Daq board

5–6 dpf zebrafish larvae were first anaesthetized in mivacurium and then mounted in 1.5% low-melting-temperature agarose in a glass- bottom dish (Mat Tek) and immersed in E3 water. Fish were imaged on an upright Nikon resonant-scanning two-photon microscope (A1RMP), with a 25x water immersion objective (NA = 1.1) and the laser (Coherent Vision II) tuned to 960 nm. To image different z planes, a piezo drive (Mad City Labs) was used at a step size of 10 μm for light-evoked activity recording. The frame rate for whole brain imaging was 7.1 Hz (Fig. 2); each stack consisted of 10 frames. For light stimulus, the blue and red light boxes were controlled by a power supply unit (TMS-lite), triggered by a 5 V TTL signal from a National Instruments DAQ board that was controlled by the Nikon Elements software.
Green light could not be used as a stimulus in the imaging experiment, due to overlap with the emission spectrum of GCaMP6f.

Zebrafish larvae were anaesthetized in mivacurium and embedded in low-melting temperature agarose (1.2–2.0% in E3; egg water: 5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl₂, 0.33 mM MgSO₄) in a glass-bottom dish (Mat Tek). They were imaged on a Nikon two-photon microscope (A1RMP), attached to a fixed stage upright microscope, using a 25× water immersion objective (NA = 1.1). The femtosecond laser (Coherent Vision II) was tuned to 920 nm for GCaMP imaging. Stacks were collected in resonant-scanning mode with a 525/50 nm bandpass emission filter and with 8× pixel averaging; single-plane images were collected in galvano-scanning mode with 2× pixel averaging.
Light stimuli were generated by 5 mm blue LEDs (458 nm peak emission) powered by a 5 V TTL signal from a control computer and synchronized with image capture using a National Instruments DAQ board, controlled by the Nikon Elements software. Light intensity at the sample was 0.13 mW/cm².
For wide-field microscopy, excitation was provided by LEDs (Cairn OptoLED) at 470 nm. Images were captured on a Zeiss Axio Examiner with a 20× water immersion objective, using a Flash4 camera (Hamamatsu) controlled by MetaMorph. After background subtraction, change in fluorescence was measured using MetaMorph.

Zebrafish larvae (aged 5–10 days-post-fertilization, dpf) were immobilized in mivacurium (1.5 mg/ml) and embedded in low-melting temperature agarose (2.0% in E3) in a glass-bottom dish (Mat Tek). They were imaged on a Nikon two-photon microscope (A1RMP), using a 25x water immersion objective (NA = 1.1). The femtosecond laser (Coherent Vision II) was tuned to 920 nm for GCaMP6f imaging. Stacks were collected in resonant-scanning mode with 2x pixel averaging. The sample size was based on [11 (link)]. Blue light stimulus was generated by 5 mm blue LEDs (458 nm peak emission), which was powered by a 5 V TTL signal from a control computer and synchronized with image capture using a National Instruments DAQ board, controlled by the Nikon Elements software. Each light pulse was 20 s long and followed by 20 s dark with a total of 4 pulses of light. Light intensity at the sample was 0.13 mW/cm².