Full-field exposure of larval zebrafish to narrow waveband LED light sources at defined power and energy for optogenetic applications

Background: Optogenetic approaches in transparent zebrafish models have provided numerous insights into vertebrate neurobiology. The purpose of this study was to develop methods to activate light-sensitive transgene products simultaneously throughout an entire larval zebrafish.New method: We developed a LED illumination stand and microcontroller unit to expose zebrafish larvae reproducibly to full field illumination at defined wavelength, power, and energy.Results: The LED stand generated a sufficiently flat illumination field to expose multiple larval zebrafish to high power light stimuli uniformly, while avoiding sample bath warming. The controller unit allowed precise automated delivery of predetermined amounts of light energy at calibrated power. We demonstrated the utility of the approach by driving photoconversion of Kaede (398 nm), photodimerization of GAVPO (450 nm), and photo activation of dL5**/MG2I (661 nm) in neurons throughout the CNS of larval zebrafish. Observed outcomes were influenced by both total light energy and its rate of delivery, highlighting the importance of controlling these variables to obtain reproducible results.Comparison with existing methods: Our approach employs inexpensive LED chip arrays to deliver narrow waveband light with a sufficiently flat illumination field to span multiple larval zebrafish simultaneously. Calibration of light power and energy are built into the workflow.Conclusions: The LED illuminator and controller can be constructed from widely available materials using the drawings, instructions, and software provided. This approach will be useful for multiple optogenetic applications in zebrafish and other models.