TY - JOUR
T1 - Optoporation and genetic manipulation of cells using femtosecond laser pulses
AU - Davis, Andrew A.
AU - Farrar, Matthew J.
AU - Nishimura, Nozomi
AU - Jin, Moonsoo M.
AU - Schaffer, Chris B.
PY - 2013/8/20
Y1 - 2013/8/20
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=84883031923&partnerID=8YFLogxK
U2 - 10.1016/j.bpj.2013.07.012
DO - 10.1016/j.bpj.2013.07.012
M3 - Article
C2 - 23972838
AN - SCOPUS:84883031923
SN - 0006-3495
VL - 105
SP - 862
EP - 871
JO - Biophysical Journal
JF - Biophysical Journal
IS - 4
ER -