Department of Biomedical Engineering, Cornell University, Ithaca, NY, USA.
Biophys J. 2013 Aug 20;105(4):862-71. doi: 10.1016/j.bpj.2013.07.012.
Femtosecond laser optoporation is a powerful technique to introduce membrane-impermeable molecules, such as DNA plasmids, into targeted cells in culture, yet only a narrow range of laser regimes have been explored. In addition, the dynamics of the laser-produced membrane pores and the effect of pore behavior on cell viability and transfection efficiency remain poorly elucidated. We studied optoporation in cultured cells using tightly focused femtosecond laser pulses in two irradiation regimes: millions of low-energy pulses and two higher-energy pulses. We quantified the pore radius and resealing time as a function of incident laser energy and determined cell viability and transfection efficiency for both irradiation regimes. These data showed that pore size was the governing factor in cell viability, independently of the laser irradiation regime. For viable cells, larger pores resealed more quickly than smaller pores, ruling out a passive resealing mechanism. Based on the pore size and resealing time, we predict that few DNA plasmids enter the cell via diffusion, suggesting an alternative mechanism for cell transfection. Indeed, we observed fluorescently labeled DNA plasmid adhering to the irradiated patch of the cell membrane, suggesting that plasmids may enter the cell by adhering to the membrane and then being translocated.
飞秒激光转染是一种将膜不可渗透的分子（如 DNA 质粒）导入培养细胞中的有效技术，但仅探索了很窄的激光范围。此外，激光产生的膜孔的动力学以及孔行为对细胞活力和转染效率的影响仍未得到充分阐明。我们在两种辐照模式下使用聚焦紧密的飞秒激光脉冲研究了培养细胞中的转染：数百万个低能量脉冲和两个更高能量的脉冲。我们定量测量了孔半径和再封闭时间作为入射激光能量的函数，并确定了两种辐照模式的细胞活力和转染效率。这些数据表明，孔大小是细胞活力的控制因素，与激光辐照模式无关。对于存活的细胞，较大的孔比较小的孔更快地再封闭，排除了被动再封闭机制。基于孔大小和再封闭时间，我们预测很少有 DNA 质粒通过扩散进入细胞，这表明细胞转染可能有替代机制。事实上，我们观察到荧光标记的 DNA 质粒附着在细胞膜的辐照区域，这表明质粒可能通过附着在膜上然后被易位进入细胞。
