Optoporation and genetic manipulation of cells using femtosecond laser pulses.

TitleOptoporation and genetic manipulation of cells using femtosecond laser pulses.
Publication TypeJournal Article
Year of Publication2013
AuthorsDavis AA, Farrar MJ, Nishimura N, Jin MM, Schaffer CB
JournalBiophys J
Volume105
Issue4
Pagination862-71
Date Published2013 Aug 20
ISSN1542-0086
KeywordsAnimals, Cell Membrane, Cell Survival, CHO Cells, Coloring Agents, Cricetinae, Cricetulus, Cytological Techniques, DNA, Lasers, Plasmids, Time Factors, Transfection
Abstract

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.

DOI10.1016/j.bpj.2013.07.012
Alternate JournalBiophys J
PubMed ID23972838
PubMed Central IDPMC3752125
Related Institute: 
Molecular Imaging Innovations Institute (MI3)

Weill Cornell Medicine
Department of Radiology
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