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Construction of a femtosecond laser microsurgery system

Nature Protocols volume 5, pages 395–407 (2010)Cite this article

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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.

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Figure 1: Optical system layout.
Figure 2: An exploded view of the dichroic mounting adapter.
Figure 3: Optical path for simultaneous epifluorescence imaging and laser axotomy.
Figure 4: An exploded view of the beam expander.
Figure 5: Use of the infrared (IR) alignment tool.
Figure 6: Ablated patterns in permanent marker on cover glass under different alignment conditions.
Figure 7: Point spread function of the laser at the focal plane.
Figure 8: Femtosecond laser microsurgery.

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Acknowledgements

We thank the following funding sources: NIH Director's New Innovator Award Program (1-DP2-OD002989–01), Packard Award in Science and Engineering, Sloan Award in Neuroscience, Lincoln Laboratory Advanced Concepts Committee, NSF Career Award, NSF Graduate Research Fellowship, 'La Caixa' Fellowship, NIH Biotechnology Training Grant and the NDSEG Fellowship.

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Author notes

  1. Joseph D Steinmeyer, Cody L Gilleland and Carlos Pardo-Martin: These authors equally contributed to this work.

Authors and Affiliations

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

    Joseph D Steinmeyer, Cody L Gilleland, Carlos Pardo-Martin, Matthew Angel, Christopher B Rohde, Mark A Scott & Mehmet Fatih Yanik

  2. Division of Health Science and Technology, Harvard University, Cambridge, Massachusetts, USA

    Carlos Pardo-Martin & Mark A Scott

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  1. Joseph D Steinmeyer
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Contributions

C.L.G. and C.B.R. developed the laser axotomy techniques described in this protocol. M.A.S. and J.D.S. developed the beam expander structure. M.A., C.B.R. and C.L.G. developed the other elements of the system. M.A.S. developed the laser alignment technique. J.D.S., C.L.G. and C.P.-M. wrote the manuscript, and M.A. and M.F.Y. commented on the manuscript at all stages.

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Correspondence to Mehmet Fatih Yanik.

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Steinmeyer, J., Gilleland, C., Pardo-Martin, C. et al. Construction of a femtosecond laser microsurgery system. Nat Protoc 5, 395–407 (2010). https://doi.org/10.1038/nprot.2010.4

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