title: Electro-microinjection of fish eggs with an immobile capillary electrode.
abstract: Microinjection with ultra-fine glass capillaries is widely used to introduce cryoprotective agents and other foreign molecules into animal cells, oocytes, and embryos. The fragility of glass capillaries makes difficult the microinjection of fish eggs and embryos, which are usually protected by a hard outer shell, called the chorion. In this study, we introduce a new electromechanical approach, based on the electropiercing of fish eggs with a stationary needle electrode. The electropiercing setup consists of two asymmetric electrodes, including a μm-scaled nickel needle placed opposite to a mm-scaled planar counter-electrode. A fish egg is immersed in low-conductivity solution and positioned between the electrodes. Upon application of a short electric pulse of sufficient field strength, the chorion is electroporated and the egg is attracted to the needle electrode by positive dielectrophoresis. As a result, the hard chorion and the subjacent yolk membrane are impaled by the sharp electrode tip, thus providing direct access to the egg yolk plasma. Our experiments on early-stage medaka fish embryos showed the applicability of electro-microinjection to fish eggs measuring about 1 mm in diameter. We optimized the electropiercing of medaka eggs with respect to the field strength, pulse duration, and conductivity of bathing medium. We microscopically examined the injection of dye solution into egg yolk and the impact of electropiercing on embryos' viability and development. We also analyzed the mechanisms of electropiercing in comparison with the conventional mechanical microinjection. The new electropiercing method has a high potential for automation, e.g., via integration into microfluidic devices, which would allow a large-scale microinjection of fish eggs for a variety of applications in basic research and aquaculture.
authors.affiliation: Institute of Industrial Science, The University of Tokyo , Tokyo 153-8505, Japan.
authors.forename: Ryo
authors.initials: R
authors.lastname: Shirakashi
authors.affiliation: Institute of Industrial Science, The University of Tokyo , Tokyo 153-8505, Japan.
authors.forename: Tatsuo
authors.initials: T
authors.lastname: Yasui
authors.affiliation: Lehrstuhl für Biotechnologie und Biophysik, Biozentrum, Universität Würzburg , Würzburg 97074, Germany.
authors.forename: Simon
authors.initials: S
authors.lastname: Memmel
authors.affiliation: Lehrstuhl für Biotechnologie und Biophysik, Biozentrum, Universität Würzburg , Würzburg 97074, Germany.
authors.forename: Vladimir L
authors.initials: VL
authors.lastname: Sukhorukov