Department of Electrical Engineering and Computer Science, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.
Nat Protoc. 2010 Mar;5(3):395-407. doi: 10.1038/nprot.2010.4. Epub 2010 Feb 11.
Femtosecond laser microsurgery is a powerful method for studying cellular function, neural circuits, neuronal injury and neuronal regeneration because of its capability to selectively ablate sub-micron targets in vitro and in vivo with minimal damage to the surrounding tissue. Here, we present a step-by-step protocol for constructing a femtosecond laser microsurgery setup for use with a widely available compound fluorescence microscope. The protocol begins with the assembly and alignment of beam-conditioning optics at the output of a femtosecond laser. Then a dichroic mount is assembled and installed to direct the laser beam into the objective lens of a standard inverted microscope. Finally, the laser is focused on the image plane of the microscope to allow simultaneous surgery and fluorescence imaging. We illustrate the use of this setup by presenting axotomy in Caenorhabditis elegans as an example. This protocol can be completed in 2 d.
飞秒激光显微手术是一种强大的方法，用于研究细胞功能、神经回路、神经元损伤和神经元再生，因为它能够选择性地在体外和体内消融亚微米级别的靶标，同时对周围组织的损伤最小。在这里，我们提供了一个逐步的协议，用于构建一个飞秒激光显微手术设置，用于使用广泛可用的复合荧光显微镜。该协议首先从飞秒激光器输出端的光束调节光学元件的组装和对准开始。然后组装并安装二向色镜安装座，将激光束引导到标准倒置显微镜的物镜中。最后，将激光聚焦在显微镜的像平面上，以允许同时进行手术和荧光成像。我们通过展示秀丽隐杆线虫中的轴突切断作为示例来说明该设置的使用。这个方案可以在 2 天内完成。
