In vivo Multiphoton Calcium Imaging in Larval Zebrafish

GCaMP6s imaging was performed on 7–8 dpf larvae embedded in 2% low-melting-point agarose without anesthetic. For RGC imaging and all receptive field mapping experiments, larvae were first paralyzed by spinal injection of alpha-bungarotoxin (Alomone Labs, Jerusalem, Israel) 30–120 min prior to imaging. Multiphoton imaging was performed on a custom Scientifica (East Sussex, UK) microscope equipped with a Chameleon titanium-sapphire laser (Coherent Inc., Santa Clara, CA, USA) tuned to 920nm for GCaMP6s excitation. All functional imaging was performed using a Nikon LWD 16x 0.8NA water immersion objective. Image acquisition rates were between 1 and 2 Hz. Visual stimuli were generated using PsychoPy software [59 (link)] and presented using an ASUS Zenbeam picoprojector (ASUStek, Inc.) equipped with Kodak Red 25 Wratten filter (Edmund Optics, USA).