Smithson Laura J, Waller Thomas J, Collins Catherine A
Department of Molecular, Cellular, and Developmental Biology, University of Michigan, Ann Arbor, Michigan 48109, USA.
Department of Molecular, Cellular, and Developmental Biology, University of Michigan, Ann Arbor, Michigan 48109, USA
Cold Spring Harb Protoc. 2025 Jun 2;2025(6):pdb.prot108171. doi: 10.1101/pdb.prot108171.
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 in larvae. 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 of larvae, use of the MicroPoint system can be adapted to other focal surgeries in other organisms.
激光显微手术是一种用于切除神经系统中特定细胞并探究动物体内这些细胞缺失所产生功能后果的强大方法。通过在动物的小区域制造局灶性损伤，激光显微手术还能够破坏神经元回路内的特定连接，并研究神经系统如何对精确形式的损伤做出反应（例如，对特定轴突或树突的损伤，已发现这种损伤会在神经元中引发不同类型的反应）。MicroPoint激光是一种脉冲染料激光，可安装在任何标准显微镜上，因此是一种价格合理的替代双光子激光的设备，可用于进行高功率局灶性消融。本方案描述了如何使用MicroPoint激光消融系统在幼虫中诱导局灶性损伤。本方案指导有机会使用已安装在合适显微镜上用于高分辨率成像并已配置使用香豆素440染料进行激光消融的MicroPoint激光的用户。该方案涵盖了如何使用激光进行手术或消融、如何更换激光染料和校准功率设置以及如何确保激光正确聚焦。虽然该方案提供了在幼虫外周神经系统中进行轴突切断术（切断轴突）的示例，但MicroPoint系统的使用可适用于其他生物体的其他局灶性手术。
