Nd1 filter

Imaging and delivery of visual stimuli followed Leong et al. 2016^{38 (link)}. Fluorescence was monitored in vivo using two-photon microscopy. We used a Leica SP 5 II equipped with the HCX APO L 20X/NA1.00 water dipping lens (Leica, Wetzlar, Germany). GCaMP6f was excited at 920 nm, and the power was ~5–8 mW at the stage. Recordings lasted ~3.5 minutes. GCaMP6f fluorescence signals were acquired with a bandpass filter (525/50m), at ~20 Hz (bidirectional scanning at 1.4 kHz, across a FOV of 128 pixels x 256 pixels, rows x columns). Pixels measured ~290 x ~290 nm. The stimulus screen subtended ~ 60° x 90° (azimuth x elevation) of the left visual field. Visual stimuli were delivered with a LightCrafter 4500 DLP (Texas Instruments, Dallas, TX, USA) using a 100 Hz frame rate. The LightCrafter was configured to use only the blue LED, then the stimulus was filtered with a 447/60 bandpass filter (Semrock, IDEX Health and Science, Rochester, NY, USA), and a ND1 filter (Thorlabs, Newton, NJ, USA). The mean radiance was ~0.04 W sr⁻¹ m⁻².

Imaging and delivery of visual stimuli were followed as in Leong et al. 2016 (link). Fluorescence was monitored in vivo using two-photon microscopy. For two-photon voltage and calcium imaging, we used a Leica SP 5 II (ASAP2f excitation @ 920 nm, ~15 mW at the stage, jRGECO1a excitation @ 1040 nm, ~15 mW at the stage). Recordings lasted ~1 h. Voltage and calcium signals were acquired at ~15 Hz (bidirectional scanning at 1.4 kHz, across a FOV of 128 pixels x 256 pixels, rows x columns). Pixels measured ~290 x ~290 nm. ASAP2f and jRGECO1a fluorescence signals were acquired with different bandpass filters (525/50m and 585/40m, respectively). The stimulus screen subtended ~ 60° x 90° (azimuth x elevation) of the left visual field. Visual stimuli were delivered with a Lightcrafter 4500 DLP, configured to deliver exclusively blue LED illumination, using a 100 Hz frame rate. The stimulus was attenuated with a 447/60 bandpass filter (Semrock), and a ND1 filter (Thorlabs). The mean radiance was 0.04 W sr⁻¹ m⁻².