title: Construction of a femtosecond laser microsurgery system.
abstract: 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.
authors.affiliation: Department of Electrical Engineering and Computer Science, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.
authors.forename: Joseph D
authors.initials: JD
authors.lastname: Steinmeyer
authors.forename: Cody L
authors.initials: CL
authors.lastname: Gilleland
authors.forename: Carlos
authors.initials: C
authors.lastname: Pardo-Martin
authors.forename: Matthew
authors.initials: M
authors.lastname: Angel
authors.forename: Christopher B
authors.initials: CB
authors.lastname: Rohde
authors.forename: Mark A
authors.initials: MA
authors.lastname: Scott
authors.forename: Mehmet Fatih
authors.initials: MF
authors.lastname: Yanik
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified; D019771:Axotomy / Q000295:instrumentation / Q000379:methods; D017173:Caenorhabditis elegans / Q000235:genetics / Q000502:physiology / Q000648:ultrastructure; D004867:Equipment Design; D053685:Laser Therapy / Q000295:instrumentation* / Q000379:methods; D008856:Microscopy, Fluorescence / Q000295:instrumentation; D008866:Microsurgery / Q000295:instrumentation* / Q000379:methods; D009416:Nerve Regeneration