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Top1. Introduction
Zebrafish has emerged as a significant model organism for molecular biology and drug research, enjoying advantages of similarities in the major gene to humans, external fertilization and development, short development period, and the transparency of embryos, making it convenient to observe the fate of individual cells during development (Monte Westerfield, 2000). Compared to other techniques of introducing foreign materials into a zebrafish embryo, such as electroporation (Rols, 2006), viral vectors (W. Walther, 2000), ultrasonic (J. Sundaram, 2004), gene gun(Lin MTS, 2000) and MEMS-based injection(Hashmi, 1995), microinjection remains the most effective in terms of injection material preparation, cell damage and most important, the efficiency of delivery.
Traditionally, zebrafish embryo injection is conducted manually, requiring a proficient operator to complete numerous repetitive tasks under microscope. To fulfill one single successful injection, firstly the embryo must be carefully moved to appropriate position by one hand with unremitting eye’s feedback. Then the micropipette held by another hand is transferred close to the embryo and finally pierced into the embryo using a gingerly controlled force, otherwise the delicate construction of embryo maybe destroyed. Further, the material must be delivered quickly employing a microinjector, usually triggered by foot pedal, therefore the force of foot also required to be steady for uniform injection volume. Coordination of eye, hand and foot as well as their respective precise control and being labor intensive are huge challenges to successful embryo injection, not to mention batch injection. The fatigue can also lead to low success rate and excessive human participation may introduce contamination. Therefore, an automated system capable of batch zebrafish embryo injection is desired.
Efforts in automating cell injection have been continuous, resulting many excellent achievements from tele-operated system (Mattos, 2007; Matsuoka, 2003) to semi-automated system(Zappe, 2006) and automated system(Zhe Lu, 2011; Yu Sun, 2002). An automated system for zebrafish (Wenhui Wang, 2007) embryo injection has been reported providing high performance. However, this two system relied on a special embryo holding device to get the embryo strictly immobilized. Similarly, the automatic batch injection system reported by Zhe Lu et al. needed to mount the petri dish on a tilted holder to fulfill injection (Zhe Lu, 2007). Various sensors, e.g. force and position sensor, have been utilized to fulfill robot-assisted microinjection(Yu Xie, 2011; Huang, 2009). Piezoelectric driven injector has also been introduced for robot-assisted microinjection on zebrafish embryos(Huang, 2011). Despite the great contribution to automating cell microinjection, these systems are dependent to kinds of extra equipment like cell holding devices and sensors or other tools, which will increase the spending, decrease the compatibility, and even may introduce unpredictable injury to delicate cell structure. Further, there’s an important issue that have not been thoroughly discussed in above mentioned works: quantitative microinjection control. Appropriate and precise injection volume is a key element of successful microinjection(Cid-Arregui, 1998). However, only a few of these systems adopt some very expensive commercial microinjectors, yet the quantitative injection effect has not been inspected in the experiment. Addressing these challenges, an automatic operating process for zebrafish embryo injection is proposed in this paper, with ability to accomplish quantitative injection on nanoliter level, and dependent only on common devices in zebrafish embryo injection. Firstly, technologies of the automatic operating process are introduced. Then the overall operating process design leveraging the key technologies of computer vision and micromanipulation robot control. Lastly, the evaluation experiment, together with the application of fluorescent material and drug investigation verified the feasibility and efficiency of the automatic operating process for zebrafish embryo injection.