light shaping through a gradient index lens for precise in-depth optogenetic photostimulation