Optical cell transfection with a sub-15 fs and a 250-fs laser

Cells can be transfected by transient laser-induced perforation of the cell membrane which allows foreign genetic material to enter the cell interior. This method (“optoporation”) has emerged as a powerful noninvasive and highly efficient cell-transfection technique. A detailed comparison of efficiencies and results, however, is often hindered by different experimental setups and lasers used in different labs [1].We combined two different lasers with the same experimental setup so that a direct comparison of the influence of the laser parameters becomes possible by treating cells, even of one common cell population, with both lasers. The first laser is a sub-15 fs Ti:sapphire oscillator with a center wavelength of 800 nm (Integral, FemtoLasers) with compensation of the group velocity dispersion in the beam path [2]. The second laser is an ultra-compact femtosecond laser (HighQ-2, Newport Spectra-Physics) emitting 250 fs pulses at 1040 nm. Differences in the experimental setups or preparation procedures that commonly make the comparison of results obtained with different lasers difficult are in this way circumvented [3]. The experimental setup consists of a modified multiphoton laser scanning microscope employing high NA focusing optics. A custom made software identifies cell positions for optoporation and controls the hardware to automatically laser-illuminate these positions [4]. In this way, the number of treatable cells is significantly increased compared to the usual manual targeting of cells. By software-controlled stitching of several microscopic field-of-views in principle all cells in a culture dish can be targeted without further user interaction. The software-based automation allows a significant increase of the number of treatable cells per time compared to a manual approach. We illustrate the experimental capabilities in CHO-cell optoporation experiments and discuss the influence of the parameters of the two lasers on post treatment survival and optoporation efficiency rates.