WO2009079474A1

WO2009079474A1 - Methods, systems, and computer readable media for facilitating automation of blastocyst microinjection

Info

Publication number: WO2009079474A1
Application number: PCT/US2008/086871
Authority: WO
Inventors: Edward Grant; Leonardo Serra Mattos; Randy Thresher
Original assignee: The University Of North Carolina At Chapel Hill
Priority date: 2007-12-14
Filing date: 2008-12-15
Publication date: 2009-06-25

Abstract

Methods, systems, and computer readable media for facilitating automation of blastocyst microinjection are disclosed. According to one method, a target blastocyst is automatically fed, from among a plurality of blastocysts, into a blastocyst microinjection area. The target blastocyst is automatically held in a fixed position within the blastocyst microinjection area and oriented so as to be suitable for injection. One or more cells are injected into the target blastocyst. After injection of the one or more cells, the target blastocyst is automatically released and removed from the blastocyst microinjection area.

Description

DESCRIPTION

METHODS, SYSTEMS, AND COMPUTER READABLE MEDIA FOR FACILITATING AUTOMATION OF BLASTOCYST MICROINJECTION

RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Patent Application Serial No. 61/007,701 filed December 14, 2007; the disclosure of which is incorporated herein by reference in its entirety.

TECHNICAL FIELD

The subject matter described herein relates to blastocyst microinjection. More specifically, the subject matter relates to methods, systems, and computer readable media for facilitating automation of blastocyst microinjection.

BACKGROUND

Microinjection of materials into target cells, such as blastocysts, has conventionally been a manual process. In conventional microinjection, trained experts place a target cell into an injection area that is viewed with the aid of a microscope. The target cell is then oriented and held in place, typically using a holding pipette exerting suction on the target cell, while the operator injects other cells, such as embryonic stem (ES) cells, through appropriate portion of the cellular wall. This is accomplished using a small needle (e.g., injection pipette) containing the material to be injected capable of penetrating the target cell wall. The injection pipette is manually controlled in the x, y, and z directions using a manual control, such as a joystick. After injection, the target cell is removed from the injection area, placed back in the pool of target cells, and a next target cell is obtained.

Conventional microinjection is prone to human error. For example, inserting the injection pipette into the cell with too much force may damage the cell making it unsuitable for further use. Inserting the injection pipette into an incorrect portion of the cell wall may also result in damage to the cell (e.g., improper orientation of the cell). Additionally, conventional microinjection is a slow and tedious process resulting in an expensive process. For example, manually orienting a cell, locating a candidate cell from among a pool of cells, and manipulating the injection pipette are slow procedures. These problems have conventionally been addressed by providing extensive training for human operators in order to build a network of experienced operators. While such operators generally do perform higher quality injections in less time than inexperienced operators, the cost of training operators is high. Accordingly, in light of these difficulties, a need exists for improved methods, systems, and computer readable media for facilitating automation of blastocyst microinjection.

SUMMARY Methods, systems, and computer readable media for facilitating automation of blastocyst microinjection are disclosed. According to one method, a target blastocyst is automatically fed, from among a plurality of blastocysts, into a blastocyst microinjection area. The target blastocyst is automatically held in a fixed position within the blastocyst microinjection area and oriented so as to be suitable for injection. One or more cells are injected into the target blastocyst. After injection of the one or more cells, the target blastocyst is automatically released and removed from the blastocyst microinjection area.

A system for facilitating automation of blastocyst microinjection is also disclosed. The system includes a feeder pipette for automatically feeding a target blastocyst from among a plurality of blastocysts into blastocyst microinjection area. A holding pipette automatically holds the target blastocyst in a fixed position within the blastocyst microinjection area and releases the target blastocyst from the blastocyst microinjection area after injection of the one or more cells. An injection pipette orients the target blastocyst so as to be suitable for injection and injects one or more cells into the target blastocyst. A collection pipette automatically removes the target blastocyst from the blastocyst microinjection area after injection of the one or more cells.

A system-on-a-chip for facilitating automation of blastocyst microinjection is also disclosed. The system includes an integrated circuit having a plurality of channels forming a fluidic communication path for blastocysts and at least one blastocyst microinjection area in the fluidic communications path. The integrated circuit forms a suction hole in the blastocyst microinjection area for holding the blastocyst in place. At least one electrode is configured to electrorotate the blastocyst so as to be suitable for injection. A microelectromechanical (MEMS) needle injects one or more cells into the target blastocyst. A sensor observes the position and orientation of the injection area.

The subject matter described herein for facilitating automation of blastocyst microinjection may be implemented using a computer program product comprising computer executable instructions embodied in a tangible computer readable medium that are executed by a computer processor. Exemplary computer readable media suitable for implementing the subject matter described herein includes disk memory devices, programmable logic devices, and application specific integrated circuits. In one implementation, the computer readable medium may include a memory accessible by a processor. The memory may include instructions executable by the processor for implementing any of the methods for routing a call described herein. In addition, a computer readable medium that implements the subject matter described herein may be distributed across multiple physical devices and/or computing platforms.

BRIEF DESCRIPTION OF THE DRAWINGS

The subject matter described herein will now be explained with reference to the accompanying drawings of which: Figure 1 is a diagram of an exemplary four-pipette system for facilitating automation of consecutive blastocyst microinjection according to an embodiment of the subject matter described herein; Figure 2A is a diagram showing exemplary steps for performing an automated microinjection task according to an embodiment of the subject matter described herein;

Figure 2B is a flow chart of an exemplary process for facilitating automation of blastocyst microinjection according to an embodiment of the subject matter described herein;

Figure 3 is a flow chart of an exemplary blastocyst delivery process for facilitating automation of blastocyst microinjection according to an embodiment of the subject matter described herein; Figure 4 is a flow chart of an exemplary blastocyst capture process for facilitating automation of blastocyst microinjection according to an embodiment of the subject matter described herein;

Figure 5 is a flow chart of an exemplary blastocyst release process for facilitating automation of blastocyst microinjection according to an embodiment of the subject matter described herein;

Figure 6 is a flow chart of an exemplary blastocyst orientation process for facilitating automation of blastocyst microinjection according to an embodiment of the subject matter described herein;

Figure 7 is a flow chart of an exemplary blastocyst collection process for facilitating automation of blastocyst microinjection according to an embodiment of the subject matter described herein;

Figure 8 is a flow chart of an exemplary process for facilitating automation of blastocyst microinjection according to an embodiment of the subject matter described herein; Figure 9 is an exemplary image data showing a blastocyst microinjection area before and after image processing for facilitating automation of blastocyst microinjection according to an embodiment of the subject matter described herein;

Figure 10 is a diagram of an exemplary control system for providing pipette motion control for facilitating automation of blastocyst microinjection according to an embodiment of the subject matter described herein; Figure 11 is a flow chart of an exemplary embryonic stem cell delivery process for facilitating automation of blastocyst microinjection according to an embodiment of the subject matter described herein;

Figure 12 is a diagram showing an exemplary system for counting of the number of delivered embryonic stem cells for facilitating automation of blastocyst microinjection according to an embodiment of the subject matter described herein;

Figure 13 is a block diagram of an exemplary system for facilitating automation of blastocyst microinjection according to an embodiment of the subject matter described herein;

Figures 14A and 14B are diagram of an exemplary physical arrangement of a system for facilitating automation of blastocyst microinjection according to an embodiment of the subject matter described herein; Figure 15A is an overhead view of a system-on-a-chip system for facilitating automation of blastocyst microinjection according to an embodiment of the subject matter described herein; and

Figure 15B is cross-sectional view of a system-on-a-chip system for facilitating automation of blastocyst microinjection according to an embodiment of the subject matter described herein.

DETAILED DESCRIPTION

Figure 1 is a diagram of an exemplary system for facilitating automation of consecutive blastocyst microinjection that includes four pipettes. Conventionally, blastocyst microinjection has been accomplished using a two-pipette system wherein a holding pipette is used to obtain a target blastocyst from a collection of possible blastocysts, held in place while an injection pipette is used to inject cells (e.g., embryonic stem cells) into the blastocyst. Upon completion of injection, the blastocyst is returned to the collection of blastocysts and a second blastocyst may be retrieved. As described above, this process is labor intensive, slow, and prone to error.

System 100 shows a four-pipette system which adds two pipettes (i.e., a feeder pipette and a collection pipette) to conventional holder pipette and injection pipette systems. Pipettes included within system 100 may be associated with micropositioners, computer-controlled micrometer syringes, and other related components (not shown) for facilitating automation of consecutive blastocyst microinjection. Referring to system 100, feeder pipette 102 may obtain a single blastocyst 104 from a collection of blastocysts and deliver blastocyst 104 to injection area 105. For example, in order to accommodate typical sizes of blastocyst 104, the diameter of feeder pipette 102 may be approximately 120um to allow for the entry of a single blastocyst without distortion. Upon entering injection area 105, blastocyst 104 may be temporarily fixed to holding pipette 106 via suction. Next, blastocyst 104 may be oriented so as to be suitable for injection. One exemplary technique may include the push-pull technique for rotating blastocyst 104 so as to orient a portion of the cell wall of blastocyst 104 opposite holding pipette 106. Injection pipette 108 may then be inserted into blastocyst 104 and one or more cells 110 may be injected into blastocyst 106. Finally, holding pipette 106 may release blastocyst 104, and collection pipette may remove blastocyst 104 from injection area 105.

The addition of feeder pipette 102 and collection pipette 112 to microinjection system 100 simplifies the design and implementation of control algorithms for automation. Simplification occurs because feeding and collecting blastocysts directly to and from the injection area avoids the need to move the XY stage to search for them. Consequently, system 100 reduces the number of control variables in the system by reducing the number of moving parts during microinjections. Additional pipettes 102 and 112 also simplify the requirements for video processing algorithms by keeping the injected and the non-injected blastocysts separated from each other. Therefore, vision algorithms are not required to differentiate between multiple blastocyst states (e.g., pre-injection and post-injection). In addition, system 100 may be capable of processing multiple consecutive blastocyst microinjections (i.e. a production line) without user intervention. Such a configuration may reduce the amount of time required for microinjections, thus contributing to improved productivity. Additional inputs for user control of feeder and collection micrometer syringes may, for example, be implemented using keys of a computer keyboard for providing additional user control in semi-automated embodiments.

In addition to the advantages described above, four-pipette system 100 described above has the potential to speedup multiple blastocyst microinjections by avoiding the need to search for the embryos (i.e., the working well) since they are kept at known locations and by avoiding the need to identify non-injected blastocysts because injected and non-injected blastocysts are kept separated from each other. System 100 was evaluated by conducting microinjection experiments under teleoperated manual control. The performed experiments showed that system 100 may be appropriate for automated blastocyst microinjections. First, it was found that applying suction to the holding pipette prior to the delivery of a blastocyst, the blastocyst was captured immediately once it was fed to the injection area, i.e., as soon as it exits the feeder pipette, a similar result was observed for blastocyst collection, i.e., the creation of suction on the collection pipette prior to the blastocyst's release from the holder causes it to move directly from one pipette to the other once it is released.

In comparative studies, the performance of a semi-automated (joystick teleoperated) embodiment of microinjection system 100 was tested against a traditional manual microinjection system. There was a dramatic reduction in operator training time and there was an associated increase in transgenic mice Chimera birth rates. Microinjection success rates, commonly between 40%-70% were greater than 80% for both the experienced expert and the novice using this new system. Similarly, the performance of a fully-automated embodiment of microinjection system 100 was tested against a traditional manual microinjection system where a high- level control system automatically penetrated and extracted the injection pipette during blastocyst microinjection. The automated system was 75% successful in the injection process and the injected blastocysts were implanted and developed to term. The automated microinjection system gave a 53.3% birth rate and yielded 20% Chimeras. Figure 2A is a diagram showing exemplary steps for performing an automated microinjection task according to an embodiment of the subject matter described herein. Referring to Figure 2A, an automate microinjection task may begin at block 200 wherein a blastocyst is delivered to the injection area. In block 202, the blastocyst may be captured (i.e., held in place by holding pipette 106) and oriented in block 204. Next, injection process 206 may be composed of several steps. For example, in blocks 208 and 210, an injection needle may approach and penetrate the target blastocyst, respectively. This process may be aided by using image analysis techniques that enhance or highlight the boundaries of the injection needle and the blastocyst cell wall, as well as control techniques for accurately controlling the movement of the injection needle. This is illustrated in block 210 as pipette motion control, which will be described in greater detail below. After penetration of the blastocyst, cells may be injected into its interior in block 214. The injection needle may then be retracted in step 216, with the help of pipette motion control 210. Finally, the blastocyst may be released in step 218, and collected with other blastocysts which have been previously injected in step 220.

Figure 2B is a flow chart of an exemplary process for facilitating automation of blastocyst microinjection according to an embodiment of the subject matter described herein. The steps shown in Figure 2B may refer to software routines executable by a processor of a computer. Referring to Figure 2B, beginning at step 222, an auto-inject command may initiate the automatic injection of consecutive blastocysts. This may be aided, as shown in step 224, by pre-loading a feeder pipette with multiple blastocysts such that the system may repeat the injection process for each blastocyst without user intervention. Initially, the injection pipette may be moved to a "home" position corresponding to a known, pre-determined location relative to the injection area as well as other components such that distances may be easily calculated for performing accurate pipette motion control.

Beginning at step 226, a procedural loop may begin by delivering a next blastocyst from among a plurality of blastocysts queued in the feeder pipette to the injection area. In step 228, the blastocyst may be captured by the holding pipette. In step 230, the blastocyst may be oriented if necessary using one or a variety of possible techniques. The blastocyst is then injected in step 230 and collected (i.e., removed from the injection area and placed in a collection area) in step 234. While the process shown in Figure 2B may be fully automated, in the event of errors, it may be advantageous to provide an opportunity between blastocyst injections to allow for user input and/or correction. Thus, step 236 allows for the user to continue and repeat the automated injection process for the next blastocyst or allow them to exit the process and perform some action. Accordingly, if the user continues the process, control returns to step 226 and alternatively, may end at step 238.

Figure 3 is a flow chart of an exemplary blastocyst delivery process for facilitating automation of blastocyst microinjection according to an embodiment of the subject matter described herein. Referring to Figure 3, at step 300, a feed blastocyst command may feed a single blastocyst into the injection area. For example, multiple blastocysts may be queued in feeder pipette and dispensed into the injection area individually. The delivery of a blastocyst to the injection area may be automated by commanding the feeder micrometer syringe to slowly rotate clockwise until a blastocyst was delivered and positively identified in the injection area. In step 302, updated image data of the blastocyst is obtained. For example, the position and orientation of the blastocyst may be determined by detecting the boundaries of the blastocyst relative to the known boundaries of the XY stage using an optical sensor, such as a microscope and associated CCD camera. In step 304, it is determined whether the blastocyst is present in the image. If the blastocyst is not present in the image, then a command is issued in step 306 to perform a clockwise step on the feeder micrometer syringe. Next, no action is performed a short amount of time in order to allow for the fluid response in step 308, after which control returns to step 302 where updated blastocyst information is obtained from the vision system (step 302). Now, if the blastocyst is present in the image (step 304), then control proceeds to step 310 where the process is completed.

Figure 4 is a flow chart of an exemplary blastocyst capture process for facilitating automation of blastocyst microinjection according to an embodiment of the subject matter described herein. Referring to Figure 4, beginning at step 400, a capture blastocyst command is issued in order to hold the blastocyst in a fixed position using suction applied by the holding pipette. In other words, as soon as a new blastocyst enters the injection area it should be captured by the holding pipette. Similar to the process described above in Figure 3, in step 402, updated image data of the blastocyst is obtained. For example, the position and orientation of the blastocyst may be determined by detecting the boundaries of the blastocyst relative to the known boundaries of the XY stage or other objects within the injection area (e.g., the holding pipette). In step 404, it is determined whether the blastocyst is next to the tip of the holding pipette. If the blastocyst is not next to the tip of the holding pipette, then a command is issued in step 406 to perform a clockwise step on the holder micrometer syringe. Next, no action is taken for a short amount of time in order to allow for the fluid response in step 408, after which control returns to step 402 where updated blastocyst information is obtained from the vision system. Now, if the blastocyst is next to the tip of the holding pipette (step 404), then control proceeds to step 410 where the process is completed.

In one example, the algorithm shown in Figure 4 assumes that a blastocyst is present in or near the injection area, and commands the holder micrometer syringe to rotate counterclockwise until the blastocyst is captured by the holding pipette. The task is deemed completed when the blastocyst is located next to the holding pipette tip, i.e., when the following equations are true:

(Xblast — Rblast) < Xholder

Equation 1

Yholder -0.25Ri)/asf < Yblast < Yholder + 0.25Rf>/asf

Equation 2

In the equations above, (Xhoider, Yhoider) are the coordinates of the holding pipette tip, (Xbiast, Ybiast) are the coordinates of the blastocyst center, and Rbiast is the blastocyst radius. All of these parameters were obtained in real-time from the vision processing system.

Figure 5 is a flow chart of an exemplary blastocyst release process for facilitating automation of blastocyst microinjection according to an embodiment of the subject matter described herein. There are basically two situations in which the blastocyst must be released from the holding pipette during orientation adjustments prior to injection and during its collection after microinjection. As may be appreciated from Equations 3-5 below, these situations may basically correspond to the inverse of the Equations 1 and 2 above for blastocyst capture. During blastocyst release, the holder micrometer syringe may be slowly rotated clockwise until the blastocyst is no longer next to the holding pipette tip. Exemplary decision equations for task completion may be given by:

(Xblast - Rblast)> Xholder

Equation 3

Yblast > Yholder + 0.25Rblast

Equation 4

Yblast < Yholder -0.25Rblast Equation 5

Thus, a blastocyst release task may be deemed completed when any one of Equations 3-5 is true. Referring to Figure 5, beginning at step 500, a release blastocyst command is issued in order to release the blastocyst from the holding pipette. In step 502, updated image data of the blastocyst is obtained. For example, the position and orientation of the blastocyst may be determined by detecting the boundaries of the blastocyst relative to the known boundaries of the XY stage or other objects within the injection area (e.g., the holding pipette). In step 504, it is determined whether the blastocyst is next to the tip of the holding pipette. If the blastocyst is not next to the tip of the holding pipette, then a command is issued in step 506 to perform a clockwise step on the holder micrometer syringe. Next, no action is taken for a short amount of time in order to allow for the fluid response in step 508, after which control returns to step 502 where updated blastocyst information is obtained from the vision system. Now, if the blastocyst is next to the tip of the holding pipette (step 504), then control proceeds to step 510 where the process is completed. Figure 6 is a flow chart of an exemplary blastocyst orientation process for facilitating automation of blastocyst microinjection according to an embodiment of the subject matter described herein. During preparation for injection, the blastocyst's orientation must be verified and adjusted. This is necessary to avoid injecting through the ICM. Here, the push-pull method of blastocyst orientation may be used for automation. This method was selected for its simplicity. As a result, the algorithm for automatic blastocyst orientation consisted of releasing and capturing the blastocyst until an acceptable orientation for injection was detected. Referring to Figure 6, beginning at step 600, an adjust blastocyst orientation command is issued in order to adjust the orientation of the blastocyst. In step 602, updated image data of the blastocyst is obtained. For example, the position and orientation of the blastocyst may be determined by detecting the boundaries of the blastocyst relative to the known boundaries of the XY stage or other objects within the injection area (e.g., the holding pipette). In step 604, it is determined whether the orientation of the blastocyst is acceptable for injection. If the orientation of the blastocyst is not acceptable for injection, then commands are issued in steps 606 and 608, respectively, to release and re-capture the blastocyst. Next, control returns to step 602 where updated blastocyst orientation information is obtained from the vision system. If the blastocyst is the orientation of the blastocyst is acceptable for injection, then control proceeds to step 610 where the process is completed.

Figure 7 is a flow chart of an exemplary blastocyst collection process for facilitating automation of blastocyst microinjection according to an embodiment of the subject matter described herein. Blastocyst collection is performed after the injection pipette retracts from the blastocyst. This task completes the microinjection cycle, so it involves releasing the blastocyst from the holding pipette and collecting it through suction on the collection pipette. Referring to Figure 7, beginning at step 700, a collect blastocyst command is issued in order to remove the blastocyst from the injection area and place it in a collection area. Ideally, this collection area serves to separate injected blastocysts from un-injected blastocyst in order to save time in identifying them. In step 702, a command is issued to perform a clockwise step on the collector micrometer syringe. A command is then issued in step 704 to release the blastocyst, where it is automatically removed from the injection area. In step 706, it is determined whether the blastocyst is present in the image of the injection area. If the blastocyst is not present in the image, then a command is issued in step 708 to perform a clockwise step on the holder micrometer syringe. Next, no action is taken for a short amount of time in order to allow for the fluid response in step 710, after which control returns to step 704 where another blastocyst release command is issued. If the blastocyst is no longer present in the injection area, then control proceeds to step 712 where the process is completed. Figure 8 is a flow chart of an exemplary process for facilitating automation of blastocyst microinjection according to an embodiment of the subject matter described herein. Referring to Figure 8, in step 800, the process of injecting a blastocyst may be initiated. Initially, the holding and injection pipettes may be aligned (horizontally) in step 802. The injection pipette may then be moved to the edge of the blastocyst's outer membrane in step 804, after which the piezo injector may be turned on in step 806. Next, in step 808, the injection pipette may be moved to the center of the blastocyst by piercing the outer membrane. The piezo injection may then be turned on in step 810, and one or more cells (e.g., embryonic stem cells) may be delivered into the blastocyst in step 812. Next, the injection pipette may be retracted from the blastocyst in step 814 and moved to its home position in step 816. The injection process is thus completed in step 818, and may be repeated automatically for a next target blastocyst.

Figure 9 shows exemplary image data of a blastocyst microinjection area before and after image processing for facilitating automation of blastocyst microinjection according to an embodiment of the subject matter described herein. Referring to Figure 9, original image 900 includes shows unprocessed image data acquired from a microscope coupled to a digital camera and displayed on a computer display. Image 900 shows objects located within the blastocyst microinjection area including holding pipette 902, target blastocyst 904, and injection pipette 906. Image 900 represents what is conventionally seen by operators trying to manually inject blastocysts. It is appreciated that while somewhat useful, a lack of contrast or other highlighting of areas of interest within the image for facilitating injection typically results in slower injections that are more prone to error.

In contrast, processed image 908 shows the result of image 900 after image analysis is performed. Holding pipette 902, target blastocyst 904, and injection pipette 906 may all be more readily seen because the contrast ratio has been increased so that objects may appear brighter and whiter than the background, which appears darker. Additionally, bounding boxes 904 and 906 may be added to image 908 to highlight the boundaries of holding pipette 902 and injection pipette 906, respectively. By adding bounding boxes 904 and 906, either a human operator or a computer-controlled micropositioner may more accurately determine the position of holding pipette 902 and injection pipette 906 and therefore may more accurately, quickly, and consistently injection target blastocyst 904.

Figure 10 is a diagram of an exemplary control system for providing pipette motion control for facilitating automation of blastocyst microinjection according to an embodiment of the subject matter described herein. One of the main system characteristics that may have a major impact on the success of blastocyst microinjections is the quality of the injection pipette motion control. Motion control for manipulating a micromanipulation robot may be based on visual servoing techniques. In one embodiment, all motion commands were may be computed solely based on visual feedback, i.e., on localization information provided by a video processing system. In such an embodiment, robot motions cause changes to captured video images, which result in changes to the vision system's output parameters. An exemplary control structure may consist of a PID (proportional-integral-derivative) control loop in which position errors are computed directly from 2-D image parameter space. For example, target coordinates and injection pipette tip coordinates may be defined with respect to an image reference frame. Position errors used for control may be obtained from the difference between those two points, as defined by Equation 6.

E = T-P

Equation 6 where T e 9i ² is the target location, and P e SR² is the pipette location, both defined in terms of image coordinates;

ηn X t Y t Equation 7

Equation 8

Referring to Figure 10, a relationship between system variables is shown, including control parameters: proportional gain (Kp), integral gain (Ki) and derivative gain (Kd). These parameters may be used for control system calibration and provide the appropriate mapping of commands generated in an image space to motion commands in a target space.

PID control equations for an exemplary 2-D visual servoing system may be given by: E i(k) = E i(k - 1) + — (E(Jc) + E(k - 1))

Eq. 8.9

(E(k) - E(k - l))

C(k) = K pl(k) + KiIi(K) + Kd

At Eq. 8.10

where Ei is the integral of the error signal; At is the sample period; and

CxCy is the motion command vector sent to the robot driver. The control system configuration shown in Figure 10 restricts the task space to 2-D during the microinjection operations. This restriction means that the robot was constrained to move along the X and Y axes only, guaranteeing that the vertical alignment of the holding and injection pipettes was not affected. Furthermore, the control system may be configured to compensate for small misalignments between the robot and the image axes in order to successfully avoided expensive system calibration.

Thus, the movement of the injection pipette may be based on the visual servoing system described above (i.e., injection pipette insertion, delivery of ES cells, and pipette retraction). The algorithm shows that, once a target coordinate is commanded, the visual servoing system takes the pipette to the desired location. In one embodiment, this positioning process is stopped, and the task is deemed completed, when the error between the target the actual pipette position is small (less than 2 pixels). Figure 11 is a flow chart of an exemplary embryonic stem cell delivery process for facilitating automation of blastocyst microinjection according to an embodiment of the subject matter described herein. The delivery of ES cells inside the blastocyst cavity was not automated in this research. Consequently, we required an operator to manually perform this task using the joystick. Referring to Figure 11 , beginning at step 1100, a deliver embryonic stem cells command is issued in order to inject one or more ES cells into the blastocyst. In step 1102, a command is issued to the vision system to being counting the number of cells delivered. The number of delivered cells are totaled in step 1104 and compared to a predetermined desired number in step 1106. If it is determined that not enough ES cells have been delivered, then a command is issued in step 1108 to perform a clockwise step on the holder micrometer syringe. Next, no action is taken for a short amount of time in order to allow for the fluid response in step 1110, after which control returns to step 1104 where an updated count of the number of delivered cells is obtained. If enough ES cells have been delivered, then control proceeds to step 1112 where the process is completed. The automated of this part of the system is deemed future work.

Getting this task automated requires the development of extra video processing algorithms to detect and count the number of delivered ES cells.

This is not a trivial task because the ES cells are very small and typically move very fast inside the injection pipette.

Figure 12 is a diagram showing an exemplary system for counting of the number of delivered embryonic stem cells for facilitating automation of blastocyst microinjection according to an embodiment of the subject matter described herein. A small section of the injection pipette may be monitored for the detection of cells passing through it. This detection may be possible by defining a small monitoring area which can only detect one cell at a time. In this way, the ES cells count can be incremented every time a new cell is detected in the defined area. For example, injection pipette 1200 may be inserted into target blastocyst 1202 and contain one or more ES cells 1204. Monitoring area 1206 may be defined at an area approximately equal to the size of one of cells 1204 and may be located at the intersection of injection pipette 1200 and the outer membrane of blastocyst 1202. As ES cells 1204 pass through monitoring area 1206, they may be counted using a vision system. This location for monitoring area 1206 is likely to be an ideal detection site because it avoids background image noise from blastocyst inner structures, and because the image focus in that part of the pipette is close to the focus on the pipette's tip.

Figure 13 is a block diagram of an exemplary system for facilitating automation of blastocyst microinjection according to an embodiment of the subject matter described herein. Referring to Figure 13, system 1310 may include blastocyst injection area 1302. For example, a Petri dish containing an area suitable for maintaining blastocysts during the microinjection process may be located on an anti-vibration table in order to increase the accuracy of each injection. Blastocyst dispensing pipette 1304 may be located near injection area 1302 so that blastocyst may be delivered to injection area 1304. When a blastocyst enters injection area 1302, holding pipette 1306 may hold the blastocyst in place. Injection pipette 1308 may injection one or more cells into the blastocyst and collection pipette 1310 may remove the blastocyst from injection area 1302 after injection. Thus, the exemplary configuration shown in system 1300 is based on four micromanipulation robots (i.e., micromanipulators 1314, 1318, 1322, and 1332) and four motorized micrometer syringes (i.e., micrometer syringes 1316, 1320, 1330, and 1334).

In the configuration shown in Figure 13, injection area 1302 may be magnified by microscope 1312 in order view objects and actions occurring inside of injection area 1302.

Dispensing pipette 1104 may be controlled by microcontroller 1314 and blastocysts may be fed into dispensing pipette 1304 using motorized micrometer syringe 1316. There is unlikely to be a need for manual control of dispensing pipette 1104, and thus, the embodiment shown in system 1300 shows a fully automated control of dispensing pipette 1304.

In contrast, holding pipette 1306 may be controlled by manual micromanipulator 1318, and may be fed by a motorized micrometer syringe

1320. This is because, as described above, it may be advantageous in some situations to allow user control of holding pipette 1306. However, ideally, fully automated control of holding pipette 1306 may be maintained.

Injection pipette 1308 may be associated with p iezo microinjector 1322 for injecting one or more cells into target blastocysts. Piezo microinjection 1322 may be controlled by micromanipulation robot 1324. Micromanipulation robot 1324 may further be associated with robot driver 1328 for sending commands to robot 1324 in order to physically manipulate its position and/or orientation. Robot driver 1328 may receive commands from motion control software located on a desktop computer for automatically manipulating robot 1324. Alternatively, robot 1324 may be controlled by manual micromanipulator 1326 for semi-automated embodiments of system 1300. Finally, motorized micrometer syringe 1330 may be associated with injection pipette 1308 for injecting one or more cells into target blastocysts. It is appreciated that micrometer syringe 1330 may also be operated using commands issued by motion control software located on a desktop computer. After injection of one or more cells, the target blastocyst may be collected and removed from injection area 1302 usi ng collection pipette 1310. Associated with collection pipette 1310 may be micropositioner 1332 and motorized micrometer syringe 1334. Similar to motorized micrometer syringe 1330, motorized micrometer syringe 1334 may be controlled using motion control software located on a desktop computer.

Blastocyst injection are 1302 may, for example, be located within a Petri dish or other suitable environment. In the embodiment shown in system 1300, injection area 1302 may be physically located on top of XY stage 1338. XY stage 1338 may include a flat platform made of glass having a rigid frame (e.g., metal) so as to provide a platform for viewing blastocysts for injection. XY stage 1338 may be manipulated by XY stage driver 1336, such as a DC servo motor and rotary encoder, for providing accurate positioning, flatness, straightness, orthogonality, and repeatability. Additionally, in order to perform digital image analysis on images of injection area 1302, charge coupled device (CCD) sensor-based camera 1340 may be associated with microscope 1312. CCD camera 1340 may obtain images of injection area 1302 and send the digital images to a desktop computer for analysis. For example, desktop computer 1342 may contain one or more software modules, stored in memory and executable by a processor, for receiving images of injection area 1302, modifying the images for distinguishing key features of blastocysts etc., displaying the images, and receiving user input for controlling micromanipulators 1314, 1318, 1322, and 1332. For example, digital images may be received by frame grabber 1342 from CCD camera 1340 and sent to image processing module 1346. Image processing module 1346 may output images to a display, such as monitor 1348 associated with computer 1342. Additionally, the captured images may be forwarded to an experiment data recorder 1350 for analyzing data extracted from the images such as the number of blastocysts injected, the number of cells injected in each blastocyst, the number of errors, etc. Also associated with experiment data recorder 1350 may be a motion control module 1352 for controlling the movement of micromanipulators 1314, 1318, 1322, and 1332. Motion control module may include instructions executable by computer 1342 for implementing one or more of the algorithms described above.

In one embodiment, a conventional inverted microscope (having multiple objectives) may be replaced by a microscope 1312 having a single objective and a motorized set of precision zoom lenses that can be directly attached to video camera 1340. Additional light necessary for illuminating injection area 1302 may be provided by a fiber optic illuminator (not shown). Finally, user commands manager module 1354 may be associated with motion control module 1352 for managing input (e.g., commands) received from the user. For example, user commands module 1354 may be connected to a keyboard 1356 and a joystick 1358 for manually manipulating micromanipulators 1318 and 1326.

Figures 14A and 14B are diagram of an exemplary physical arrangement of a system for facilitating automation of blastocyst microinjection according to an embodiment of the subject matter described herein. Referring to the view shown in Figure 12A, XY stage 1338 may support a Petri dish or other suitable container for implementing a blastocyst injection area. An inverted microscope 1312 may be located above XY stage 1338 and associated with CCD camera 1340 for obtaining image of the injection area. Motorized micrometer syringes 1316, 1320, 1330, and 1334 are located in proximity to XY stage 1338 in order to, for example, dispense blastocysts into the injection area, hold and orient the blastocysts for injection, and remove the blastocyst from the injection area after injection. Manual micromanipulators 1318 and 1326 may be located in a position suitable for use by a user for controlling injection pipette 1308 and holding pipette 1306. Piezo injection 1322 may be associated with injection pipette 1306 for injecting one or more cells into a target blastocyst. It is further appreciated that, as described above, piezo injection 1322 may be connected to motion control module 1352 located separately on desktop computer 1342.

Referring to Figure 14B, Petri dish 1400 may be located centrally on top of XY stage 1338. Surrounding Petri dish 1400 may be feeder pipette 1304, holding pipette 1306, injection pipette 1308, and collection pipette 1310.

Figure 15A is an overhead view of a system-on-a-chip system for facilitating automation of blastocyst microinjection according to an embodiment of the subject matter described herein. Referring to Figure 15A, system-on-a-chip 1500 may include integrated circuit 1502 having four channels 1504, 1506, 1508, and 1510. The injection area may be located at the intersection of channels 1504, 1506, 1508, and 1510. Each of channels 1504-1510 may be filled with a fluid suitable for sustaining the viability of the blastocyst during injection. Because the size of channels 1504-1510 is small (e.g., channel diameters of around 100 nanometers to several hundred micrometers), microfluidic properties dominate the behavior of the fluid in the channel. The behavior of fluids at the microscale can differ from macrofluidic behavior in that factors such as surface tension, energy dissipation, and fluidic resistance start to dominate. For example, in microfluidic conditions, the Reynolds number, which characterizes the presence of turbulent flow, is extremely low, and thus the flow will remain laminar (i.e., two fluids joining will not mix readily via turbulence, so diffusion alone must cause the two fluids to mingle). In Figure 15A, blastocyst 1512 may enter the injection area via channel 1504. Once inside in the injection area, blastocyst 1512 may be held in place using micro-suction hole 1510 located beneath the injection area. Blastocyst 1512 may then be oriented using electrorotation applied by electrode 1516. Electrorotation (ER) is the circular movement of an electrically polarized particle, such as blastocyst 1512 through manipulation of an applied electric field. Once properly oriented, MEMS needle 1518 may be used to inject one or more cells into blastocyst 1512. For example, MEMS needle 1518 may be a silicon tube having a diameter so as to enable only one cell to be injected at a time.

Figure 15B is cross-sectional view of a system-on-a-chip system for facilitating automation of blastocyst microinjection according to an embodiment of the subject matter described herein. Referring to Figure 15B₁ blastocyst 1512 may enter the injection are via feeder channel 1304 (i.e., the left). Micro-suction hole 1514 may be located centrally within the injection area and directly below blastocyst 1512. Optical sensor 1318 may be located above blastocyst 1512 for capturing image data of the injection area. It is appreciated that optical sensor 1518 replaces microscope 1312 in other embodiments, and may therefore be easier, cheaper, and more accurate. It is also appreciated that while an optical sensor is shown in system 1500, other sensors may also be used without departing from the scope of the subject matter described herein. In order to illuminate blastocyst 1512, a light source may be located opposite optical sensor 1518, such as light emitting diode (LED) 1520. Electrodes 1522 may be used to orient blastocyst 1512 using 3-D electrorotation. MEMS needle 1518 may be inserted into blastocyst 1512 in order to inject one or more cells. Finally, blastocyst 1512 may be removed from the injection area via collection channel 1506 (i.e., the right) using fluid flow within the channel. During the injection process, blastocyst 1512 may be cooled using the Peltier effect. One advantage of the system-on-a-chip embodiment shown in Figures 15A and 15B includes eliminating the need for a microscope and a micromanipulator.

It will be understood that various details of the subject matter described herein may be changed without departing from the scope of the subject matter described herein. Furthermore, the foregoing description is for the purpose of illustration only, and not for the purpose of limitation, as the subject matter described herein is defined by the claims as set forth hereinafter.

Claims

CLAIMS What is claimed is:

1. A method for facilitating automation of blastocyst microinjection, the method comprising: automatically feeding a target blastocyst from among a plurality of blastocysts into a blastocyst microinjection area; automatically holding the target blastocyst in a fixed position within the blastocyst microinjection area; orienting the target blastocyst so as to be suitable for injection; injecting one or more cells into the target blastocyst; and automatically releasing and removing the target blastocyst from the blastocyst microinjection area after injection of the one or more cells.

2. The method of claim 1 wherein orienting the target blastocyst includes using a push-pull technique.

3. The method of claim 1 wherein orienting the target blastocyst includes semi-automatically orienting the target blastocyst using a graphical user interface (GUI) and a user input device to vary the orientation of the target blastocyst.

4. The method of claim 1 wherein orienting the target blastocyst includes automatically orienting the target blastocyst using image analysis.

5. The method of claim 1 wherein injecting one or more cells into the target blastocyst includes injecting one or more embryonic stem cells.

6. A system for facilitating automation of blastocyst microinjection, the system comprising: a feeder pipette for automatically feeding a target blastocyst from among a plurality of blastocysts into blastocyst microinjection area; a holding pipette for automatically holding the target blastocyst in a fixed position within the blastocyst microinjection area and releasing the target blastocyst from the blastocyst microinjection area after injection of the one or more cells; an injection pipette for orienting the target blastocyst so as to be suitable for injection and injecting one or more cells into the target blastocyst; and a collection pipette for automatically removing the target blastocyst from the blastocyst microinjection area after injection of the one or more cells.

7. The system of claim 6 comprising a magnifier for enlarging an image of the injection area.

8. The system of claim 6 wherein the magnifier includes one of a single objective microscope, a multi-objective microscope, a fixed lens microscope, and a zooming lens microscope.

9. The system of claim 6 comprising a camera for recording images of the injection area and converting the images to digital image data.

10. The system of claim 6 comprising one or more micropositioners for manipulating the position of at least one of the feeder pipette and the dispensing pipette.

11. The system of claim 6 comprising one or more micromanipulators for manipulating the position of at least one of the holding pipette and the injection pipette.

12. The system of claim 11 wherein the one or more micromanipulators are manually controllable by a human operator.

13. The system of claim 6 comprising a pipette control module for manipulating at least one of the holding pipette and the injection pipette.

14. The system of claim 6 comprising one or more syringes associated with at least one of the feeder pipette, the holding pipette, the injection pipette, and the collection pipette.

15. The system of claim 6 comprising a syringe control module for manipulating the one or more syringes.

16. The system of claim 6 comprising a display for displaying image data of the injection area.

17. The system of claim 6 comprising an image analysis module for determining boundaries of objects within the injection area.

18. The system of claim 6 comprising an input device for receiving input from a user.

19. The system of claim 6 wherein the injection pipette orients the target blastocyst using a push-pull technique.

20. The system of claim 6 wherein the injection pipette orients the target blastocyst semi-automatically using a graphical user interface (GUI) and a user input device to vary the orientation of the target blastocyst.

21. The system of claim 6 wherein the injection pipette orients the target blastocyst automatically using image analysis.

22. The system of claim 6 wherein injection pipette injects one or more embryonic stem cells into the target blastocyst.

23. A system for facilitating automation of blastocyst microinjection, the system comprising: an integrated circuit having a plurality of channels forming a fluidic communication path for blastocysts and at least one blastocyst microinjection area in the fluidic communications path, the integrated circuit forming a suction hole in the blastocyst microinjection area for holding a target blastocyst in place; at least one electrode for electrorotating the target blastocyst so as to be suitable for injection; a microelectromechanical (MEMS) needle for injecting one or more cells into the target blastocyst; and a sensor for observing the position and orientation of the injection area.

24. The system of claim 23 wherein the sensor includes an optical sensor.

25. The system of claim 23 comprising a light source located opposite the injection area for illuminating the blastocyst.

26. The system of claim 23 comprising an input device for receiving input from a user.

27. The system of claim 23 wherein the at least one electrode orients the target blastocyst automatically using image analysis.

28. The system of claim 23 wherein injection pipette injects one or more embryonic stem cells into the target blastocyst.

29. A computer readable medium comprising computer executable instructions embodied in a tangible computer readable medium and when executed by a processor of a computer performs steps comprising: automatically feeding a target blastocyst from among a plurality of blastocysts into a blastocyst microinjection area; automatically holding the target blastocyst in a fixed position within the blastocyst microinjection area; orienting the target blastocyst so as to be suitable for injection; injecting one or more cells into the target blastocyst; and automatically releasing and removing the target blastocyst from the blastocyst microinjection area after injection of the one or more cells.