Extended Data Fig. 3: Schematic of the setup for calcium imaging.

a, Top view of the embedding preparation for 2-photon imaging of juvenile zebrafish. To enable active respiration, agarose columns are cut out in front of the mouth and gills. The tail is also freed to improve oxygen uptake through the skin. Oxygenated water in the imaging chamber is constantly renewed with a peristaltic pump. b, Side view of the preparation and remote focusing system. The imaging chamber, consisting of a small petri dish, is placed in a large petri dish filled with water. Diffusive paper serving as a screen and a small spacer are placed between the large and small petri dish. The large petri dish is placed on a custom-made sample holder. A cold mirror is placed under the preparation to reflect projector images onto the screen. The input beam to the remote focusing system (red), passes through a half-wave plate and is reflected by a polarizing beam splitter. The beam is enlarged by two lenses, passes through a quarter-wave plate, and is focused by an objective onto a mirror mounted to a custom piezo stage. The piezo moves the mirror and thus adjusts the effective focal distance of the reflected beam, which ultimately changes the collimation of the beam at the main objective, changing the focus. The second pass through the quarter-wave plate on the return trip results in a change of polarization compared with the input beam, so the reflected beam now continues straight through the polarizing beam splitter, reaching the microscope. To bypass the remote focusing path, the input half-wave plate can be rotated so the input beam instead passes through the polarizing beam splitter, hits a mirror and passes through a quarter-wave plate twice, and then is reflected into the microscope. The detection path is standard and is not depicted.