An alternative method for delivering exogenous material into developing zebrafish embryos

Abstract

Non-invasive manipulation of multicellular systems is important for medical and biological research. The ability to introduce, remove, or modify molecules in the intracellular environment is pivotal to our understanding of cellular structure and function. Herein, we report on an alternative method for introducing foreign material into developing embryos using the application of femtosecond (fs) laser pulses. When intense fs laser pulses are focused to a sub-micron spot, transient pores are formed, providing a transport pathway for the delivery of exogenous material into embryonic cells. In this study, zebrafish embryos were used as a model system to demonstrate the non-invasiveness of this applied delivery tool. Utilizing optically induced transient pores chorionated and dechorionated zebrafish embryos were successfully loaded with a fluorescent reporter molecule (fluorescein isothiocyanate), Streptavidin-conjugated quantum dots or DNA (Simian-CMV-EGFP). Pore formation was independent of the targeted location, with both blastomere-yolk interface and blastomere pores competent for delivery. Long-term survival of laser manipulated embryos to pec-fin stage was 89% and 100% for dechorionated and chorionated embryos, respectively. To our knowledge, this is the first report of DNA delivery into zebrafish embryos utilizing fs laser pulses.

Evolutionary crossroads in developmental biology: annelids

“…A recent alternative approach to microinjection has made use of lasers to photoporate material into individual cells of the small marine polychaete P. lamarckii (Torres-Mapa et al, 2011). This approach has the potential to enable fine-scale manipulations of the early embryogenesis of annelids, as well as other taxa (Kohli et al, 2007;Kohli and Elezzabi, 2008).…”

Surgical applications of femtosecond lasers

“…Proof of principle experiments for ablation in zebrafish were published in 2003 and 2007 [79,134]. A couple of papers specify the optimal laser parameters for femtosecond laser optoporation of embryonic cell membranes and the resulting short and long-term viabilities, which are generally above 90% [135,136]. In another study, two-photon calcium imaging identified specific spinal projection neurons whose activity correlate with stimuli evoking turns.…”

Application of dynamic diffractive optics for enhanced femtosecond laser based cell transfection

“…Usually a Titanium-Sapphire laser at a wavelength of approximately 800 nm and pulse duration of $100 fs is tightly focused on the cellular membrane creating a sub-micron pore that lasts a fraction of a second. The unmatched precision of the treatment has enabled a number of challenging biological experiments such as the transfection of embryonic zebrafish [7] and stem cells [8]. It was also shown that a transcription factor Elk1 mRNA optoinjected into the soma of primary neurons causes a different cellular response to mRNA injected into the dendrites [9].…”