Performing Chromophore-Assisted Laser Inactivation in Drosophila Embryos Using GFP

Chromophore-assisted laser inactivation (CALI) is an optogenetic technique in which light-induced release of reactive oxygen species triggers acute inactivation of a protein of interest, with high spatial and temporal resolution. At its simplest, selective protein inactivation can be achieved via the genetic fusion of the protein to a photosensitizer such as EGFP, and using standard optical setups such as laser scanning confocal microscopes. Although use of CALI in Drosophila is relatively recent, this technique can be a powerful complement to developmental genetics, especially in vivo as it allows visualization of the immediate consequences of local protein inactivation when coupled to time-lapse microscopy analysis. In addition to providing examples of protocols, this chapter is intended as a conceptual framework to support the rational design of CALI experiments.