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Nat Protoc. 2010 Mar;5(3):395-407. doi: 10.1038/nprot.2010.4. Epub 2010 Feb 11.

Construction of a femtosecond laser microsurgery system.

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  • 1Department of Electrical Engineering and Computer Science, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.


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.

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