Permeabilization and cell surgery using femtosecond laser pulses: an emerging tool for cellular manipulation

Abstract

Non-invasive manipulation of live cells is important for cell-based therapeutics. Herein, we report on the application of femtosecond laser pulses for cellular manipulation, and the generation of optical pores for cytoplasmic delivery of non-reducing cryoprotectants. Under precise laser focusing, we demonstrate membrane surgery on live mammalian cells, and ablation of focal adhesions adjoining fibroblast cells. In both studies, the morphology of the cell post-laser treatment was maintained with no visible collapse or disassociation. When mammalian cells were suspended in a hyperosmotic cryoprotectant solution, focused femtosecond laser pulses were used to transiently permeabilize live cells for sucrose uptake. To verify the cytoplasmic uptake, the volumetric response of cells in 0.2, 0.3, 0.4, and 0.5 M cryoprotective sucrose was measured using video microscopy. From membrane integrity assays, we determined that optimal cell survival of 91.5 ± 8% is achieved using 0.2 M sucrose, with a decline in survival at higher concentrations. Using diffusion analysis for a porous membrane, the intracellular accumulation of cryoprotective sucrose was theoretically determined. At a diffusion length of 10 um, > 70% of the extracellular osmolarity was estimated to be intracellularly delivered following closure of the transient pore. We anticipate that our study will have important applications for biopresevation, and profound implications for surgery and cell-isolation.