Sachihiro Youoku

Automated high-throughput micro-injection system for floating cells

We have developed an automated microinjection system that captures many floating cells and controls capillary positions precisely. To capture many cells simultaneously, we constructed an array of holes on a 10 x 10 mm silicon-based substrate. The hole diameter is 3 μm because our target cells are 10 - 20 μm in diameter. A suction pump connected to the bottom of the multi-hole silicon chip draws the medium into the holes using a slight vacuum, so cells are caught there. Using an initial prototype chip having 121 holes, we captured over 90 cells in a single sweep. Automated microinjection requires precise control of the capillary positions, so images of the capillary and holes on the chip are observed using a microscope with a CCD camera located above the biological medium. The 3D positions of these elements are accurately measured by processing these images. The capillary and the chip are mounted on automatic stages individually controlled with this position data. Using these techniques, this system can microinject about one cell per second. Its success rate for microinjection is 61% for PC12 cells.

Direct core monitoring technique for passive optical alignment between multichannel silicon waveguide and single-mode fiber array

We propose a passive optical alignment method between multichannel silicon waveguides with spot size converters and a single-mode fiber array. In this method, highly accurate positional and angular alignment is realized by a novel monitoring technique of the waveguide and the fiber in one field of view. We achieve a simultaneous accurate multichannel coupling using a developed assembly system.

Automated microinjection system for adherent cells

We have developed an automated microinjection system that can handle more than 500 cells an hour. Microinjection injects foreign agents directly into cells using a micro-capillary. It can randomly introduce agents such as DNA, proteins and drugs into various types of cells. However, conventional methods require a skilled operator and suffer from low throughput. The new automated microinjection techniques we have developed consist of a Petri dish height measuring method and a capillary apex position measuring method. The dish surface height is measured by analyzing the images of cells that adhere to the dish surface. The contrast between the cell images is minimized when the focus plane of an object lens coincides with the dish surface. We have developed an optimized focus searching method with a height accuracy of ±0.2 um. The capillary apex position detection method consists of three steps: rough, middle, and precise. These steps are employed sequentially to cover capillary displacements of up to ±2 mm, and to ultimately accomplish an alignment accuracy of less than one micron. Experimental results using this system we developed show that it can introduce fluorescent material (Alexa488) into adherent cells, HEK293, with a success rate of 88.5%.

Development of microchannel device for automated micro-injection

We have developed a microchannel device and a technique for automated microinjection, through which DNA molecules can be delivered to living cells. Microinjection is a reliable way of introducing DNA and various compounds for new drugs into many kinds of cells. However, it is tedious because all the operations, such as holding each cell with a micropipette, positioning it, and injecting the materials, have to be done manually. This is why we have developed the microchannel device and use it in conjunction with the cell manipulation and trapping techniques. Cells flow in a suspension liquid and are trapped when suctioned through a microhole at the bottom of the microchannel. We can automatically trap cells and inject individual DNA molecules. The microchannel device is made of a 100 x 50 mm cross section of silicon rubber. A micro hole is drilled to a minimum diameter of 3mm by excimer laser ablation on a polycarbonate plate. A glass capillary filled with DNA is inserted in the trapped cell from the upper side of the microchannel. We verified the basic operation of the microchannel device in an experiment using white blood corpuscles (K562 cell line) of about 15mm in diameter.