Is 3 really better than 2? In vivo functional calcium imaging of cortical circuits by two- and three-photon microscopy

This study explores the capability of three-photon functional imaging to resolve functional cortical architecture in mice. By a quantitative, back-to-back comparison in awake head-fixed mice, we demonstrate that three-photon imaging is equally capable compared to two-photon imaging at superficial layers (II/III) and allows functional assessment of deep cortical layers (V/VI), beyond the reach of conventional two-photon microscopy.