Simultaneous two-photon imaging and two-photon manipulation of neural activity in freely behaving mice

Simultaneous two-photon imaging and two-photon manipulation of neural activity in freely- behaving mice The ability to record and manipulate neural activity in freely-behaving mice is a key step to understand the neural circuits under natural behavior. One-photon miniscope has become a mature technology and it can

perform calcium imaging and optogenetics simultaneously while the mice freely behave. Though it has enabled many new discoveries in neuroscience, the ability to image and manipulate neural activity deeper into the tissue and in higher spatial specificity can further advance the field and enable studying many new

questions. Compared to one-photon, two-photon techniques can access much deeper tissue and have a much higher spatial specificity. However, it has been challenging to integrate high performance optics into a compact footprint miniscope to perform simultaneous two-photon imaging and two-photon optogenetics,

due to the excessive challenges in optical and mechanical design. Here, we propose a new miniscope that can simultaneously perform two-photon calcium imaging and two- photon optogenetics in freely-behaving mice. We innovatively integrate two different beam forming techniques in the miniscope for the imaging beam and optogenetics beam. This unique combination

enables a very compact mechanical design and a high optical performance. Crucially, we will achieve a high-spatiotemporal-resolution in imaging, and patterned stimulation in optogenetics where a group of user-selected neurons could be simultaneously photostimulated. This allows us to manipulate the ensemble activity while monitoring the response of the neural circuit, all in cellular resolution. The entire

device can have a dimension