Goniometers

We used the same imaging system as described previously (Kaifosh et al., 2013 (link); Lovett-Barron et al., 2014 (link)) with an 8kHz resonant galvanometer (Bruker). Approximately 150-250 mW of laser power was used during imaging (measured under the objective), with adjustments in power levels to accommodate varying window clarity. To optimize light transmission, we adjusted the angle of the mouse's head using two goniometers (Edmund Optics, ±10 degree range) such that the imaging window was parallel to the objective. All images were acquired with a Nikon 40× NIR water-immersion objective (0.8 NA, 3.5 mm WD). Green (GCaMP6f) fluorescence was acquired using an emission cube set (green, HQ525/70m-2p; red, HQ607/45m-2p; 575dcxr, Chroma Technology) taking images (512 × 512 pixels) at ∼30 Hz covering 160 μm × 160 μm area. The emitted photons were captured with a photomultiplier tube (GaAsP PMT, Hamamatsu Model 7422P-40), and a dual stage preamp (1.4 × 10⁵ dB, Bruker) was used to amplify signals prior to digitization.

Imaging was conducted using a microscope setup which consists of 8 kHz resonant galvanometer (Bruker) mounted to a mirror-based multi-photon microscopy system (Prairie Technologies) and an ultra-fast pulsed laser beam (920-nm wavelength; Chameleon Ultra II, Coherent, 20–40-mW average power at the back focal plane of the objective) controlled with an electro-optical modulator (Conoptics, Model 302 RM). GCaMP fluorescence was excited through a 40x water immersion objective (Nikon NIR Apo, 0.8 NA, 3.5 mm WD) and fluorescence signals detected with photomultiplier tubes (Hamamatsu 7422P-40), acquired with PrairieView software (Prairie) at 30fps frame rate (512×512 pixels, 1.3 μm/pixel). A custom dual stage preamp (1.4×10⁵ dB, Bruker) was used to amplify signals prior to digitization. Two goniometers (Edmund Optics) were used to adjust the angle of each mouse’s head in order to achieve the same imaging plane over multiple sessions.