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.
Bibliographical noteFunding Information:
acknowleDGMents 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.
All Science Journal Classification (ASJC) codes
- Biochemistry, Genetics and Molecular Biology(all)