Patent ID: 12233539

DETAILED DESCRIPTION

The present invention now will be described more fully hereinafter with reference to the accompanying drawings, in which some, but not all embodiments of the invention are shown. Indeed, this invention may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will satisfy applicable legal requirements. Like numbers refer to like elements throughout. It is to be understood that this invention is not limited to the particular methodology and protocols described, as such may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention.

Many modifications and other embodiments of the invention set forth herein will come to mind to one skilled in the art to which the invention pertains having the benefit of the teachings presented in the foregoing description and the associated drawings. Therefore, it is to be understood that the invention is not to be limited to the specific embodiments disclosed and that modifications and other embodiments are intended to be included within the scope of the appended claims. Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation.

As used herein the singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “an actuator” can include a plurality of such actuators, and so forth.

All technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this invention belongs unless clearly indicated otherwise.

Ranges can be expressed herein as from “about” one particular value, and/or to “about” another particular value. When such a range is expressed, another aspect includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms another aspect. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint.

As used herein, the terms “optional” or “optionally” mean that the subsequently described event or circumstance may or may not occur, and that the description includes instances where said event or circumstance occurs and instances where it does not.

The word “or” as used herein means any one member of a particular list and also includes any combination of members of that list.

I. Overview

The present invention comprises systems and methods for orienting objects (e.g., extracted embryos) in desired orientations for further processing. In exemplary aspects, the disclosed systems and methods can be used to position extracted plant embryos in a desired orientation for contacting a growth medium. Optionally, in these aspects, the disclosed systems and methods can be used to singulate the extracted plant embryos and evaluate the orientation of the singulated embryos to determine if the orientation of the embryos needs to be adjusted to permit further processing. Optionally, the disclosed systems and methods can be used to transport extracted embryos from a first location (e.g., a petri dish or singulation apparatus) to a second location (e.g., a tray containing a growth medium) while ensuring that the embryos are positioned in a desired orientation at the second location. It is contemplated that, unless otherwise stated, any of the steps of the disclosed methods can be performed in an automated fashion.

As used herein, the term “automated” refers to the use of mechanical, electrical, software, imaging, vision-based and/or other known automation-based technologies to augment processes typically performed by human interaction.

In exemplary aspects, it is contemplated that the actuators of the disclosed systems can be positioned in operative communication with at least one controller800, such as, for example and without limitation, a programmable logic controller (PLC) or a computer having a processor as is known in the art. In these aspects, it is contemplated that the processor of the controller(s) can be configured to activate the actuators of the disclosed systems and assemblies in an automated manner.

II. Systems and Methods of Singulating Extracted Embryos and Other Objects

In exemplary aspects, and with reference toFIGS.1,7A, and8A, disclosed herein is a singulation apparatus300. As further described herein, the singulation apparatus300can “singulate” objects (e.g., embryos) to permit processing of individual objects (e.g., embryos), one at a time. In these aspects, the singulation apparatus300can comprise a fluid bath303, a screen302having a shape configured for receipt within the fluid bath, at least one actuator304coupled to the screen, and a camera301. In these aspects, the actuator304can be configured to selectively move the screen302relative to a vertical axis. It is contemplated that the actuator304can be a conventional linear actuator, such as, for example and without limitation, a mechanical actuator, a hydraulic actuator, a pneumatic actuator, a piezoelectric actuator, or an electromechanical actuator. In exemplary aspects, the actuator304can be a pneumatic cylinder. In other exemplary aspects, the screen302can have a mesh structure as is known in the art to permit flow of fluid through the openings defined by the mesh structure. In these aspects, it is contemplated that the mesh structure can have a selected mesh size, such as 20×20 or 40×40 (holes/inch). However, it is contemplated that any desired mesh size can be used, provided the mesh size is large enough to reduce surface tension of an aqueous solution but not so large that the embryo is lodged within or passes through the mesh. In further aspects, the screen302can be moveable about and between a submerged position within the fluid bath303and an elevated position above the fluid bath. In additional aspects, the camera301can be positioned above the fluid bath303relative to the vertical axis. In these aspects, when the screen302is in the elevated position above the fluid bath, the camera301can be configured to produce an image of objects (e.g., embryos) positioned on the screen. Exemplary images of embryos on the screen302are shown inFIG.7B.

In exemplary aspects, the fluid bath can have a substantially rectangular or square perimeter. However, it is contemplated that any desired shape (e.g., a round perimeter shape) can be used. Similarly, it is contemplated that the screen can have any shape that is complementary to the shape of the fluid bath.

As shown inFIG.7A, it is contemplated that the singulation apparatus300can comprise a structure that supports the liquid bath in an elevated position relative to a floor surface or other horizontal surface. Optionally, in exemplary aspects, the support structure can comprise a plurality of support legs extending between the horizontal surface and an undersurface of the fluid bath303and/or between the horizontal surface and an undersurface of a platform supporting the fluid bath.

In exemplary aspects, the screen302of the singulation apparatus300can be positioned on and/or coupled to an upper surface of a support element308that is configured for selective movement relative to the vertical axis. In these aspects, the actuator304of the singulation apparatus300can be coupled to the support element308such that the actuator is capable of imparting motion to the support element and, consequently, the screen302. In exemplary aspects, the actuator304can be coupled to the support element308by at least one arm306that is coupled to the actuator and that extends to the support element. Optionally, in these aspects, the actuator304can be coupled to a horizontal platform305that is configured for selective movement relative to the vertical axis, and the arms306can be coupled to the platform and extend from the platform to the support element308. In further aspects, the support element308can define at least one engagement structure309that is configured for selective engagement with the arms. It is contemplated that each engagement structure309of the support element308can be a recess, a projection, an opening, a slot, or other conventional engagement means that is configured to engage a corresponding portion of an arm306. Optionally, in some aspects, the arms306can be shaped to at least partially extend under the support element308to provide additional support and stability to the support element (and the screen302). For example, as shown inFIG.7A, when the actuator304is positioned below the fluid bath303, it is contemplated that the arms306can have a “C” or “G” type shape, with a distal end of each arm optionally extending underneath a portion of the support element308. In further exemplary aspects, the support element308can comprise at least one opening to allow fluid to reach the screen302.

In exemplary aspects, the singulation apparatus300can further comprise a controller800positioned in communication with the camera301. In these aspects, the controller800can comprise a processor configured to analyze the image of the screen302produced by the camera301to identify singulated embryos on the screen. In one aspect, the processor of the controller800can be configured to identify contours of objects (e.g., embryos) on the screen302to determine the presence of singulated objects (e.g., embryos) on the screen. In another aspect, the controller800can be configured to direct the actuator304to move the screen about and between the submerged position and the elevated position.

In use, the singulation apparatus300can singulate objects (e.g., previously extracted embryos) for use in downstream processes as further disclosed herein. In exemplary aspects, a method for singulating objects (e.g., embryos) can comprise positioning a plurality of objects (e.g., embryos) on the screen of the singulation apparatus. In these aspects, it is contemplated that the objects (e.g., embryos) can be placed on the screen in either a manual or automated fashion. In another aspect, the method can further comprise activating the actuator to move the screen to the submerged position within the fluid bath. Optionally, in this aspect, the fluid bath can contain an aqueous solution. Optionally, it is contemplated that the liquid bath can be filled with at least one of water, solution, buffer, or liquid gel. In an additional aspect, the method can further comprise activating the actuator to move the screen to the elevated position. In a further aspect, the method can further comprise activating the camera to produce an image of the objects (e.g., embryos) on the screen. In another aspect, the method can further comprise processing the image to identify singulated objects (e.g., embryos) on the screen. In still another aspect, the method can comprise removing the singulated objects (e.g., embryos) from the screen. Optionally, in this aspect, the singulated objects (e.g., embryos) can be removed from the screen by a vacuum nozzle.

In exemplary aspects, the method can further comprise repeating the steps of: activating the actuator to move the screen to the submerged position within the fluid bath; activating the actuator to move the screen to the elevated position; activating the camera to produce an image of the embryos on the screen; processing the image to identify singulated objects (e.g., embryos) on the screen; and removing the singulated objects (e.g., embryos) from the screen until a desired number of singulated objects (e.g., embryos) are removed from the screen. Thus, in use, each time the screen is moved from the submerged position to the elevated position, the processor can identify singulated objects (e.g., embryos) and initiate removal of the singulated objects (e.g., embryos). Then, the process can be repeated as necessary until a desired number of singulated objects (e.g., embryos) have been identified and removed from the screen. In operation, it is contemplated that the objects (e.g., embryos) on the screen will settle to a more singulated state following withdrawal of the screen from the fluid bath.

In exemplary aspects, the processor of the controller can identify singulated embryos on the screen by applying one or more threshold parameters to the image. Such threshold parameters can include size parameters, shape parameters, color parameters, and the like. In various aspects, the processor of the controller can identify singulated embryos in an automated manner by identifying the embryos on the screen and applying the threshold parameters to determine the locations of singulated embryos.

As shown inFIG.9, it is contemplated that the at least one controller800disclosed herein can function as a central controller of each of the components of the system100disclosed herein. However, it is also contemplated that the disclosed singulation apparatus can have its own dedicated controller that can optionally be positioned in communication with other controllers of the system100.

III. Systems and Methods of Adjusting the Orientation of an Extracted Embryo or Other Object

With reference toFIGS.1-6C, disclosed herein is an object orientation apparatus200,500. In exemplary applications, and as further disclosed herein, the object orientation apparatus200,500can be used to position extracted embryos (e.g., plant embryos) in desired orientations. However, it is contemplated that the disclosed object orientation apparatus200,500can also be used to position other objects in desired orientations. In exemplary aspects, the object orientation apparatus can comprise first and second nozzle assemblies208a,208b,508a,508b.

In exemplary aspects, each of the first and second nozzle assemblies208a,208b,508a,508bcan comprise a respective vacuum nozzle202a,202b,502a,502band a respective actuation subassembly205a,205b,505a,505b. In these aspects, each of the vacuum nozzles202a,202b,502a,502bcan have a respective longitudinal axis212a,212b,512and a respective distal end216a,216b,516a,516b. In use, each vacuum nozzle202a,202b,502a,502bcan be configured to apply suction to retain an object against the distal end216a,216b,516a,516bof the vacuum nozzle. In exemplary aspects, each vacuum nozzle202a,202b,502a,502bcan be configured to apply a suction force that is at a sufficiently strong level to retain an embryo against the distal end216a,216b,516a,516bof the vacuum nozzle but at a sufficiently low level to avoid damage to the embryo. In another aspect, within each nozzle assembly208a,208b,508a,508b, the vacuum nozzle202a,202b,502a,502bis rotatable by the actuation subassembly205a,205b,505a,505b. In this aspect, the vacuum nozzles202a,202b,502a,502bof the first and second nozzle assemblies208a,208b,508a,508bare selectively rotatable to an object-transfer position in which the longitudinal axis of the first vacuum nozzle202a,502ais aligned with the longitudinal axis of the second vacuum nozzle202b,502band the distal end216a,516aof the first vacuum nozzle is positioned proximate the distal end216b,516bof the second vacuum nozzle.

In exemplary aspects, each of the first and second vacuum nozzles202a,202b,502a,502bcan be configured for compliant movement relative to its longitudinal axis. Optionally, in these aspects, the first and second vacuum nozzles202a,202b,502a,502bcan be telescopic nozzles that are configured for “free” movement relative to their longitudinal axes. In use, the compliancy of the vacuum nozzles202a,202b,502a,502bcan provide tolerance for engaging objects (e.g., embryos) as disclosed herein. For example, it is contemplated that the compliancy of the vacuum nozzles202a,202b,502a,502bcan permit engagement of objects (e.g., embryos) when the objects are not positioned in an ideal (e.g., perfectly aligned) position for engagement.

In exemplary aspects, the vacuum nozzles202a,202b,502a,502bcan comprise stainless steel. However, it is contemplated that other materials can be used. For example, in alternative aspects, it is contemplated that the vacuum nozzles202a,202b,502a,502bcan comprise rubber suction cups.

In further aspects, the actuation subassembly205a,205b,505a,505bof each nozzle assembly208a,208b,508a,508bcan comprise at least one rotational actuator coupled to the vacuum nozzle. Optionally, in these aspects, the at least one rotational actuator of each actuation subassembly205a,205b,505a,505bcan comprise a plurality of rotational actuators that are configured to effect rotational movement of the nozzle assembly208a,208b,508a,508brelative to a plurality of rotational axes, such as for example, and without limitation, two, three, four, five, six, or more axes. In further aspects, the actuation subassembly205a,205b,505a,505bof each nozzle assembly208a,208b,508a,508bcan further comprise at least one axial actuator configured to effect axial movement of the vacuum nozzle of the nozzle assembly relative to at least one axis.

In various aspects, and with reference toFIG.9, the object orientation apparatus200,500can further comprise at least one controller800that is communicatively coupled to the actuation subassemblies205a,205b,505a,505bof the first and second nozzle assemblies208a,208b,508a,508b. It is contemplated that the at least one controller800can be configured to control rotation of the vacuum nozzles202a,202b,502a,502bof the first and second nozzle assemblies208a,208b,508a,508b. In exemplary aspects, it is contemplated that the at least one controller of the object orientation apparatus200,500can function as a central controller of each of the components of the system100disclosed herein. However, it is also contemplated that the disclosed object orientation apparatus200,500can have its own dedicated controller that can optionally be positioned in communication with other controllers of the system100.

In the object-transfer position, as shown inFIGS.3B and6B, the longitudinal axes of the first and second vacuum nozzles202a,202b,502a,502bcan be substantially parallel to a transverse axis240that is perpendicular to a vertical axis. As used herein, the term “substantially parallel” refers to orientations in which the longitudinal axes of the first and second vacuum nozzles202a,202b,502a,502bare both within about 10 degrees of being parallel to the transverse axis240. In exemplary aspects, the longitudinal axes of the first and second vacuum nozzles202a,202b,502a,502bcan be parallel to the transverse axis240.

More generally, in further exemplary aspects, in the object-transfer position, it is contemplated that the longitudinal axes of the first and second vacuum nozzles202a,202b,502a,502bcan be positioned in any orientation in which the distal ends216a,216b,516a,516bof the first and second vacuum nozzles are positioned sufficiently close from one another to permit the transfer of an object (e.g., embryo) from one vacuum nozzle to the other vacuum nozzle. In these aspects, it is contemplated that the longitudinal axes of the first and second vacuum nozzles202a,202b,502a,502bcan be positioned parallel to the transverse axis240or at any desired acute angle relative to the transverse axis240.

In exemplary aspects, and as shown inFIGS.3A,6A, and6C, the vacuum nozzles202a,202b,502a,502bof the first and second nozzle assemblies208a,208b,508a,508bcan be selectively rotatable to respective object release/retrieval positions in which the vacuum nozzle of each vacuum nozzle assembly is angled downwardly from the transverse axis240. Optionally, in the object release/retrieval position of each of the vacuum nozzle assemblies208a,208b,508a,508b, the longitudinal axis of the vacuum nozzle202a,202b,502a,502bis positioned at a selected acute angle relative to the transverse axis240. Alternatively, in the object release/retrieval position of each of the vacuum nozzle assemblies208a,208b,508a,508b, the longitudinal axis of the vacuum nozzle202a,202b,502a,502bcan be perpendicular to the transverse axis240.

Optionally, in exemplary aspects and as shown inFIGS.4A-6C, each nozzle assembly508a,508bcan further comprise a cartridge510a,510bthat is configured to selectively engage the vacuum nozzle502a,502bof the nozzle assembly such that movement of the cartridge imparts a corresponding movement to the vacuum nozzle. In these aspects, a portion of the cartridge510a,510bcan be selectively engageable by the actuation subassembly505of the nozzle assembly508a,508b. Optionally, in additional aspects, each nozzle assembly508a,508bcan further comprise a guide tube568that has a proximal portion569with an at least partially threaded outer surface and is configured to receive a portion of the vacuum nozzle502a,502bof the nozzle assembly508a,508b. In these aspects, the cartridge510a,510bcan define a first threaded bore that is configured to threadedly engage the threaded outer surface of the guide tube568. Optionally, in further optional aspects, each nozzle assembly508a,508bcan further comprise a vacuum tube564. In these aspects, the cartridge510a,510bcan define a vacuum port560configured to receive suction from a vacuum source. As shown inFIGS.4A-4B, the vacuum tube564can be positioned in communication with the vacuum port560and the guide tube568to provide suction to the vacuum nozzle502a,502bof the nozzle assembly508a,508b. In exemplary aspects, and as shown inFIG.4B, it is contemplated that the vacuum tube564can optionally be provided with a proximal threaded portion565that is configured for receipt within a portion of the vacuum port560. In further exemplary aspects, it is contemplated that each cartridge510a,510bcan define a respective blow-off port562. It is further contemplated that the vacuum port560and the blow-off port562can both be positioned in fluid communication with the vacuum tube564. In use, the vacuum port can be positioned in fluid communication with a positive pressure source to thereby permit application of positive pressure through the vacuum tube and the vacuum nozzle. In exemplary aspects, the application of positive pressure through the blow-off port562(and the vacuum tube564and the nozzle502a,502b) can ensure that an object retained by the nozzle is released from the nozzle. It is further contemplated that the blow-off port562can be provided at an interior location relative to the vacuum port560to ensure that positive pressure applied through the blow-off port562can remove all contaminants from within the cartridge assembly. Optionally, in further exemplary aspects, it is contemplated that the cartridge510a,510bcan be shaped to leave at least a portion of the vacuum tube564exposed and accessible from outside the cartridge. In these aspects, it is contemplated that a distal portion of the vacuum tube564can be coupled to a collar566, with the vacuum tube564having a shape and/or curvature that is configured to bias the collar566(e.g., through a spring force) to a bottomed-out position in which the collar abuts the proximal portion569as shown inFIGS.4A-4B.

In further exemplary aspects, and with reference toFIGS.5A-5B, each actuation subassembly505can comprise a cartridge gripper580that is configured to selectively engage a proximal (top) portion of a respective cartridge510a,510b. In these aspects, the cartridge gripper580can comprise a pair of opposed panels582a,582bthat are moveable toward and away from each other relative to an axis parallel to the transverse axis240, to and from an engaged position (to engage the cartridge) and a disengaged position (no contact with the cartridge). Each panel582a,582bcan define respective locating pins584that are configured for engagement with corresponding alignment recesses517defined in the proximal portion of the cartridge510a,510b. The first panel582acan define a blow-off fitting586and corresponding opening that is configured for communication with the blow-off port562of the cartridge, and the second panel can define a vacuum fitting588and corresponding opening that is configured for communication with the vacuum port560of the cartridge. The openings for the vacuum fitting588and the blow-off fitting586can be provided with suitable sealing members590. In use, the actuation subassembly505can selectively move the panels582a,582babout and between the engaged and disengaged positions. Thus, it is contemplated that one cartridge can be exchanged (e.g., automatically exchanged) for another cartridge if desired. It is further contemplated that cartridges can be exchanged as needed to account for damage to cartridge/nozzle components and/or to permit periodic sterilization of used cartridges.

Optionally, in exemplary aspects, and as shown inFIG.6A, the object orientation apparatus500can further comprise first and second robots600a,600b. In these aspects, it is contemplated that the first robot600acan have an arm coupled to the first nozzle assembly508aand the second robot600bcan have an arm coupled to the second nozzle assembly508b. It is further contemplated that the arm of the first robot600acan be configured for selective movement to impart corresponding movement to the first nozzle assembly508aand that the arm of the second robot600bcan be configured for selective movement to impart corresponding movement to the second nozzle assembly508b. It is further contemplated that the first and second robots600a,600bcan be communicatively coupled to the at least one controller800. It is contemplated that the at least one controller800that is coupled to the first and second robots600a,600bcan function as a central controller of each of the components of the system100disclosed herein. However, it is also contemplated that the disclosed robots600a,600bcan have their own dedicated controller(s) that can optionally be positioned in communication with other controllers of the system100. In exemplary aspects, the robots disclosed herein can be configured for rotational and/or axial movement relative to a plurality of axes, such as, for example and without limitation, six axes. Optionally, in these aspects, it is contemplated that the robots can comprise a Selective Compliance Assembly Robot Arm (SCARA) apparatus as is known in the art, such as SCARA robots manufactured by Epson Robots (Carson, CA).

Alternatively, in other optional aspects, and as shown inFIGS.1-3B, the object orientation apparatus200can further comprise a base assembly206. In these aspects, the first and second nozzle assemblies can be independently rotationally coupled to the base assembly. Optionally, as shown inFIG.1, it is contemplated that the object orientation apparatus200can further comprise a robot that is coupled to the base assembly206.

In one aspect, the first nozzle assembly208acan comprise a first plate220athat is secured to the vacuum nozzle202aand at least one rotational actuator of the first nozzle assembly and rotationally coupled to the base assembly206such that rotation of the first plate effects a corresponding rotation of the vacuum nozzle. In this aspect, the second nozzle assembly208bcan comprise a second plate220bthat is secured to the vacuum nozzle202band at least one rotational actuator of the second nozzle assembly and rotationally coupled to the base assembly206such that rotation of the second plate effects a corresponding rotation of the second vacuum nozzle.

In another aspect, the base assembly206can comprise a transverse arm244and first and second nozzle assembly supports242a,242bthat are slidably coupled to the transverse arm. In this aspect, the first plate220acan be rotationally coupled to the first nozzle assembly support242a, and the second plate220bcan be rotationally coupled to the second nozzle assembly support242b. It is further contemplated that the first and second nozzle assembly supports242a,242bcan be selectively and independently moveable relative to the transverse axis240to move the vacuum nozzles202a,202bof the first and second nozzle assemblies208a,208brelative to the transverse axis. In a further aspect, the object orientation apparatus200,500can further comprise first and second transverse actuators respectively secured to the first and second nozzle assembly supports242a,242b. In this aspect, the at least one controller800can be configured to control movement of the first and second nozzle assembly supports242a,242brelative to the transverse axis240.

Optionally, in various aspects, it is contemplated that the vacuum nozzles202a,202bcan be slidably coupled to the first and second plates220a,220bto thereby permit axial movement of the vacuum nozzles. In these aspects, it is contemplated that the plates220a,220bcan define respective slide elements203a,203bthat are coupled to the vacuum nozzles and permit axial movement of the vacuum nozzles relative to the support plates. As shown inFIGS.2A-2B, the slide elements203a,203bcan be oriented parallel to the longitudinal axes of the vacuum nozzles to permit compliant movement of the nozzles relative to their longitudinal axes. It is contemplated that the slide elements203a,203bcan be configured to provide minimal inertial interference with axial movement of the vacuum nozzles. In further aspects, it is contemplated that the nozzle assemblies208a,208bcan comprise an actuator201a,201bthat is configured to effect axial movement of the vacuum nozzles202a,202b. Optionally, in these aspects, it is contemplated that the actuators201a,201bcan comprise a blow down mechanism for delivering pressurized air. Alternatively, in other aspects, it is contemplated that the actuators201a,201bcan comprise a pneumatic cylinder or other conventional mechanical actuator. As shown inFIGS.2A-2B, it is contemplated that the vacuum nozzles202a,202bcan have an elbow structure that is supported by a housing, with the housing being slidably coupled to the plates220a,220b.

In various exemplary aspects, the object orientation apparatus200,500can further comprise at least one camera107positioned in communication with the at least one controller800. In these aspects, the at least one camera107can be configured to produce at least one image of an object (e.g., embryo) retained by the vacuum nozzle202a,502aof the first nozzle assembly208a,508a. An exemplary side elevational view of an embryo held against the distal end of a vacuum nozzle is provided inFIG.8B. In operation, the at least one controller800can be configured to determine an orientation of the object (e.g., embryo) based upon the at least one image produced by the at least one camera. It is contemplated that the at least one controller800can use conventional optical recognition techniques to evaluate the shape of the retained object to determine the orientation of the object. In exemplary aspects, the at least one controller can be configured to determine if the orientation of the object corresponds to a desired orientation of the object. In response to determining the orientation of the object does not correspond to the desired orientation, the at least one controller800can be configured to selectively activate the at least one rotational actuator of each actuation subassembly205a,205b,505a,505bto move the first and second vacuum nozzle assemblies208a,208b,508a,508bto the object-transfer positions. When the actuation subassemblies205a,205b,505a,505bof the nozzle assemblies208a,208b,508a,508balso comprise at least one linear (axial) actuator configured to effect axial movement of the vacuum nozzle202a,202b,502a,502bof the nozzle assembly relative to at least one axis, the controller can be configured to selectively activate the at least one axial actuator of each actuation subassembly to move the first and second vacuum nozzle assemblies to the object-transfer positions.

In various aspects, the object orientation apparatus can further comprise at least one vacuum source positioned in communication with the at least one controller800and the vacuum nozzles202a,202b,502a,502bof the first and second nozzle assemblies208a,208b,508a,508b. In these aspects, the at least one controller800can be configured to selectively and independently adjust the application of vacuum force to the vacuum nozzles202a,202b,502a,502bof the first and second nozzle assemblies208a,208b,508a,508b. In further aspects, the object orientation apparatus can further comprise at least one positive pressure source positioned in communication with the at least one controller800and the vacuum nozzles202a,202b,502a,502bof the first and second nozzle assemblies208a,208b,508a,508b. In these aspects, the at least one controller800can be configured to selectively and independently adjust the application of positive to the vacuum nozzles202a,202b,502a,502bof the first and second nozzle assemblies208a,208b,508a,508b.

In exemplary aspects, the disclosed object orientation apparatus200,500can be used in a method for positioning an object (e.g., an embryo) in a desired orientation. In these aspects, the method can comprise selectively activating the actuation subassembly of the first nozzle assembly to position the vacuum nozzle of the first nozzle assembly in the object release/retrieval position. In another aspect, the method can comprise applying suction through the vacuum nozzle of the first nozzle assembly to retain an object (e.g., embryo) against the distal end of the vacuum nozzle. In a further aspect, the method can comprise producing an image of the object (e.g., embryo) using the camera. In still another aspect, the method can comprise determining an orientation of the object (e.g, embryo) using the controller.

In exemplary aspects, the method can further comprise using the controller to compare the orientation of the object (e.g., embryo) to a desired orientation of the object (e.g., embryo). In response to determining that the orientation of the object corresponds to the desired orientation, the controller can selectively activate at least one actuator of the actuation subassembly of the first nozzle assembly to return the first nozzle assembly to the object release/retrieval position. Optionally, with the first nozzle assembly in the object release/retrieval position, the controller can cause the vacuum source to cease application of suction. In conjunction with ceasing application of suction, the controller can also activate a positive pressure source to apply positive pressure through the vacuum nozzle of the first nozzle assembly to ensure that the object is detached from the distal end of the vacuum nozzle and delivered to a selected location, such as, for example, growth media. In response to determining that the orientation of the object (e.g., embryo) does not correspond to the desired orientation, the controller can selectively activate at least one actuator of the actuation subassemblies of the first and second nozzle assemblies to move the first and second nozzle assemblies to the object-transfer position. With the first and second nozzle assemblies in the object-transfer position, the controller can: activate a vacuum source to apply suction through the vacuum nozzle of the second nozzle assembly to retain the object (e.g., embryo) against the distal end of the vacuum nozzle of the second nozzle assembly; and activate a positive pressure source to apply positive pressure through the vacuum nozzle of the first nozzle assembly to detach the object from the distal end of the vacuum nozzle of the first nozzle assembly. More specifically, with the first and second nozzle assemblies in the object-transfer position and the object retained against the distal end of the first nozzle assembly, the controller can initially activate a vacuum source to apply negative pressure through the vacuum nozzle of the second nozzle assembly and also, either concurrently or shortly thereafter, cause a vacuum source to cease application of negative pressure through the first nozzle assembly. Next, the controller can activate a positive pressure source to apply positive pressure through the vacuum nozzle of the first nozzle assembly as the second nozzle assembly is moved away from the object-transfer position. It is contemplated that the positive pressure applied through the vacuum nozzle can be sufficient to completely eliminate any negative pressure within the vacuum tube and/or nozzle assembly while remaining low enough to avoid disturbance of objects (e.g., embryos) that have already been placed in a desired location (e.g., growth media) as further disclosed herein. With the object retained against the distal end of the vacuum nozzle of the second nozzle assembly, the controller can selectively activate at least one actuator of the actuation subassembly of the second nozzle assembly to move the second nozzle assembly to the object release/retrieval position. With the second nozzle assembly in the object release/retrieval position, the controller can cause the vacuum source to cease application of suction. In conjunction with ceasing application of suction, the controller can also activate a positive pressure source to apply positive pressure through the vacuum nozzle of the first nozzle assembly to ensure that the object is detached from the distal end of the vacuum nozzle and delivered to a selected location, such as, for example, growth media.

IV. Systems and Methods of Singulating and Adjusting the Orientation of Extracted Embryos or Other Objects

In exemplary aspects, and with reference toFIGS.1,6A-6C,8A, and9, it is contemplated that the disclosed singulation apparatus300and the disclosed object orientation apparatus200,500can be provided together as part of a system100. As shown inFIG.1, in addition to the singulation apparatus300and the object orientation apparatus200,500, the system100can further comprise petri dish assemblies101,102, growth containers (e.g., STERIVENT tray assemblies manufactured by Duchefa Biochemie)103,104, a background screen105, at least one plating station106, and a camera107. In exemplary aspects, it is contemplated that the robots disclosed herein can be configured to selectively control movement of the petri dishes and growth containers to various locations within the system, including, for example and without limitation, a lid removal station, an inspection/plating station, and an input/output stacking station. The at least one controller800of the system100can comprise additional cameras and/or optical/visualization equipment as required to permit analysis and identification of selected characteristics of embryos positioned within the petri dishes and growth containers within the system. In exemplary aspects, these cameras and/or optical/visualization equipment can provide information that the controller800can use to determine appropriate movements of the robots within the system. For example, if the cameras and/or optical visualization equipment identifies embryos in a petri dish that have selected characteristics, the robot can be activated to retrieve those selected embryos from the petri dish and transfer the embryos to a second location for further processing.

In exemplary aspects, and with reference toFIGS.6A-6C, it is contemplated that embryos can first enter the system100by being placed directly within a singulation apparatus300as disclosed herein or by being positioned within a petri dish that is provided to the system.

In exemplary aspects, the petri dish can contain selection media, such as, for example and without limitation, a selection medium useful in a conventional doubled haploid process. In these aspects, it is contemplated that the double haploid process can optionally be used in conjunction with plant breeding process as are known in the art. Optionally, the selection medium can comprise an antimitoticor chromosome doubling agent (e.g., colchicine, oryzalin, or trifluralin) as is known in the art. It is contemplated that the placement of an embryo in the doubling media can cause the doubling of the chromosomes of the embryo. After identification of the doubled haploids using conventional camera and visualization systems, the doubled haploids can be selectively transferred from the petri dish to a growth container (e.g., a STERIVENT tray assembly) as disclosed herein, and germination can begin.

FIG.6Adepicts the initial step of transferring an embryo from a petri dish to a growth container, such as, for example and without limitation, a STERIVENT tray assembly. It is contemplated that a similar process can be followed to transfer a singulated embryo from a singulation apparatus as disclosed herein to a growth container. In either case, the growth container can comprise a selected growth medium. It is contemplated that an embryo positioned in the growth medium as disclosed herein can be used in plant breeding as is known in the art. Generally, in aspects, it is contemplated that following positioning of the embryo into or onto the growth medium, the resulting tissue can be used in plant breeding applications as are known in the art.

Following removal of the lid of a selected petri dish (in the case of embryos plated on a petri dish) or following singulation (in the case of embryos positioned on the screen of a simulation apparatus), the at least one controller800can determine the locations of the embryos that are to be transferred to a second location (e.g., a culture container) and selectively control movement of the first nozzle assembly to engage a selected embryo. Following engagement with the selected embryo, the at least one controller can determine the orientation of the engaged embryo and transfer the engaged embryo to a second nozzle assembly (FIG.6B) as appropriate. As shown inFIG.6C, when the embryo is positioned in the correct orientation for growth, the at least one controller can effect movement of the appropriate nozzle assembly to place the embryo in a desired location within the growth media of a culture container.

All publications and patent applications mentioned in the specification are indicative of the level of those skilled in the art to which this invention pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, certain changes and modifications may be practiced within the scope of the appended claims.