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

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

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