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Femtosecond optical transfection of individual mammalian cells

Nature Protocols volume 8, pages 1216–1233 (2013)Cite this article

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Abstract

Laser-mediated gene transfection into mammalian cells has recently emerged as a powerful alternative to more traditional transfection techniques. In particular, the use of a femtosecond-pulsed laser operating in the near-infrared (NIR) region has been proven to provide single-cell selectivity, localized delivery, low toxicity and consistent performance. This approach can easily be integrated with advanced multimodal live-cell microscopy and micromanipulation techniques. The efficiency of this technique depends on an understanding by the user of both biology and physics. Therefore, in this protocol we discuss the subtleties that apply to both fields, including sample preparation, alignment and calibration of laser optics and their integration into a microscopy platform. The entire protocol takes ∼5 d to complete, from the initial setup of the femtosecond optical transfection system to the final stage of fluorescence imaging to assay for successful expression of the gene of interest.

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Figure 1: Experimental setup.
Figure 2: Experimental setup for the generation of a Bessel beam.
Figure 3: Back-reflection of the NIR beam from the glass-air interface on the coverslip or bottom of a Petri dish.
Figure 4: Examples of transient gas bubbles formed on cellular membrane.
Figure 5: Typical images acquired in the optoinjection efficiency and viability study.
Figure 6: Examples of cell-targeted optical transfection of genes.

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Acknowledgements

This work is supported by the UK Engineering Physical Sciences Research Council (EPSRC). We would like to acknowledge Roslin Cellab for providing the human embryonic stem cells through a SUPA start-up grant. M.A. acknowledges the support of an EPSRC-funded 'Rising Star' Fellowship and the SULSA. K.D. is a Royal Society Wolfson Merit Award holder. F.J.G.-M. acknowledges the support of the R.S. MacDonald Charitable Trust, SU2P and The 'BRAINS' 600th anniversary appeal. Both K.D. and F.J.G.-M. acknowledge the support of E. Killick. We are grateful to all current and past members of the Biophotonics group at University of St. Andrews who contributed to the development and optimization of this protocol.

Author information

Author notes

  1. Maciej Antkowiak and Maria L Torres-Mapa: These authors contributed equally to this work.

  2. Kishan Dholakia and Frank J Gunn-Moore: These authors jointly directed this work.

Authors and Affiliations

  1. Scottish Universities Life Sciences Alliance (SULSA), School of Biology, University of St. Andrews, St. Andrews, UK.,

    Maciej Antkowiak, Maria L Torres-Mapa & Frank J Gunn-Moore

  2. Scottish University Physics Alliance (SUPA), School of Physics and Astronomy, University of St. Andrews, St. Andrews, UK.,

    Maciej Antkowiak, Maria L Torres-Mapa, David J Stevenson & Kishan Dholakia

Authors
  1. Maciej Antkowiak
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  2. Maria L Torres-Mapa
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  5. Frank J Gunn-Moore
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Contributions

M.A. and M.L.T.-M. obtained the results reported in the protocol and wrote the manuscript. M.A., M.L.T.-M., D.J.S., K.D. and F.J.G.-M. edited and approved the manuscript. K.D. and F.J.G.-M. led the project.

Corresponding authors

Correspondence to Kishan Dholakia or Frank J Gunn-Moore.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Video 1

Examples of transient gas bubbles formed on cellular membrane. Left: a long-lasting bubble indicates an irradiation overdose. Right: a small (<2 μm) bubble indicates good beam alignment and correct irradiation parameters. (AVI 11528 kb)

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Antkowiak, M., Torres-Mapa, M., Stevenson, D. et al. Femtosecond optical transfection of individual mammalian cells. Nat Protoc 8, 1216–1233 (2013). https://doi.org/10.1038/nprot.2013.071

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