Et510 80m 2p

We used the same imaging system as described previously (29) . Briefly in vivo two photon imaging was performed using a dual galvanometric and resonant laser scanning two-photon microscope (Ultima, Bruker), coupled to a tunable Ti:Sapphire laser (MaiTai eHP DeepSee, Spectraphysics) pulsed at a 80 MHz repetition rates and <70fs pulse width along with dispersion compensation. GCaMP fluorophore was excited at 920 nm, using a resonant scanning Xgalvanometer (8kHz) paired with a 6mm standard scanning Y-galvanometer. The scanning system was mounted on movable objective Ultima microscope, equipped with an orbital nosepiece coupled to a 16X, 0.8 NA, 3 mm water immersion objective (Nikon) and a piezo drive for angled imaging and ultrafast volumetric scanning. Imaging was performed at a scan speed of 29 fps, using 512x512 frame size (1.012 µm/ pixel resolution). Fluorescence signal was separated into green channel and red channel using a dichroic mirror (T565lpxr, Chroma Technology) and emission filters (green, ET510/80m-2p; red, ET605/70m-2p, Chroma Technology), respectively. Each emission signal was detected using high-sensitivity GaAsP photomultiplier tubes (model 7422PA-40 PMTs, Hamamatsu).

Imaging was performed using a two-photon 8-kHz resonant scanner (Ultima, Bruker) with a 16x, 0.8 NA water-immersion objective (Nikon). Excitation was performed at 920 nm with an 80 MHz pulsed laser (Mai Tai DeepSee, Spectra Physics). GCaMP6f emission fluorescence was collected with a GaAsP photomultiplier tube (7422P-40, Hamamatsu) following red and green channel separation with a filter cube consisting of a dichroic mirror (T565lpxr, Chroma Technology) and filters (green, ET510/80m-2p; red, ET605/70m-2p, Chroma Technology). Images were acquired at a 30 Hz frame rate, 512x512 pixel resolution, and 1.5x digital zoom corresponding to a field size of 555 µm x 555 µm.