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.

Date Published: 20 February 2008
PDF: 9 pages
Proc. SPIE 6854, Optical Interactions with Tissue and Cells XIX, 685413 (20 February 2008); doi: 10.1117/12.760078

Published in SPIE Proceedings Vol. 6854:
Optical Interactions with Tissue and Cells XIX
Steven L. Jacques; William P. Roach; Robert J. Thomas, Editor(s)