Scottish Universities Life Sciences Alliance (SULSA), School of Biology, University of St. Andrews, St. Andrews, UK.
Nat Protoc. 2013 Jun;8(6):1216-33. doi: 10.1038/nprot.2013.071. Epub 2013 May 30.
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.
激光介导的哺乳动物细胞基因转染最近已经成为一种强大的替代更传统的转染技术。特别是，使用在近红外（NIR）区域中工作的飞秒脉冲激光已被证明可提供单细胞选择性、局部递送、低毒性和一致的性能。这种方法可以很容易地与先进的多模态活细胞显微镜和微操作技术集成。该技术的效率取决于用户对生物学和物理学的理解。因此，在本方案中，我们讨论了适用于这两个领域的细微差别，包括样品制备、激光光学的对准和校准及其集成到显微镜平台中。从飞秒光学转染系统的初始设置到荧光成像的最终阶段，整个方案大约需要 5 天才能完成，以检测感兴趣基因的表达是否成功。
