Laser Microsurgery inDrosophilaLarvae Using the MicroPoint Ablation System

Laser microsurgery is a robust method to ablate specific cells in the nervous system and probe the functional consequences of their loss in the animal. By introducing focal lesions to small locations in the animal, laser microsurgery also enables disruptions of specific connections within neuronal circuits and the study of how the nervous system responds to precise forms of damage (for instance, damage to specific axons or dendrites, which have been found to evoke different kinds of responses in neurons). The MicroPoint laser is a pulsed dye laser that can be mounted onto any standard microscope, hence is an affordable alternative to two-photon lasers for providing high powered focal ablations. This protocol describes how to use a MicroPoint laser ablation system to induce focal injuries inDrosophilalarvae. This protocol guides a user who has access to a MicroPoint laser that has already been installed onto an appropriate microscope for high-resolution imaging and configured for laser ablation using Coumarin 440 dye. The protocol covers how to use the laser to carry out surgeries or ablation, how to change the laser dye and calibrate the power settings, and how to make sure the laser is properly focused. While the protocol provides an example of axotomy (axon severing) in the peripheral nervous system ofDrosophilalarvae, use of the MicroPoint system can be adapted to other focal surgeries in other organisms.