title: Two-photon voltage imaging using a genetically encoded voltage indicator.
abstract: Voltage-sensitive fluorescent proteins (VSFPs) are a family of genetically-encoded voltage indicators (GEVIs) reporting membrane voltage fluctuation from genetically-targeted cells in cell cultures to whole brains in awake mice as demonstrated earlier using 1-photon (1P) fluorescence excitation imaging. However, in-vivo 1P imaging captures optical signals only from superficial layers and does not optically resolve single neurons. Two-photon excitation (2P) imaging, on the other hand, has not yet been convincingly applied to GEVI experiments. Here we show that 2P imaging of VSFP Butterfly 1.2 expresssing pyramidal neurons in layer 2/3 reports optical membrane voltage in brain slices consistent with 1P imaging but with a 2-3 larger ΔR/R value. 2P imaging of mouse cortex in-vivo achieved cellular resolution throughout layer 2/3. In somatosensory cortex we recorded sensory responses to single whisker deflections in anesthetized mice at full frame video rate. Our results demonstrate the feasibility of GEVI-based functional 2P imaging in mouse cortex.
authors.affiliation: RIKEN Brain Science Institute, Hirosawa, Wako-City, 351-0198 Saitama, Japan.
authors.forename: Walther
authors.initials: W
authors.lastname: Akemann
authors.forename: Mari
authors.initials: M
authors.lastname: Sasaki
authors.forename: Hiroki
authors.initials: H
authors.lastname: Mutoh
authors.forename: Takeshi
authors.initials: T
authors.lastname: Imamura
authors.forename: Naoki
authors.initials: N
authors.lastname: Honkura
authors.forename: Thomas
authors.initials: T
authors.lastname: Knöpfel
mesh_terms: D000818:Animals; D002540:Cerebral Cortex / Q000502:physiology*; D005071:Evoked Potentials / Q000502:physiology*; D005260:Female; D008164:Luminescent Proteins; D051379:Mice; D059906:Neuroimaging; D009474:Neurons / Q000502:physiology; D017785:Photons; D011247:Pregnancy; D013003:Somatosensory Cortex / Q000502:physiology
chemical_list: D008164:Luminescent Proteins