Fig. 4. Two-photon and three-photon imaging in mice.

a, Simultaneous two-photon imaging (through a prism) and mesoscopic wide-field imaging in awake, head-fixed mice obtained a larger FOV with the Cousa. Time-averaged two-photon image obtained through the prism show the difference in FOV compared with a commercially available 20 mm air objective. Time series for neurons imaged with two-photon excitation through the Cousa objective and microprism were used to detect cell-centered networks for 74 neurons in one mouse. b, Top: the Cousa objective enables in vivo functional imaging of cochlear hair cells. The mouse is held supine for imaging of IHCs and OHCs. We imaged with both the Cousa objective and a conventional objective, the Nikon ×20/0.4 NA, 19 mm WD (TU Plan ELWD 20X), with the same laser power (30 mW). Middle: for calcium imaging in IHCs, the Cousa was used with 30 mW and the Nikon was used with 70 mW, to obtain minimal usable signal levels for both at 5.7 frames per s. In response to sound stimulation, IHCs exhibited responses only during Cousa imaging. Bottom: fluorescent particles (diameter 5.63 µm) faded rapidly when imaged with the Nikon, and maintained fluorescence when imaged with the Cousa. a.u., arbitrary units. c, Left: the Cousa supports large FOV three-photon imaging, with a 20 mm WD. The vasculature across the entire 4 mm² region is visible after an intravenous injection of Texas Red dextran. Right: higher zoom single z plane three-photon images from a second mouse with dual channel imaging of Texas Red dextran (magenta) and THG (cyan) in cortex and white matter.