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We report the application of high-intensity femtosecond laser pulses as a novel tool for manipulating biological specimens. When femtosecond laser pulses were focused to a near diffraction-limited focal spot, cellular material within the laser focal volume was surgically ablated. Several dissection cuts were made in the membrane of live mammalian cells, and membrane surgery was accomplished without inducing cell collapse or disassociation. By altering how the
laser pulses were applied, focal adhesions joining live epithelial cells were surgically removed, resulting in single cell
isolation. To further examine the versatility of this reported tool, cells were transiently permeabilized for introducing
foreign material into the cytoplasm of live mammalian cells. Localizing focused femtosecond laser pulses on the
biological membrane resulted in the formation of transient pores, which were harnessed as a pathway for the delivery of
exogenous material. Individual mammalian cells were permeabilized in the presence of a hyperosmotic cryoprotective
disaccharide. Material delivery was confirmed by measuring the volumetric response of cells permeabilized in 0.2, 0.3,
0.4 and 0.5 M cryoprotective sugar. The survival of permeabilized cells in increasing osmolarity of sugar was assessed
using a membrane integrity assay. Further demonstrating the novelty of this reported tool, laser surgery of an aquatic
embryo, the zebrafish (Danio rerio), was also performed. Utilizing the transient pores that were formed in the embryonic
cells of the zebrafish embryo, an exogenous fluorescent probe FITC, Streptavidin-conjugated quantum dots or plasmid
DNA (sCMV) encoding EGFP was introduced into the developing embryonic cells. To determine if the laser induced
any short- or long-term effects on development, laser-manipulated embryos were reared to 2 and 7 days post-fertilization
and compared to control embryos at the same developmental stages. Light microscopy and scanning electron microscopy
were used to compare whole body mosaics of the developed larvae. Key developmental features that were compared
included the olfactory pit, dorsal, ventral and pectoral fins, notochord, otic capsule and otic vesicle. No differences in the
morphology and placement of the fore-, mid- and hindbrains were observed.
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