Dual optoled power supply

Calcium fluorescence recordings were carried out at 15 DIV using the cell-permeant calcium sensitive dye Fluo4-Acetoxymethyl ester (Fluo4-AM) (Invitrogen, Thermo Fisher), similar to [23 (link)]. Fluo4 1 mM dissolved in DMSO was added to the culture medium to a final concentration of 1 μM and incubated for 20 min at 37 °C. After incubation, cultures were placed in a 35-mm-diameter glass bottom chamber (P35G-0-14-C; MatTek Corporation) for recording. Recordings were carried out in pH-stable (7.4) external medium (EM). EM consisted of HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), 10 mM; NaCl, 128 mM; KCl, 4 mM; glucose, 10 mM; sucrose, 45 mM; CaCl₂, 2 mM; and MgCl₂, 1 mM. The recording chamber was mounted on an Olympus IX70 inverted microscope equipped with a Hamamatsu Orca Flash 4.0 V2 (Digital CMOS camera C11440-22CU) camera and Dual OptoLED power supply (Cairn Research Ltd) as a source of light. Fluorescence images were acquired at 20 frames per second (fps) at room temperature (RT) with a × 5 objective.

Longer wavelength experiments were conducted using a similar setup as above with the following changes. The monochromator used to generate longer wavelengths was unable to also generate short‐wavelength light needed for the larvae to swim into the water column (see Section 3). To be sure that larvae were responding to longer wavelengths, we used an LED UV light controlled by a dual OptoLED power supply (Cairn Research) which peaked at 370 nm. The output of the LED was measured using a spectrophotometer at the point of the cuvette and manually adjusted until it was set to the same approximate total intensity as the long wavelengths presented to the animals. Animals were exposed to 5 min of UV light, then the test wavelength (575, 600, 625, 650, 675, or 700 nm), or darkness for 1 h (Figure 2). We saw no difference in swimming behavior in preliminary tests with 1 or 2 h in the dark, therefore only 1 h of darkness was used for experiments. For wavelengths 575 and 600 nm, behavioral response reached equilibrium (all animals swam to the bottom of the cuvette) well before an hour and some trials were stopped earlier than an hour.