Imaging Neuronal Activity in Awake Mice

Three mice were head-restrained under the objective 4–13 days after the implantation of the cranial windows and performed the same motor task during TPLSM imaging as was used during 8K-SDCLM imaging. Two-photon images were acquired using an FVMPE-RS system (Olympus, Tokyo, Japan) with a broadly tunable ultrafast laser (InSight DS-OL; Spectra Physics, CA, USA) tuned to 940 nm. The back aperture of the objective (XLPLN25XWMP2; back aperture diameter, 15.1 mm; numerical aperture [NA], 1.05; Olympus) was underfilled with the diameter-shortened (7.2 mm) laser beam to reduce the effective excitation NA of the objective^{14 (link)}. The dimensions of the FOV were 512 × 512 pixels (127.2 × 127.2 µm) for all imaging fields. The imaging fields were at 40–60 µm depth below the cortical surface. The laser power was adjusted to maintain a relatively constant fluorescent intensity from the axonal boutons (5.94–11.9 mW). A series of 10,800 continuous images was acquired at 30 Hz. A 570 nm dichroic mirror (FV30-FGR; Olympus) and a bandpass filter at 495–540 nm were used. For XYZ imaging (Fig. 5i), three planes with an interval of 8 µm in the same horizontal field were sequentially imaged in three mice. For each plane, the dimensions of the FOV were 512 × 256 pixels (84.9 × 42.4 µm) and a series of 1860 or 2230 images was acquired at 6.2 Hz.