Abstract:
An automated cell injection system and method are described, which can perform automatic, reliable, and high-throughput cell injection of foreign genetic materials, proteins, and other compounds. The system and method overcome the problems inherent in traditional manual injection that is characterized by poor reproducibility, human fatigue, and low throughput. The present invention is particularly suited for zebrafish embryo injection but can be readily extended to other biological injection applications such as mouse embryo,  drosophila  embryo, and  C. elegans  injections, capable of facilitating high-throughput genetic research at both academic and industry levels. A novel vacuum based cell-holding device is also provided.

Description:
This application claims the benefit of Canadian Patent Application No. 2,560,352, filed 21 Sep. 2006. 
     FIELD OF THE INVENTION 
     The present invention relates to cell manipulation, automation, micromanipulation and microrobotics. 
     BACKGROUND OF THE INVENTION 
     Recent advances in molecular biology, such as the creation of transgenic organisms, demonstrate that increasingly complex micromanipulation strategies are required for manipulating individual biological cells. In order to create transgenic organisms such as those for cancer studies, genetic materials need to be injected into cells. Conventionally, cell injection has been conducted manually; however, long training, low throughput, and low success rates from poor reproducibility in manual operations call for the elimination of direct human involvement and fully automated injection systems. 
     The zebrafish has emerged as an important model organism for development and genetic studies, due to the similarities in gene structures to the human being, external fertilization and development, short development period, and the transparency of embryos making it easy to observe the fate of individual cells during development. The recent growth in the number of laboratories and companies using zebrafish in vertebrate developmental genetics has been exponential. The injection of thousands of zebrafish embryos is required on a daily basis in a moderate-sized zebrafish genetics laboratory/company, for applications such as embryonic development studies and mutation screening to identify genes. The laborious manual injection task easily causes fatigue in injection technicians and hinders performance consistency and success rates. The current manual technology is not capable of meeting the needs of such high-throughput applications. 
     Currently, no automated, high-throughput zebrafish embryo injection systems are available. Many attempts have been made to leverage existing technologies, such as microrobotics and MEMS (microelectromechanical systems), to facilitate the process of cell injection. Microrobot-assisted (i.e. teleoperated) cell injection systems have been developed, where microrobots/micromanipulators are controlled by the operator to provide “steady hand” and conduct “human-in-loop” cell injections. (See R. Kumar, A. Kapoor, and R. H. Taylor, “Preliminary experiments in robot/human cooperative microinjection,” Proc. IEEE International Conf. on Intelligent Robots and Systems, pp. 3186-3191, Las Vegas, 2003; and H. Matsuoka, T. Komazaki, Y. Mukai, M. Shibusawa, H. Akane, A. Chaki, N. Uetake, and M. Saito, “High throughput easy microinjection with a single-cell manipulation supporting robot,”  J. of Biotechnology , Vol. 116, pp. 185-194, 2005.) Although the microrobots can to a certain extent facilitate cell injection by a human operator without long training, the human involvement still exists in the process of cell injection, resulting in a low throughput and reproducibility. 
     A visually servoed microrobotic mouse embryo injection system has been developed, using a holding micropipette for immobilizing a single mouse embryo, and a visually servoed microrobot for automated cell injection. (See Y. Sun and B. J. Nelson, “Biological cell injection using an autonomous microrobotic system,”  Int. J. of Robot. Res ., Vol. 21, pp. 861-868, 2002.) However, switching from one embryo to another was conducted manually, and thus, injection was time consuming. 
     A semi-automated MEMS-based high-throughput  drosophila  embryo injection system was reported recently, where a MEMS microneedle was used as an injector. (See S. Zappe, M. Fish, M. P. Scott, and O. Solgaard, “Automated MEMS-based  drosophila  embryo injection system for high-throughput RNAi screens,” Lap Chip, Vol. 6, pp. 1012-1019, 2006.) A 3-DOF scanning stage was used for locating randomly dispersed embryos that were ‘glued’ on a glass slide, and another 3-DOF motion stage with the injector mounted was employed for injection. One drawback of this system is that manual alignment of the two stages was required before injection. The large alignment error would greatly influence the injection performance. More importantly, the low stiffness of the MEMS injector requires that the hard embryo chorion be removed in order to facilitate the injection, which may affect subsequent embryonic development, making the system unsuitable for zebrafish or mouse embryo injection. Additionally, randomly dispersing embryos slows down the injection speed due to the embryo searching process. 
     A commercial cell injection system has been developed for oocyte injection of  Xenopus laevis  (frog), where oocytes are manually loaded into a standard 96 well plate, an x-y stage is responsible for positioning target cell to the operation area, and a z-motor with an injection micropipette mounted conducts cell injection (ROBOOCYTE™ by Multi Channel Systems MCS GmbH). In this system, introducing oocytes into regular patterned wells is conducted manually, which is tedious and time consuming. The injection accuracy was sacrificed due to the open-loop operation. Without feedback, such as vision, integrated into the control system to improve the positioning accuracy and monitor the injection process, the injection performance is sacrificed and robustness not warranted. 
     U.S. Patent Application No. 20050250197 to Ando et al. discloses a microinjection apparatus and corresponding operation methods. A silicon microfabricated device integrating suction holes is proposed for cell trapping. The deformation of the thin silicon membrane due to an applied suction pressure is compensated for by measuring the height of the membrane with a detection-mark focusing technique. The silicon substrate is not optically transparent, making the observation, monitoring, and control of the injection process difficult. 
     U.S. Patent Application No. 20050250197 also proposes two methods for measuring the vertical distance between the micropipette tip and substrate surface, using the mirror effects of well-polished silicon surface. The methods intend to determine the height information by focusing on certain features, which will be effective only when the depth of focus is small. However, the size of zebrafish embryos requires a relatively low microscopy magnification that inherently has a large depth of focus (hundreds of micrometers). Thus, the detection methods proposed are not suitable to use for zebrafish embryo injection. 
     Targeting high-throughput cell injection, MEMS-based microneedle arrays have been developed to perform parallel cell injection. The paper “An array of hollow micro-capillaries for the controlled injection of genetic materials into animal/plant cells” (K. Chun, G. Hashiguchi, H. Toshiyoshi, H. Fujita, Y. Kikuchi, J. Ishikawa, Y. Murakami, and E. Tamiya, in Proc. IEEE Conf. MEMS, 1999, pp. 406-411) describes a microneedle array-based cell injection system, including a microneedle array injector and a microchamber array for cell trapping. 
     U.S. Pat. No. 5,262,128 to Leighton et al., U.S. Pat. No. 5,457,041 to Ginaven et al., and U.S. Pat. No. 6,558,361 to Yeshurun also disclose microneedle array designs for cell injection use. Although the concept of using microneedle arrays for parallel cell injection is appealing, solutions to several critical issues do not exist. First, precisely aligning microneedles with regularly positioned cells is difficult. Manual alignment (in-plane or x-y alignment) through microscopic observation from an off-optical-axis angle cannot guarantee a high accuracy. Second, determining the vertical distance (out-of-plane or z) between microneedle tips and cells is difficult. Size differences from one cell to another (e.g., zebrafish embryos can differ by 200-300 cm) make vertical alignment/positioning impossible. Automation is not an option. Third, particularly for zebrafish embryo injection, the size of zebrafish embryos requires microneedles with a tip length of ˜600 μm and outer diameter of 5-10 μm throughout the 600 μm length. The injection needles also must be strong enough without buckling under hundreds of microNewton penetration forces during zebrafish embryo injection. These requirements for microneedles make the selection of MEMS-based solutions inappropriate. In summary, parallel injection with MEMS microneedle arrays is not applicable to zebrafish embryo injection. 
     It should be understood that despite their relatively large size (˜600 μm and ˜1.2 mm including chorion), zebrafish embryos have a delicate structure and can be easily damaged. They are also highly deformable, making the automatic manipulation task difficult. Therefore specific difficulties in achieving automated zebrafish embryo injection include: (i) the ability to quickly (i.e. seconds) immobilize a large number of zebrafish embryos into a regular pattern; (ii) the ability to automatically and robustly identify cell structures for vision-based position control (i.e. visual servoing) and account for size differences across embryos; and (iii) the ability to co-ordinately control two motorized positioning devices to achieve robust, high-speed zebrafish embryo injection. 
     In view of the foregoing, what is needed is a system and method for cellular injection that overcomes the limitations of the prior art, such that the system and method feature automation, robustness, high-throughput (including sample positioning), high success rates, and high reproducibility. 
     SUMMARY OF THE INVENTION 
     In one aspect, the present invention is a system for automated cellular injection comprising: a first positioner control device operable to control motion of a first positioner, the first positioner connected to a holding device operable to immobilize one or more cells in a desired position, the one or more cells including a target cell; a second positioner control device operable to control motion of a second positioner, the second positioner connected to an injection means, the injection means having a tip; a pressure unit connected to the injection means, the pressure unit operable to pass a desired deposition volume of a material at a desired injection pressure to the injection means; and a microscope means for viewing the position of the injection means relative to the holding device; wherein the first positioner control device, the second positioner control device, the pressure unit and the microscope means are linked to a host computer, the host computer including control software for motion control and image processing that enables a user to inject the material into the target cell through the tip of the injection means. 
     In another aspect, the present invention is a method for automated cellular injections comprising immobilization, control sequence, and computer vision recognition. According to this method, a number of cells are positioned on a holding device and viewed through a microscope means. Each cell is recognized and centered in the field of view, and the injection means tip is moved to a “switching point” for the target cell, as defined herein. The tip penetrates the chorion of the target cell and deposits material into the cytoplasm of the target cell. The next cell is then brought into the field of view. The cell is recognized, and injection process is repeated until all cells in the batch are injected. In yet another aspect, the present invention provides a contact detection method to establish a home position for the injection means, e.g., a micropipette, in order to avoid unwanted contact between the micropipette tip and the cells when shifting between cells in the injection order. Upon retraction from the cell, the tip is moved to the home position. 
     The present invention allows for precise, highly reproducible deposition of foreign materials into a cell or a yolk of an embryo. Although the present description discusses depositing material into the cytoplasm center for embryos, it should be understood that the present invention is readily adaptable to allow for the deposition of material into other parts of a cell or embryo, as desired. 
     The present invention overcomes the problems of poor reproducibility, human fatigue, and low throughput inherent with traditional manual injection techniques. Besides automating cell injection by replacing human operation with high reliability and success rates, the present invention also provides high reproducibility and enables genuine high-throughput genetic research. The system and method have been implemented for the injection of zebrafish embryos, but can be readily extended to automated injection of other biological entities, such as mouse embryos,  drosophila  embryos, and  C. elegans.    
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
       A detailed description of the preferred embodiments is provided herein below by way of example only and with reference to the following drawings, in which: 
         FIG. 1  illustrates a schematic diagram of components of the high-throughput automated cellular injection system. 
         FIGS. 2(   a )-( d ) illustrate schematic diagrams of a zebrafish embryo holding device from a top view, bottom view, A-A section view and B-B section view, respectively. 
         FIG. 3  illustrates coordinate frames in the present invention. 
         FIG. 4  illustrates an image projection model relating camera/image frames. 
         FIGS. 5(   a )-( h ) illustrate micropipette motion sequences for injecting each embryo. 
         FIGS. 6(   a )-( e ) illustrate embryo injection sequences and through-hole configuration. 
         FIG. 7  illustrates automatic injection control flow. 
         FIG. 8  illustrates a micropipette moving in the image plane. 
         FIG. 9  illustrates contact between micropipette tip and embryo holding device. 
         FIGS. 10(   a )-( e ) illustrate image processing steps for embryo structure recognition. 
         FIG. 11  illustrates convexity defects between the cell contour and its convex hull. 
         FIG. 12  illustrates parallel task execution of the two positioners. 
         FIG. 13  illustrates alternative injection control flow with an on-line pixel size calibration step. 
         FIG. 14  illustrates an example control program interface. 
     
    
    
     In the drawings, one embodiment of the invention is illustrated by way of example. It is to be expressly understood that the description and drawings are only for the purpose of illustration and as an aid to understanding, and are not intended as a definition of the limits of the invention. 
     DETAILED DESCRIPTION OF THE INVENTION 
     With reference to  FIG. 1 , a system in accordance with the present invention comprises the following main components: 
     (i) Two motorized positioning devices (herein termed ‘positioner’)  1 ,  2 , such as multi-DOF motorized positioning stages or microrobots/micromanipulators that control the motion of embryos and micropipette  8 , respectively. 
     (ii) Control software unit running on a host computer  12  for motion control and image processing. 
     (iii) Positioner control device  13 ,  14  connected to or mounted on the host computer  12  to physically provide control signals to the two positioners  1 ,  2  and the pressure unit  11  (component viii). 
     (iv) An embryo holding device  7  placed on one positioner  1 . 
     (v) An injection means in the form of a micropipette  8  (e.g., glass capillary or microfabricated needle) attached to the second positioner  2 . The tip of the micropipette  8  is preferably about 100 to 800 μm long, and more preferably about 600 μm long, and preferably about 5 to 10 μm in diameter, as an example. The dimensions of a suitable injection means will vary depending on the structure of the target. 
     (vi) An optical microscope (objective  9  and base  3 ). 
     (vii) A CCD/CMOS camera  4  mounted on the optical microscope. 
     (viii) A computer-controlled pressure unit  11 . 
     (ix) A vibration isolation table  15  to minimize vibration (optional). 
     Although this particular configuration of the system relates to the injection of material into zebrafish embryos, it should be expressly understood that this is an illustrative example only and the present invention is readily adaptable for the automated injection of other biological entities such as mouse embryos,  drosophila  embryos, and  C. elegans , or any other appropriate cell as would be recognized and understood by a person of skill in the art. As would be appreciated by a person of skill in the art, the precise techniques of cell immobilization and cell structure would vary for different biological entities. 
     An embryo holding device  7 , either microfabricated or conventionally machined, is used to position a large number of zebrafish embryos into regular patterns.  FIG. 2  shows one example vacuum-based device. The device described in  FIG. 2  has two parts: embryo sucking structure  20 , a flat piece  200  glued on the bottom of the embryo sucking structure  20 . Arrays of through holes  201  are used to immobilize zebrafish embryos with negative pressure applied through the air outlet  202  on the side wall of the chamber  205 . When a large number of zebrafish embryos are dispersed onto the device, each hole immobilizes a single embryo, and the non-trapped embryos are flushed away. Materials to use for constructing the cell holding device are ideally optically transparent, biocompatible, and easy for machining (e.g., polycarbonate). 
     The diameter of the through holes  201  is between 0.4 mm and 0.5 mm, for example. This through hole size is particularly suitable for zebrafish embryos. For mouse embryos, for example, the hole diameter would be smaller, about 20-40 μm, for example. Preferably, the negative pressure applied to immobilizing embryos should be low enough not to cause damage or negative effects for embryonic development. For example, the negative pressure is 0.5-7.5 InHg. 
     A reservoir  204  contains culture media/solution throughout the injection process. A slope  210  on the bottom surface of the holes  201  can be created in order for air bubbles to escape more readily such that they do not stick to the bottom surface. The three airflow channels  208  along the bottom surface are for inducing air to smoothly flow out of the chamber  205  via the air outlet  202 . The air outlet  202  is positioned higher than the slope  210  to guarantee that the slope  210  is submerged in culture media/solution. The steps  209  are created such that the cell holding device can be fixed by two clamps under the microscope. 
     The coordinate frames of the system used in  FIG. 3  and  FIG. 4  are summarized in Table 1. 
     
       
         
               
             
               
               
             
           
               
                 TABLE 1 
               
             
             
               
                   
               
               
                 Coordinate frames (FIG. 3 and FIG. 4) of the system. 
               
             
          
           
               
                 Symbol 
                 Coordinate frame 
               
               
                   
               
               
                 e 
                 End-effector coordinate frame X e -Y e -Z e  attached 
               
               
                   
                 to positioner 2 (micropipette 8 as the end-effector) 
               
               
                 t 
                 Target coordinate frame X t -Y t -Z t  attached to 
               
               
                   
                 positioner 1 that controls the motion of embryos 
               
               
                 c 
                 Camera coordinate frame X c -Y c -Z c   
               
               
                 i 
                 Coordinate frame x i -y i  (or x-y) for the image plane 
               
               
                   
               
             
          
         
       
     
     A point P=(x,y,z) in the camera frame X c -Y c -Z c  is mapped to a point p=(u,v) in the image plane x-y via 
                 [           s   x         0           0         s   y           ]     ⁡     [         u           v         ]       =     [         x           y         ]           
where s x  and s y  are fixed scale factors or pixel size in x-axis (s x ) and y-axis (s y ) respectively that can be either calibrated off-line manually or on-line automatically as discussed later. They will be referred to as s thereafter.
 
Overall Injection Method
 
     A large number of zebrafish embryos are first positioned in a regular pattern on the embryo holding device  7 . The embryos are brought into focus with an auto-focusing algorithm. A vision-based contact detection algorithm determines the vertical positions of the micropipette tip and the top surface of the holding device  7 . Each embryo is recognized and centered in the field of view; simultaneously, the micropipette tip is moved to a switching point. The tip penetrates the chorion  51  and deposits genetic materials into the cytoplasm  52  of the target cell. Upon retreating out of the embryo, the tip is moved to the home position. In the meanwhile, the next embryo is brought into the field of view. The embryo is recognized, and injection process is repeated until all embryos in the batch are injected. 
     Micropipette Motion Control Sequence for Injecting Each Embryo 
     ‘Cytoplasm’ in this invention refers to the combination of the yolk and the cells of an embryo, e.g., a zebrafish embryo. As shown in  FIGS. 5(   a )-( d ), when the micropipette tip is nearly horizontal, the two principal planes  53  and  54  (crossing the cytoplasm center O, parallel to the X e -Z e  and X e -Y e  plane, respectively) overlap at two points. The point that is closer to micropipette  8  is referred to as the switching point S. The motion sequence of the micropipette  8  for injecting one embryo is as follows:
     1. Move from home position (home position, described later in control flow  702  and  703 , is above and to the right of the chorion  51 ) to the switching point S simultaneously along all three axes with positioner  2  ( FIG. 5(   b )). In the meanwhile, the embryo cytoplasm center O is brought to the center of the field of view by positioner  1 .   2. Penetrate the chorion  51  and move to the cytoplasm center O along the X e  direction only ( FIG. 5(   c )). Upon reaching center O, a pre-specified amount of genetic materials (e.g., DNA or morpholinos) is deposited by the computer-controlled pressure unit  11 .   3. Retreat from the cytoplasm center O beyond the switching point S along the X e  direction only ( FIG. 5(   d )).   4. Move to home position ( FIG. 5(   a )). Simultaneously, the next embryo is brought into the field of view by positioner  1 .   

     This invention allows for precise, highly reproducible deposition of foreign materials into the cell or the yolk. The following description assumes that one desires to deposit foreign materials into the cytoplasm center for every embryo. 
     When the micropipette tip has a significant tilting angle (e.g., &gt;5°) as shown in  FIG. 5(   e )-( h ), micropipette motion control sequence can be made slightly different from the above-mentioned in order to minimize cellular damage. The two different steps are: (i) principal plane  54  still crosses cytoplasm center O but is parallel to micropipette tip  8  from the side view; and (ii) during penetration and retraction, the micropipette moves along the direction of its principle axes ( FIG. 5  ( g )-( h )), instead of purely along the X e  direction. 
     Although the following description corresponds to the case shown in  FIG. 5(   a )-( d ), the invention can also be implemented as shown in  FIG. 5(   e )-( h ). 
     Injection Path Planning 
     Denote the pitch (i.e., spacing between two adjacent holes  201 ) along the X t  and Y t  directions as Δx and Δy. Denote the number of embryos along the X t  and Y t  directions as m and n. Starting with the first embryo ( FIG. 6(   a )-( d )), positioner  1  is controlled to travel along the path shown in dashed lines for sequential injection of the entire batch of embryos. Of the four paths when embryos are arranged in regular grids (most often m&gt;2, n&gt;2), given Δy&gt;Δx, path  61  shown in  FIG. 6(   a ) is the shortest. Given Δx&gt;Δy, path  62  shown in  FIG. 6(   b ) is the shortest. In the case of Δy=Δx, the four paths have the same total travel distance. In order to increase throughput, the shortest path should be taken. 
     The through holes  201  can also be arranged into other patterns other than those shown in  FIG. 6(   a )-( d ). For example, every six nearest holes  201  to the middle one can be such configured that they form an equilateral hexagon ( FIG. 6(   e )). Such a configuration achieves a maximum number of holes for a given device surface area, which can be adopted for the purpose of maximizing the number of embryos for each batch. 
     Injection Control Flow 
     After a batch of zebrafish embryos are immobilized on the cell holding device  7 , fully automated operation starts according to the control flow as described in  FIG. 7 . 
     Embryo Auto-Focusing  701 : 
     Prior to autonomous injection, the embryos need to be brought into focus. This auto-focusing step  701  only needs to be conducted once for each batch of embryos. Embryos are servoed by positioner  1  upwards (or downwards) by a certain distance (e.g., 5 mm) to cross the focal plane. An autofocusing algorithm (e.g., Tenenbaum gradient) is used to locate the focal plane by constantly calculating the focus measure for each frame of image. The embryos are moved to the focal plane that corresponds to the maximum (or minimum) focus measure. 
     Identification of Micropipette Tip ROI (Region of Interest)  702 : 
     This step is to locate the tip of the micropipette  8  for use in contact detection  703 . The micropipette  8  controlled by positioner  2  moves only along the Y e  direction. The moving micropipette that stands out in the image subtracted from the background is recognized (i.e., a region of interest  81  around the tip of the micropipette, shown in  FIG. 8  is identified). Upon identification, the coordinates of the tip both in the image plane x-y and in the end-effector frame X e -Y e -Z e  are determined. The x-coordinate and y-coordinate in the image plane x-y, X e -coordinate and Y e -coordinate in the end-effector frame X e -Y e -Z e  are taken as the lateral components of the home position of the micropipette tip. 
     Contact Detection  703  Using Computer Vision Feedback: 
     This step is to automatically align the tip of the micropipette  8  with the embryo cytoplasm center O in the vertical direction. In this procedure, the top surface of the cell holding device  7  serves as the reference plane. The micropipette  8  moves only along the Z e  direction. Upon the establishment of the contact between the micropipette tip and the top surface, further vertical motion of the micropipette tip along the Z e  direction results in lateral movement along the X e  direction. As shown in  FIG. 9 , the micropipette tip is located at point a (initial contact) and b (after contact) in the surface plane. Before and after contact, the micropipette tip changes its x coordinate in the image plane x-y vs. time (i.e., image frame number), resulting in a V-shaped curve. The peak of the V-shaped curve represents the contact position along the vertical direction between the micropipette tip and the top surface of device  7 . 
     After contact detection, the Z e -coordinate of the switching point S is determined by moving upwards with respect to the contact position by half of the embryo diameter, e.g., 0.5-0.6 mm. The Z e -coordinate of the home position of the micropipette tip is determined by moving upwards with respect to the contact position by the embryo diameter, e.g., 1.0-1.2 mm. 
     Upon the completion of  702  and  703 , the home position of the micropipette tip both in the x-y image plane and the X e -Y e -Z e  frame has been automatically determined and will be fixed for use in the following procedures of injecting all embryos within the batch. 
     Moving to the Home Position  704 : 
     After  702  and  703 , positioner  2  following a position control law (e.g., PID) moves the micropipette tip upwards and laterally to its home position determined in  702  and  703  from the vertical contact position in order to prevent the micropipette from crashing with embryos in between injections. 
     Embryo Recognition  705 : 
     The objectives of this step are to identify the cytoplasm center O ( FIG. 10(   a )), the distance from the center O to the switching point S along the x direction, and the injection angle γ between the x axis and the principal axis  104  ( FIG. 10  ( e )). 
     The embryo recognition steps are summarized in Table 2. The complete recognition process typically takes 16 ms on a PC (3.0 GHz CPU and 1 GB memory). 
     
       
         
               
             
               
               
             
           
               
                 TABLE 2 
               
             
             
               
                   
               
               
                 Embryo recognition 705. 
               
             
          
           
               
                 Step # 
                 Processing 
               
               
                   
               
               
                 1 
                 Pre-processing 
               
               
                 2 
                 Chorion 51 recognition 
               
               
                 3 
                 Cytoplasm 52 recognition 
               
               
                 4 
                 Determination of injection angle γ 
               
               
                   
               
             
          
         
       
         
         
           
             (1) Pre-processing. This step is to obtain a de-noised binary image. The image is first convolved with a low-pass Gaussian filter for noise suppression. Then the gray-level image is binarized to a black-white image using an adaptive thresholding method (e.g., setting a local threshold for each pixel as the mean value of its local neighbours). The binary image is eroded to remove small areas that are not of interest and then, dilated to connect broken segments that originally belong to one object. An example image after pre-processing is shown in  FIG. 10(   b ). 
             (2) Chorion  51  recognition. Of many connected objects in the binary image, the one with the maximum area is taken as the chorion  51 . In  FIG. 10(   c ), the chorion  51  is circumvented by its minimum enclosing circle  101 . 
             (3) Cytoplasm  52  recognition. The second largest object in the image shown in  FIG. 10(   b ) is the cytoplasm  52 . Its boundary is represented by a chain code contour. In some cases, the boundary of the cytoplasm  52  is not fully connected (i.e., a rotated ‘C’ shape with an opening other than a fully closed ‘O’ shape). Thus, a convex hull of the contour is used for further processing. 
           
         
       
    
     A region R is convex if and only if for any two points x 1 , x 2 εR, the complete line segment x 1 x 2  with end points x 1  and x 2  is inside the region R. The convex hull of a region is the smallest convex region H that satisfies the condition R ⊂ H. 
     The constructed convex hull of the contour serves as the initial curve for ‘snakes’, which will form a closed curve that represents the contour of cytoplasm  52 . The obtained closed contour  102  by snake tracking is shown in  FIG. 10(   d ). The centroid of the contour is recognized as the cytoplasm center O. 
     The switching point S is then determined as the intersect point of the minimum enclosing circle  101  and the horizontal line passing through the cytoplasm center O, as shown in  FIG. 10(   d ).
         (4) Determination of injection angle γ. Fitting the contour  102  of the cytoplasm  52  into an ellipse  103  using a least squares method results in the major axis of the fitted ellipse  103 , which is taken as the principal axis  104  ( FIG. 10(   e )).       

     In order to determine the injection angle γ that represents the cell orientation, the yolk and the cell must be distinguished. The contour  102  is intercepted into two parts (cell part and yolk part) by the minor axis of the fitted ellipse  103 . Define the area difference between a contour and its convex hull as the convexity defect. The convexity defects ( FIG. 11 ) for the yolk part and cell part are calculated. Based on the fact that the yolk part always has a much more circular shape than the cell part (i.e., smaller defect), the contour with a greater defect is recognized as the cell part, thus, the cell part and yolk part are recognized.  FIG. 10(   e ) shows the principal axis  104  starting from the cytoplasm center O(+) and ending on the cell contour (x). The injection angle γ is the angle between the x axis and the principal axis  104 . As the injection angle γ represents cell orientation, the recognition of γ can also be important for automatically rotating embryos. For example, the angle can be constantly recognized in each frame of image as visual feedback for rotating an embryo such that the yolk part or the cell part can be rotated closed to or away from the micropipette tip. 
     The following two tasks  7061  and  7062  are performed in parallel after task  705 . 
     Centering Embryo  7061 : 
     According to calibrated pixel size s and the distance between the cytoplasm center O and the image center in the image plane, positioner  1  is controlled with a position control law to move the embryo into the image center. 
     Moving the Micropipette Tip to Switching Point  7062 : 
     In parallel with centering embryo  7061 , the micropipette  8  is then moved by positioner  2  from home position to the switching point S by a position control law (e.g., PID). 
     Entry into the Embryo  707 : 
     The micropipette tip is controlled to start from the switching point S to arrive at the cytoplasm center O by a position control law at an appropriate speed that does not cause embryo lysis. 
     Genetic Material Deposition  708 : 
     Based on a desired deposition volume, the micropipette tip size (inner diameter) and specified injection pressure level determine the positive pressure pulse length (i.e., pressure ‘on’ time). Injection pressure is maintained high for the determined time period through the computer-controlled pressure unit  11 , precisely depositing a desired volume of genetic materials at the cytoplasm center O. 
     Exiting from the Embryo  709 : 
     Controlled by positioner  2 , the micropipette  8  is retracted out of the embryo by a position control law at an appropriate speed that does not cause embryo lysis. 
     The following two tasks  7101  and  7102  are performed in parallel. 
     Moving the Next Embryo into the Field of View  7101 : 
     This step brings the next embryo into the field of view (the image plane x-y) according to the pitches between adjacent through holes  201  of the embryo holding device  7 . Traveling the relative displacement (Δx or Δy) is executed by an appropriate position control law, driven by positioner  1 . 
     Moving Micropipette to the Home Position  7102 : 
     In parallel with bringing the next embryo into the filed of view  7101  with positioner  1 , positioner  2  following a position control law moves the micropipette tip upwards and laterally to its home position determined in  702  and  703 . 
     Repeat  705 - 7061 - 7062 - 707 - 708 - 709 - 7101 - 7102  for Each Embryo: 
     In order to achieve the highest throughput, for injecting each embryo, the two positioners  1 ,  2  perform tasks in parallel whenever possible, as shown in  FIG. 12 . Performing tasks in parallel operation is an effective approach to enhance the efficiency of the system. 
     An Alternative Injection Control Flow 
     The control flow described in  FIG. 7  requires a prior knowledge of pixel size s that is obtained through off-line pixel size calibration. The pixel size s varies with different microscopy magnifications that are typically determined by microscope objectives, couplers, and the camera. In order to eliminate the magnification/hardware dependence, on-line calibration can be conducted to automatically determine the pixel size. Accordingly, the control flow is modified ( FIG. 13 ), particularly, for the operation on the first embryo when on-line pixel calibration is conducted. 
     Comparing the control flow shown in  FIG. 7  and the flow shown in  FIG. 13 , one can see that  7061  is replaced with task  712 . Also note that  712  is not performed in parallel with  7062 . 
     Centering Embryo, Visual Servo Control  712 : 
     Unlike  7061 ,  712  visually servos the cytoplasm center O to the center of the field of view. The cytoplasm center O recognized in step  705  is selected as the image feature for tracking and a visual tracking method (e.g., sum-squared-difference) is applied. The cytoplasm center O is continuously tracked, providing visual feedback to the image-based visual servo control loop. Based on the visual tracking results (i.e., pixel displacement in the image plane x-y) and the position feedback from positioner  1  (i.e., travelling distance in the frame X t -Y t -Z t ), the pixel size s is calibrated on line. 
     System Robustness Enhancement 
     Error-free operation is critical to warrant the commercial viability of the system. From the perspective of robustness enhancement, the system features an error protection mechanism. Table 3 summarizes potential errors that can occur during operation and their detection methods. When any error is detected, the system is halted with alarms sounded to alert the user and detailed error messages reported to the user. 
     In control software design that implements the control flow described in  FIG. 7  or  FIG. 13 , the detection methods must also be implemented as integrative components for system robustness enhancement. 
     
       
         
               
             
               
               
             
           
               
                 TABLE 3 
               
             
             
               
                   
               
               
                 Potential errors and corresponding detection methods. 
               
             
          
           
               
                 Error description 
                 Detection method 
               
               
                   
               
               
                 1. Hardware problems: 
                 Query camera, positioner control device, communication channel upon 
               
               
                 failure in establishing 
                 launching the control software. 
               
               
                 communication with 
               
               
                 controllers, error in 
               
               
                 positioner control device, 
               
               
                 etc. 
               
               
                 2. Lighting problem: light 
                 Calculate the average gray-level and standard deviation of a random frame 
               
               
                 off or poor illumination 
                 of image. If the two parameters are both lower than a pre-set threshold 
               
               
                   
                 value, for example, both are too low, lighting problem most probably exist. 
               
               
                 3. Failure in detecting 
                 In most cases, within several frames (e.g., 5–10, depending on the positioner 
               
               
                 micropipette tip ROI in 
                 2 speed and microscopy magnification) the micropipette tip can be detected. 
               
               
                 702 
                 If the tip is not detected within 20 frames of images, failure most probably 
               
               
                   
                 has occurred. 
               
               
                 4. Error in contact 
                 The micropipette tip can be too small a feature to be recognized with a 
               
               
                 detection 703 
                 noisy background. If the tip cannot be constantly detected in any frame of 
               
               
                   
                 image when the tip is being lowered down, an error should have occurred. 
               
               
                 5. Embryo recognition 
                 In each of the following cases, an error is detected: 
               
               
                 error 
                 (1) If the radius of the minimum enclosing circle for the chorion is too 
               
               
                   
                 large, for example, exceeding half of the image width in pixel, or is too 
               
               
                   
                 little. 
               
               
                   
                 (2) If the recognized cytoplasm center is outside the minimum enclosing 
               
               
                   
                 circle. 
               
               
                   
                 (3) If the recognized cytoplasm center is too close to the chorion. 
               
               
                   
                 (4) If the fitted ellipse of the cytoplasm contour has a minor axis greater 
               
               
                   
                 than ⅔ diameter of the minimum enclosing circle. 
               
               
                 6. Micropipette tip 
                 After injecting a number of embryos (e.g., a batch of 25), micropipette 
               
               
                 clogging 
                 clogging should be inspected. The pressure unit 11 pushes genetic 
               
               
                   
                 materials (e.g., DNA) out of the micropipette tip to form a sphere. Based 
               
               
                   
                 on image processing, if no sphere is formed or a reduced radius is identified 
               
               
                   
                 compared to the clogging-free case, clogging should have occurred. 
               
               
                   
                 Alternatively, for each embryo within a batch, negative pressure applied by 
               
               
                   
                 the pressure unit 11 can be measured constantly (e.g., with a pressure sensor 
               
               
                   
                 integrated in the pressure path), an abnormal pressure value indicates partial 
               
               
                   
                 or complete clogging of the micropipette. Using the pressure monitoring 
               
               
                   
                 approach, micropipette breakage can also be detected. 
               
               
                 7. Micropipette buckling 
                 Buckling can occur under one of the following conditions: (1) micropipette 
               
               
                   
                 tip is misaligned with the embryo; (2) embryo is not fully immobilized; (3) 
               
               
                   
                 in rare cases, chorion of the embryo is exceptionally stiff and much harder 
               
               
                   
                 to penetrate. Micropipette buckling can be detected by visually monitoring 
               
               
                   
                 if the micropipette contour changes from a straight line to a curve pattern. 
               
               
                 8. Reaching motion limit 
                 The first method is to constantly check the position feedback from the 
               
               
                 of positioners 
                 positioners. The second method is to monitor overall image changes when 
               
               
                   
                 the positioners are supposed to move. Little or no image pattern changes 
               
               
                   
                 indicate that the positioners could have reached limit. 
               
               
                 9. On-line calibration 
                 Before on-line calibration is conducted, if the cytoplasm center of the first 
               
               
                 error 
                 embryo is exactly at or extremely close to the image plane center, on-line 
               
               
                   
                 calibration can result in significant error (divide-by-zero). 
               
               
                   
               
             
          
         
       
     
     The system is capable of automatically inject embryos sequentially for a complete batch. It also allows only injecting selected embryos within a batch. For example, in one user-friendly control interface shown in  FIG. 14 , area  141  provides an interactive means for the user to select embryos from a batch for injection by clicking the circles (circle positions correspond to embryo positions), besides displaying the current operation status (different color indicates completed, on-going, or to be conducted). 
     It will be appreciated by those skilled in the art that other variations of the preferred embodiment may also be practiced without departing from the scope of the invention. 
     The high-throughput automated cellular injection system described herein has at least the following general advantages:
     i) high success rate;   ii) high reproducibility (because the embryo structure is fully recognized, the deposition target can be selected other than the cytoplasm center O);   iii) high-throughput;   iv) fast embryo immobilization;   v) low-cost, biocompatible, optically transparent embryo holding device that produces high image quality for image processing/pattern recognition;   vi) fully automatic contact detection, facilitating precise alignment of the micropipette tip and embryo center in height;   vii) optimized embryo injection path to shorten positioners&#39; total travel distance;   viii) robust image processing methods;   ix) optical platform (e.g., microscopy magnification) independence, enabled by the on-line pixel size calibration technique;   x) automated, precise material deposition using a computer controlled pressure unit;   xi) enhanced robustness due to error detection mechanisms; and   xii) user-friendly control program interface providing operation flexibility and process monitoring.