2P-FENDO-II: A fiber bundle microscope for all-optical, large field-of-view brain studies in freely moving mice.

All-optical strategies enable identification of functional neuronal ensembles with calcium imaging and replay/alter their spatiotemporal activity with optogenetics to decipher their behavioral implications. We previously developed a fiber-coupled microscope enabling two-photon (2P) functional imaging and 2P holographic photostimulation with near-single-cell resolution in freely moving mice: 2P-FENDO. Here, we present a significantly optimized 2P-FENDO-II system that achieves a four-times-larger field of view and a more homogeneous light distribution across the field of view, both for imaging and photostimulation, while achieving better flexibility and thus optimal adaptation to the study of freely moving mice. We demonstrate the performance and versatility of 2P-FENDO-II in experiments targeting the somatosensory cortex, the visual cortex, or the cerebellar cortex, in which we show concomitant calcium imaging with jGCaMP7s and optogenetic control with ChRmine. These enhancements establish 2P-FENDO-II as a groundbreaking tool for all-optical interrogation of neuronal circuits on large volume in naturalistic situations.