Optoporation and Genetic Manipulation of Cells Using Femtosecond Laser Pulses

Abstract

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.

Temporal Airy pulses control cell poration

“…Interestingly, it was shown that large membrane laser-induced holes are, counter-intuitively, associated with a faster resealing dynamics compared to small holes, suggesting an active resealing mechanism triggered by large membrane damages. 28 Our results can be compared to recent measurements on cell optoporation by pairs of 100 fs BWL pulses at 1 kHz from an amplified Ti:Sapphire laser. 28 The authors report successful poration and transfection with cell viability figures comparable to those obtained in pJ-level oscillator-based experiments.…”

“…28 Our results can be compared to recent measurements on cell optoporation by pairs of 100 fs BWL pulses at 1 kHz from an amplified Ti:Sapphire laser. 28 The authors report successful poration and transfection with cell viability figures comparable to those obtained in pJ-level oscillator-based experiments. 14 As those measurements were performed by BWL pulses, we could expect by spectral pulse shaping similar-or better-transfection rate and increased viability thanks to the 25% reduction in energy and 88% reduction in peak intensity associated with TAP+.…”

Analysis of poration-induced changes in cells from laser-activated plasmonic substrates

“…Both dyes have previously been used in literature to investigate flow in-and outside of cells [37,38]. We labeled C2C12 cells with 1 µM Calcein AM in PBS for 10 min at 37° C and washed once to reduce background fluorescence.…”

Dynamics of laser-induced bubble pairs

“…The use of laser-based methods for cell transfection is an active area of research, because they can selectively target individual cells within a mixed cell population and specific cells of interest within a cell cluster (or possibly even in living tissue). Various methods for opto-poration of the cell membrane have been developed in recent years (Tirlapur & König 2002;Vogel et al 2005;Baumgart et al 2008;Antkowiak et al 2010;Stevenson et al 2010;Antkowiak et al 2013a,b;Davis et al 2013). Usually, a laser pulse is focused onto the cell membrane in order to create a hole.…”

“…This implies the possibility of achieving a combination of a massive and slow jet opposed by a thin and fast jet that could be useful for various applications, for instance micropumping and opto-injection for cell transfection Stevenson et al 2010). The use of laser-based methods for introducing genetic materials or other substances into living cells has become a very active area of research, and various techniques for opto-poration of the cell membrane have been developed (Tirlapur & König 2002;Vogel et al 2005;Baumgart et al 2008;Stevenson et al 2010;Antkowiak et al 2013a,b;Davis et al 2013). They usually rely on creating one or several small holes in the cell membrane, and share the drawback that material transport into the cell relies on diffusion, which is a slow process.…”