We used a galvo-galvo-resonant two-photon microscope (Thorlabs Bergamo II, Vidrio RMR Scanner) with a fast piezoelectric objective scanner (Physik Instrumente P725) and a 20×/1.0 NA objective (XLUMPLFLN20XW, Olympus) for volumetric imaging. We used a Chameleon Vision-S Ti-Sapphire femtosecond laser tuned to 940 nm for two-photon GCaMP excitation. Emission was collected on GaAsP PMT detectors (Hamamatsu) through a 525-nm bandpass filter (Thorlabs). We used ScanImage 2018 software51 (Vidrio Technologies) to control the microscope, and imaging data were collected in ScanImage using National Instruments PXIe-6341 hardware.
The imaging region for all experiments was 256×128 pixels, with 12 slices in the z-axis for each volume (3–5 μm per slice) resulting in a ~10 Hz volumetric scanning rate. For EPG, PFNd, PFNv, SpsP, and IbSpsP imaging experiments, we imaged the PB. For LNO2 and LNO1 imaging experiments, we imaged the NO. For hΔB imaging experiments, we imaged the FB.
The imaging region for all experiments was 256×128 pixels, with 12 slices in the z-axis for each volume (3–5 μm per slice) resulting in a ~10 Hz volumetric scanning rate. For EPG, PFNd, PFNv, SpsP, and IbSpsP imaging experiments, we imaged the PB. For LNO2 and LNO1 imaging experiments, we imaged the NO. For hΔB imaging experiments, we imaged the FB.