Femtosecond optical transfection of cells:viability and efficiency

Abstract

Photoporation is a rapidly expanding technique for the introduction of macromolecules into single cells. However, there remains no study into the true efficiency of this procedure. Here, we present a detailed analysis of transfection efficiency and cell viability for femtosecond optical transfection using a titanium sapphire laser at 800 nm. Photoporation of 4000 Chinese Hamster ovary cells was performed, representing the largest optical transfection study reported to date. We have investigated a range of laser fluences at the cell membrane and, at 1.2 microJ/cm(2), have found an average transfection efficiency of 50 +/- 10%. Contrary to recent literature, in which 100% efficiency is claimed, our measure of efficiency accounts for all irradiated cells, including those lost as a result of laser treatment, thereby providing a true biological measure of the technique.

Enhancement and optimization of plasmid expression in femtosecond optical transfection

“…We observed a significant enhancement in the net transfection efficiency for Chinese Hamster Ovary (CHO-K1) cells when transfected in the presence of Nupherin-neuron. In contrast to the previously reported results, we achieved a transfection efficiency of $180% using the definition used in [8]. This obtained efficiency is approximately a 3 fold enhancement over previous work.…”

Surgical applications of femtosecond lasers

“…Later, femtosecond laser pulses of significantly lower energy were employed to open smaller pores for the delivery of foreign DNA, drugs, or stains into plant or animal cells [107,108]. A number of papers (e.g., [109,110]) have since characterized the increased cell viability (about 80%), superior transfection efficiency (about 60%), and cellular volume exchanged during femtosecond optoporation. Efforts to facilitate the application of this technique are proceeding along several fronts, including nondiffracting light beams to simplify targeting of the cell membrane [111] and optical fiber-mediated transfection to simplify delivery of pulses from fiber laser systems [112].…”

Three-dimensional opto-fluidic devices fabricated by ultrashort laser pulses for high throughput single cell detection and processing

“…Efforts towards miniaturizing electroporation have adopted manipulation of microelectrodes, micropipette and capillaries [15] or microfluidic device-based methods [16,17]. Laser-assisted photoporation has been demonstrated using various laser sources [18][19][20].…”