A Fully Automated Robotic System for Microinjection of Zebrafish Embryos

As an important embodiment of biomanipulation, injection of foreign materials (e.g., DNA, RNAi, sperm, protein, and drug compounds) into individual cells has significant implications in genetics, transgenics, assisted reproduction, and drug discovery. This paper presents a microrobotic system for fully automated zebrafish embryo injection, which overcomes the problems inherent in manual operation, such as human fatigue and large variations in success rates due to poor reproducibility. Based on computer vision and motion control, the microrobotic system performs injection at a speed of 15 zebrafish embryos (chorion unremoved) per minute, with a survival rate of 98% (n = 350 embryos), a success rate of 99% (n = 350 embryos), and a phenotypic rate of 98.5% (n = 210 embryos). The sample immobilization technique and microrobotic control method are applicable to other biological injection applications such as the injection of mouse oocytes/embryos and Drosophila embryos to enable high-throughput biological and pharmaceutical research

Portable Thermoelectric Refrigerator

This project created a versatile thermoelectric refrigerator that can be used via a wall outlet. The product is durable and effective. The refrigerator is functional in any ambient temperature, and uses thermoelectric cooling, and work from a standard 120V power outlet. By understanding existing products’ limitations and strengths, this project produced a product that outperforms what’s currently on the market. Many of the existing products’ operating temperature is dependent on the ambient temperature of the surroundings, which is a large drawback. Additionally, most of them take a long time to reach their lowest temperature, around 3 hours. One team member, Josh DiMaggio, did research with the Cal Poly Physics department over the Summer of 2017 on the effectiveness of Peltier devices in refrigeration. Josh discovered that when Peltiers are thermally in series they can create a much larger temperature difference. A Peltier is a thermoelectric cooling device, therefore satisfying the design requirement. Key design challenges included the following: heat transfer through the insulation, power draw, thermodynamic efficiency, structure strength, intuitive interface, along with other crucial factors. Following design, the refrigerator was built, tested, and the preliminary analysis was validated against the test results. There are plenty of existing patents regarding thermoelectric cooling, but only a few like what this project achieved. The final design is presented in this document

Recognition of zebrafish embryo structures.

<p>(A) After pre-processing. (B) Recognized chorion, cytoplasm center, switching point, and yolk/cell interface.</p

The structure of a zebrafish embryo.

<p>Although the embryo is relatively large, it is highly deformable and care must be taken in injection to avoid cell damage.</p

Control program interface with an array of embryos immobilized on the embryo holding device.

<p>The embryo image was taken under 0.7×. For fully automated injection, the system-level command buttons enable the user to start, pause/resume, terminate, and reset the system.</p

Statistics of ntl-MO injection.

<p>Statistics of ntl-MO injection.</p

Vacuum-based embryo holding device.

<p>Embryos are immobilized on individual through holes via a negative pressure. Extra embryos are flushed off the device. (A) Picture of a device (5×5 holes). (B) An array of immobilized embryos with continuous injection path labeled.</p

Automatic cell injection system.

<p>Microrobot-A and microrobot-B, which are three-degrees-of-freedom motorized micromanipulators with a travel of 25 mm and a 0.04 µm positioning resolution along each axis, control the position of embryos and micropipette, respectively. The system obtains visual feedback through the camera and microscope. The computer-controlled pico-injector provides positive pressure for material deposition.</p

Statistics of fluorescent dye injection.

<p>Statistics of fluorescent dye injection.</p

Illustration of the automated injection flow.

<p>Except for the task of bringing next embryo into the field of view (from E to F), control of both microrobots is based on “looking-then-moving”. Top row: 3-D view. Bottom row: microscopic (image) 2-D view. (A) The vertical height of the micropipette tip is determined with a computer vision approach. This step is required only once at the beginning of one batch. (B) Micropipette at the <i>home</i> position. The white curve outlines the recognized cytoplasm contour. The white dot represents the cytoplasm center. (C) Embryo is brought to the center of the field of view. Micropipette is positioned at the <i>switching</i> point. (D) Micropipette tip penetrates the embryo and deposits materials at a pre-set destination in a specified volume. (E) Micropipette retracts out of the embryo. (F) Micropipette returns to the <i>home</i> position, and the next embryo is brought into the field of view. From (B) to (C), and from (E) to (F), the two microrobots move in parallel to increase injection throughput.</p