Herein, we demonstrate the application of high-intensity femtosecond (fs) laser pulses for performing laser surgery on the embryonic cells of developing zebrafish (Danio rerio). When fs laser pulses were focused onto individual blastomeres, transient pores were formed exposing the extracellular space to the intracellular environment. Utilizing the transient pores as a pathway for delivery of exogenous material, both chorionated and dechorionated zebrafish embryos
were successfully loaded with a fluorescent reporter molecule (fluorescein isothiocyanate (FITC)). Streptavidin-conjugated
quantum dots or plasmid DNA (Simian-CMV-EGFP). Both FITC and quantum dots were found to disperse throughout the blastomere cells as the embryo developed. Gene expression was seen in 24 hour post-fertilized embryos, with fluorescence observed in the notochord, floor plates, somites and tails of the larvae. We also determined the survivability of laser-manipulated embryos by rearing zebrafish from early to mid cleavage stage (2-cell to 8/16-cell) to pec-fin stage. Survival rates of 89 and 100 % were found for dechorionated and chorionated embryos, respectively.
The zebrafish (Danio rerio) is an attractive model system that has received wide attention for its usefulness in the study
of development and disease. This organism represents a closer analog to humans than the common invetebrates Drosophila melanogaster and Caenorhabditis elegans, making this species an ideal model for human health research. Non-invasive manipulation of the zebrafish has been challenging, owing to the outer proteinaceous membrane and
multiple embryonic barriers. A novel tool capable of manipulating early cleavage stage embryonic cells would be important for future advancements in medial research and the aquaculture industry. Herein, we demonstrate the laser surgery of early cleavage stage (2-cell) blastomere cells using a range of average laser powers and beam dwell times. Since the novelty of this manipulation tool depends on its non-invasive application, we examined short- and long-term laser-induced developmental defects following embryonic surgery. Laser-manipulated embryos were reared to 2 and 7 days post-fertilization and compared to control embryos at the same developmental stages. Morphological analysis was performed using light microscopy and scanning electron microscopy. Developmental features that were examined included the antero- and dorsal-lateral whole body views of the larvae, the olfactory pit, dorsal, ventral and pectoral fins, notochord, pectoral fin buds, otic capsule, otic vesicle, neuromast patterning, and kinocilia of the olfactory pit rim and cristae of the lateral wall of the ear. Laser-manipulated embryos developed normally relative to the controls, with developmental patterning and morphology at 2 and 7 days indistinguishable from control larvae.
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.
In this article we investigate laser-assisted ablation of Hydroxy Ethyl Methacrylate (Hydro-gel) material using 30
femtosecond laser pulses delivered from a Ti:Sapphire multipass amplifier with a repetition rate of 1 kHz.
Measurements of the crater depth, width and removed volume as a function of laser pulse energy and pulse number
were made for stationary and translated ablation. Based on laser fluence, crater profile, collateral damage and translation
speed, optimal laser parameters for efficient micromachining were defined. The results presented in this paper will be
important for future developments in laser ophthalmology, where insight into the optimal laser parameters for corneallike
surgery will decrease both processing time and collateral tissue damage.
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.
Access to the requested content is limited to institutions that have purchased or subscribe to SPIE eBooks.
You are receiving this notice because your organization may not have SPIE eBooks access.*
*Shibboleth/Open Athens users─please
sign in
to access your institution's subscriptions.
To obtain this item, you may purchase the complete book in print or electronic format on
SPIE.org.
INSTITUTIONAL Select your institution to access the SPIE Digital Library.
PERSONAL Sign in with your SPIE account to access your personal subscriptions or to use specific features such as save to my library, sign up for alerts, save searches, etc.