Molecule transfer into mammalian cells by single sub‐nanosecond laser pulses

A rapid, precise, and viability‐retaining method for cytoplasmic molecule delivery is highly desired for cell engineering. Routine methods suffer from low throughput, lack of selectivity, requirement of helper compounds, predominant endosomal delivery, and/or are restricted to specific molecule classes. Photonic cell manipulation bears the potential to overcome these drawbacks. Here we investigated mammalian cell manipulation by single sub‐nanosecond laser pulses. Axial beam waist positioning close to a cell monolayer induced culture vessel damage and zones of cell ablation. Cells at margins of ablation zones exhibited uptake of membrane‐impermeant fluorophores and GFP expression plasmids. Increasing Rayleigh‐length and beam waist diameter reduced the sensitivity to axial defocusing and resulted in robust molecule transfer. Serial application of single pulses focused over a moving cell monolayer yielded quantitative molecule transfer to cells at rates up to 40%. Our results could be basic to spatially and temporally controlled single laser pulse‐mediated marker‐free high throughput cell manipulation. Laser light bears potential for spatially and temporally controlled cell engineering, which could significantly improve cell‐based therapy or targeted cell manipulation in microsystems engineering approaches such as organ‐on‐chip. This study presents systematic investigations on the reversible perforation of cells with single laser pulses and thus provides starting points for the development of advanced biomedical applications.