Engineers and biologists in the Ultrafast Optics and Nanophotonics Laboratory at the University of Alberta (Edmonton, Canada) have demonstrated the advantages of using femtosecond lasers to manipulate molecules and cellular material inside living cells. In experiments published in Biotechnology and Bioengineering, Vikram Kohli, Abdulhakem Elezzabi, and colleagues used femtosecond laser pulses to perform nanosurgery on living zebrafish embryos to introduce exogenous material into the embryonic cells (see figure).1 Their findings should enhance researchers’ ability to noninvasively manipulate molecules in intracellular environments, a critical component in advancing understanding of cellular structure and function.
“The application of lasers to individual cells has been done, but there has been no research into what happens afterward—how the cell reintegrates into the system,” says Elezzabi, professor and Canada Research Chair, Department of Electrical and Computer Engineering. “The zebrafish is the perfect model and is very well used in human health research. They are externally fertilized so they already exist in their natural environment, whereas other cells used in research have to be removed from their environment for research, which can affect them in ways we don’t even know.”
According to Elezzabi and Kohli, several methods for introducing foreign substances into developing embryos have been used. However, microinjection and electroporation, the most commonly used techniques, can be invasive and have some limitations. These limitations are what prompted the researchers to use femtosecond laser pulses to demonstrate the permeabilization of embryos for the delivery of foreign material such as fluorescent probes, quantum dots, and plasmid DNA via transient pores.
By focusing femtosecond laser pulses (10–15 fs/pulse) through the chorion of a zebrafish embryo, researchers at the University of Alberta (Canada) were able to create laser-induced transient pores at the blastomere-yolk interface of the embryo and in individual blastomere cells. The resulting pores were used as delivery pathways to introduce a fluorescent reporter molecule into the cells.
Surrounding the developing embryo is a noncellular layer, the chorion, which protects the embryo from the environment. In their experiments, the University of Alberta team focused femtosecond laser pulses (10–15 fs, 80 MHz rep rate, 40–200 mW power) from a Kerr lens modelocked Ti:sapphire laser to an 800 nm spot beyond the chorion, along the blastomere-yolk interface. The resulting pores were used as delivery pathways to introduce a fluorescent reporter molecule into the blastomere cell.
The laser-manipulated embryos were then tracked to monitor their short- and long-term survival rates and to see if the embryos developed normally after exposure to the laser energy. According to Kohli, the results indicate that femtosecond lasers show great promise for functional studies in cell biology and biological systems, including cryopreservation.
“The whole goal of our research is to provide a foundation for future research in single cells,” he says. “We have shown that zebrafish embryos can be successfully laser-porated to provide a transport pathway for the delivery of exogenous materials. But we also want to determine if this technology can be applied to more complex systems for developmental biologists. We want to merge the science and the engineering to come up with a novel model that can assist others in developing tools and applications for nanosurgery.” —KK
REFERENCE
V. Kohli et al., Biotechnology and Bioengineering 98, 1230 (2007).