Patent Publication Number: US-2020284756-A1

Title: Pens for Biological Micro-Objects

Description:
CROSS REFERENCE TO RELATED APPLICATION(S) 
     This application is a non-provisional (and thus claims the benefit) of U.S. provisional patent application Ser. No. 61/720,956 (filed Oct. 31, 2012), which is incorporated by reference herein in its entirety. 
    
    
     BACKGROUND 
     In bioscience fields, activities of biological micro-objects such as cells are often studied and analyzed. For example, cells that produce at least a minimum number of clones or secrete desired materials can be utilized in the production of medicines or in the study of diseases. It can thus be advantageous to identify cells that produce clones at or above a minimum rate or that secrete certain materials. Embodiments of the present invention are directed to improved micro-fluidic devices and processes for placing selected biological micro-objects into holding pens, conditioning the micro-objects in the pens, monitoring biological activity of the micro-objects in the pens, and/or moving the micro-objects whose biological activity meets a predetermined threshold from the pens for further use or processing. 
     SUMMARY 
     In some embodiments, a method of processing biological micro-objects can include actively placing individual biological micro-objects in interior spaces of holding pens in a micro-fluidic device and providing a flow of a first liquid medium to the pens over a time period. The method can also include, while providing the flow, impeding direct flow of the first medium from the flow into the interior spaces of the holding pens. 
     In some embodiments, a micro-fluidic apparatus can include a housing and holding pens. The housing can be disposed on a base, and the housing can include a flow path for a first liquid medium. The holding pens can be disposed within the housing, and each pen can comprise an enclosure enclosing an interior space. The enclosure can be structured to hold a biological micro-object suspended in a second liquid medium and impede a direct flow of the first medium into the second medium in the interior space. 
     A method of processing biological micro-objects can include creating virtual holding pens in a micro-fluidic device by directing a pattern of light in the form of the holding pens into the micro-fluidic device and thereby activating dielectrophoresis (DEP) electrodes. The method can also include placing individual biological micro-objects into the holding pens, where each of the holding pens isolates any one or more of the individual micro-objects in the holding pen from all of the micro-objects outside of the holding pen. The method can also include providing the micro-objects in the holding pens with a common flow of a liquid medium over a time period. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG. 1A  illustrates an example of a micro-fluidic device according to some embodiments of the invention. 
         FIG. 1B  is a side, cross-sectional view of the device of  FIG. 1A . 
         FIG. 1C  is a top, cross-sectional view of the device of  FIG. 1A . 
         FIG. 2  shows a cross-sectional side view of the device of  FIG. 1A  illustrating an example of the device configured with an optoelectronic tweezers (OET) device according to some embodiments of the invention. 
         FIG. 3  is a top, cross-sectional partial view of the device of  FIG. 1A  configured with the OET device of  FIG. 2  and virtual pens according to some embodiments of the invention. 
         FIG. 4  is a top, cross-sectional partial view of the device of  FIG. 1A  configured with the OET device of  FIG. 2  and pens that are physical and/or virtual according to some embodiments of the invention. 
         FIG. 5A  illustrates an example of a micro-fluidic structure disposed on a base that defines a fluidic channel and pens according to some embodiments of the invention. 
         FIG. 5B  is a top, cross-sectional view of the micro-fluidic structure and base of  FIG. 5A . 
         FIG. 6A  illustrates an example of a micro-fluidic structure disposed on a base that defines fluid channels and pens according to some embodiments of the invention. 
         FIG. 6B  is a top, cross-sectional view of the micro-fluidic structure and base of  FIG. 6A . 
         FIG. 7  illustrates an example of a variation of the pens shown in  FIG. 6B  according to some embodiments of the invention. 
         FIGS. 8A, 8B, 9, and 10  illustrate examples of alternative configurations of pens according to some embodiments of the invention. 
         FIGS. 11A and 11B  illustrated selecting and moving a micro-object using a light trap according to some embodiments of the invention. 
         FIGS. 12A and 12B  show selecting and moving a micro-object using a virtual barrier according to some embodiments of the invention. 
         FIG. 13A  illustrates an example of a micro-fluidic structure disposed on a base that defines a fluid chamber and pens according to some embodiments of the invention. 
         FIG. 13B  is a top, cross-sectional view of the micro-fluidic structure and base of  FIG. 13A . 
         FIGS. 14 and 15  show examples of alternative configurations of the pen array of  FIG. 13B  according to some embodiments of the invention. 
         FIG. 16  shows an example of a process that includes placing biological micro-objects into pens in a micro-fluidic device according to some embodiments of the invention. 
         FIG. 17  illustrates a process showing an example of operation of the device of  FIG. 1A  configured with the OET device of  FIG. 2  according to some embodiments of the invention. 
         FIGS. 18A-18C  illustrate an example of processing cells in accordance with a step of  FIG. 17 . 
         FIG. 19  illustrates an example of expanding pens as clones grown in the pens according to a step of  FIG. 17 . 
         FIG. 20  illustrates an example of turning off pens in which clones are growing too slowly and flushing the clones in those pens away according to a step of  FIG. 6 . 
         FIG. 21  illustrates an example of placing daughter clones in new pens according to a step of  FIG. 17 . 
         FIG. 22  illustrates a process showing another example of operation of the device of  FIG. 1A  configured with the OET device of  FIG. 2  according to some embodiments of the invention. 
     
    
    
     DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS 
     This specification describes exemplary embodiments and applications of the invention. The invention, however, is not limited to these exemplary embodiments and applications or to the manner in which the exemplary embodiments and applications operate or are described herein. Moreover, the Figures may show simplified or partial views, and the dimensions of elements in the Figures may be exaggerated or otherwise not in proportion for clarity. In addition, as the terms “on,” “attached to,” or “coupled to” are used herein, one element (e.g., a material, a layer, a substrate, etc.) can be “on,” “attached to,” or “coupled to” another element regardless of whether the one element is directly on, attached, or coupled to the other element or there are one or more intervening elements between the one element and the other element. Also, directions (e.g., above, below, top, bottom, side, up, down, under, over, upper, lower, horizontal, vertical, “x,” “y,” “z,” etc.), if provided, are relative and provided solely by way of example and for ease of illustration and discussion and not by way of limitation. In addition, where reference is made to a list of elements (e.g., elements a, b, c), such reference is intended to include any one of the listed elements by itself, any combination of less than all of the listed elements, and/or a combination of all of the listed elements. 
     The words “substantially” and “generally” mean sufficient to work for the intended purpose. The term “ones” means more than one. 
     The term “cell” refers to a biological cell. The term “clones,” with reference to cells, means cells that are identical because each cell was grown from the same parent cell. Clones are thus all “daughter cells” of the same parent cell. 
     As used herein, the term “biological micro-object” includes biological cells and compounds such as proteins, embryos, plasmids, oocytes, sperms, genetic material (e.g., DNA), transfection vectors, hydridomas, transfected cells, and the like as well as combinations of the foregoing. 
     As used herein a dielectrophoresis (DEP) electrode refers to a terminal on or a region of an inner surface of a chamber for containing a liquid medium at which DEP forces in the medium sufficient to attract or repel micro-objects in the medium can be selectively activated and deactivated. 
     The term “flow,” as used herein with reference to a liquid or gas, includes without limitation a continuous, pulsed, periodic, random, intermittent, or reciprocating flow of the liquid or gas. A “convection flow” is a flow of a liquid or gas that is driven by pressure. A “diffusive flow” or “diffusion” is a flow of liquid or gas that is driven by random thermal motion. The term “diffusive mixing” as used with respect to two or more liquid or gas media means the mixing of the media due to spontaneous intermingling of the media as a result of random thermal motion. The term “substantially,” as used herein with respect to “convection flow,” diffusive flow,” “diffusion,” or “diffusive mixing,” means more than fifty percent. 
     The term “deterministic,” when used to describe selecting or placing a micro-object, means selecting or placing a specifically identified and desired micro-object from a group of micro-objects. Deterministically selecting or placing a micro-object thus does not include randomly selecting or placing merely any one of the micro-objects in a group or sub-group of micro-objects. 
     As used herein, the meaning of the term “processing” a micro-object includes any one or more of the following: moving (e.g., in a flow of liquid medium, with an OET device, or the like), sorting, and/or selecting one or more of the micro-objects; modifying one or more of the micro-objects, wherein examples of such modifying include growing populations of micro-objects that are cells or other living biological entities, fusing two or more such micro-objects together, and transfecting one or more micro-objects; monitoring micro-objects; monitoring growth, secretions, or the like of micro-objects that are cells or other living biological entities; and/or conditioning micro-objects that are cells or other living biological entities. 
     Embodiments of the invention include deterministically placing individual biological micro-objects in holding pens in a micro-fluidic device. A flow of a first liquid medium can be provided to the pens, but the pens can be structured to impede a direct flow of the first medium into a second medium in the pens while allowing diffusive mixing of the first medium in the flow and the second medium in the pens. 
       FIGS. 1A-1C  illustrate an example of a micro-fluidic device  100  according to some embodiments of the invention. As shown, the micro-fluidic device  100  can comprise a housing  102 , an electrode mechanism  108 , and a monitoring mechanism  118 . As also shown, the housing  102  can comprise an interior chamber  110  for holding one or more liquid media  114  in which a plurality of biological micro-objects  116  can be suspended. The media  114  can be disposed on an inner surface  120  of the chamber  110 . A plurality of holding pens  112  for the micro-objects  116  can be disposed in the chamber  110 . As will be seen, each pen  112  can be a virtual pen, a physical pen, and/or a combination virtual/physical pen. 
     The media  114  in the device  100  can comprise, for example, a first medium  122  and a second medium  124 . The first medium  122  can be media  114  that is in the flow path  126 , and the second medium  124  can be media  114  that is inside the holding pens  112 . The first medium  122  can be the same type of medium as the second medium  124 . Alternatively, the first medium  122  can be a different type of medium than the second medium  124 . 
     The housing  102  can comprise an enclosure that defines the chamber  110 . As shown, the housing  102  can also comprise one or more inlets  104  through which media  114  and micro-objects  116  can be input into the chamber  110 . There can be one or more flow paths  126  in the chamber  110  for the media  114 . For example, as illustrated in  FIG. 1C , the chamber  110  can comprise a flow path  126  for media  114  from the inlet  104  to the outlet  106 . 
     An inlet  104  can be, for example, an input port, an opening, a valve, a channel, or the like. The housing  102  can also comprise one or more outlets  106  through which media  114  and micro-objects  116  can be removed. Micro-objects  116  can alternatively be removed from the housing  102  in other ways. For example, as noted below, a needle-like aspirator (not shown) can pierce the housing  102 , and one or more micro-objects  116  can be removed with the aspirator. An outlet  106  can be, for example, an output port, an opening, a valve, a channel, or the like. As another example, the outlet  106  can comprise a droplet outputting mechanism such as any of the outputting mechanisms disclosed in U.S. patent application Ser. No. 13/856,781 filed Apr. 4, 2013 (attorney docket no. BL1-US). All or part of the housing  102  can be gas permeable to allow gas (e.g., ambient air) to enter and exit the chamber  110 , for example, to sustain the biological micro-objects  116  in the chamber  110 . For example, a flow of gas can be applied to the gas permeable portion of the housing  102 . For example, a pulsed, regulated, or otherwise controlled flow of gas can be applied as needed (e.g., when testing indicates that micro-objects (e.g., cells) in the housing  102  need gas). 
     Although not shown, the device  100  can comprise sensors or similar components that detect relevant conditions of media  114  or the chamber  110  such as temperature, the chemical composition of media  114  (e.g., the level of dissolved oxygen, carbon dioxide, or the like in media  114 ), the pH of media  114 , osmolarity of media  114 , or the like. The housing  102 , for example, can comprise such sensors or components, which can be configured with a controller (not shown) to control input of media  114  through the inlet  104  to maintain constant or controllably adjust certain conditions (such as the conditions identified above) of media  114 . 
     The electrode mechanism  108  (shown in  FIG. 1B ) can be configured to create selectively electrokinetic forces on micro-objects  116  in media  114 . For example, the electrode mechanism  108  can be configured to selectively activate (e.g., turn on) and deactivate (e.g., turn off) dielectrophoresis (DEP) electrodes at the inner surface  120  of the chamber  110  on which media  114  is disposed. The DEP electrodes can create forces in media  114  that attract or repel micro-objects  116 , and the electrode mechanism  108  can thus select and/or move one or more of the micro-objects  116  in media  114 . For example, in some embodiments, the electrode mechanism  108  can be configured such that hardwired electrical connections to the DEP electrodes at the inner surface  120  can activate and deactivate the individual DEP electrodes. In other embodiments, the individual DEP electrodes at the inner surface  120  can be optically controlled. An example comprising an optoelectronic tweezers mechanism is illustrated in  FIG. 2  and discussed below. 
     For example, the electrode mechanism  108  can include one or more optical (e.g., laser) tweezers devices and/or one or more optoelectronic tweezers (OET) devices (e.g., as disclosed in U.S. Pat. No. 7,612,355, which is incorporated in its entirety by reference herein). As yet another example, the electrode mechanism  108  can include one or more devices (not shown) for moving a droplet of media  114  in which one or more of the micro-objects  116  are suspended. Such devices (not shown) can include electrowetting devices such as optoelectronic wetting (OEW) devices (e.g., as disclosed in U.S. Pat. No. 6,958,132). The electrode mechanism  108  can thus be characterized as a DEP device in some embodiments. 
     The monitoring mechanism  118  can comprise any mechanism for observing, identifying, or detecting individual micro-objects  116  in media  114 . In some embodiments, the monitoring mechanism  118  can also comprise a mechanism for monitoring biological activity or a biological state of micro-objects  116  in the pens  112 . 
     As shown in  FIG. 2 , the monitoring mechanism  118  can comprise an imaging device  220 . For example, the imaging device  220  can comprise a camera or similar device for capturing images of micro-objects  116  in the chamber  110 , including in the pens  112 . As also shown, a controller  218  can control the imaging device  220  and process images captured by the imaging device  220 . Although shown as disposed below the device  102  in  FIG. 2 , the imaging device  220  can be disposed in other locations such as above or to the side of the device  102 . 
     As also shown in  FIG. 2 , the electrode mechanism  108  can comprise an OET device. For example, as shown, the electrode mechanism  108  can comprise a first electrode  204 , a second electrode  210 , an electrode activation substrate  208 , and a power source  212 . As shown, media  114  in the chamber  110  and the electrode activation substrate  208  can separate the electrodes  204 ,  210 . A pattern of light  216  from the light source  214  can selectively activate a desired pattern of individual DEP electrodes at the inner surface  120  of the chamber  110 . That is, light in the light pattern  216  can reduce the electrical impedance of the electrode activation substrate  208  at a pattern of small “electrode” regions of the inner surface  120  of the chamber  110  to less than the impedance of the media  114 . The foregoing creates an electric field gradient in the media  114  from the electrode region of the surface  120  to the first electrode  204 , which in turn creates local DEP forces that attract or repel nearby micro-objects  116 . Different patterns of individual DEP electrodes that attract or repeal micro-objects  116  in media  114  can thus be selectively activated and deactivated at many different such electrode regions at the inner surface  120  of the chamber  110  by different light patterns  216  projected form a light source  214  (e.g., a laser source or other type of light source) into the micro-fluidic device  100 . 
     In some embodiments, the electrode activation substrate  208  can be a photoconductive material, and the inner surface  120  can be featureless. In such embodiments, the DEP electrodes can be created anywhere and in any pattern on the inner surface  120  of the chamber  110  in accordance with the light pattern  126  (see  FIG. 2 ). Examples are illustrated in the aforementioned U.S. Pat. No. 7,612,355 in which the undoped amorphous silicon material  24  shown in the drawings of the foregoing patent can be an example of photoconductive material that can compose the electrode activation substrate  208 . 
     In other embodiments, the electrode activation substrate  208  can comprise a circuit substrate such as a semiconductor material comprising a plurality of doped layers, electrically insulating layers, and electrically conductive layers that form semiconductor integrated circuits such as known in semiconductor fields. In such embodiments, electric circuit elements can form electrical connections between electrode regions at the inner surface  120  of the chamber  110  and the second electrode  210  that can be selectively activated and deactivated by changing patterns of the light pattern  216 . When not activated, each electrical connection can have high impedance such that the voltage drop from a corresponding electrode region at the inner surface  120  of the chamber  110  to the second electrode  210  is greater than the voltage drop from the first electrode  204  through media  114  to the corresponding electrode region. When activated by light in the light pattern  216 , however, each electrical connection can have low impedance such that the voltage drop from a corresponding electrode region at the inner surface  120  of the chamber  110  to the second electrode  210  is less than the voltage drop from the first electrode  204  through media  114  to the corresponding electrode region, which activates a DEP electrode at the corresponding electrode region as discussed above. DEP electrodes that attract or repeal micro-objects  116  in media  114  can thus be selectively activated and deactivated at many different “electrode” regions at the inner surface  120  of the chamber  110  by the light pattern  216 . Non-limiting examples of such configurations of the electrode activation substrate  208  include the phototransistor-based OET device  200  illustrated in FIGS. 21 and 22 of U.S. Pat. No. 7,956,339. 
     In some embodiments, the first electrode  204  can be part of an upper wall  202  of the housing  102 , and the electrode activation substrate  208  and second electrode  210  can be part of a lower wall  206  of the housing  102  generally as illustrated in  FIG. 2 . As shown, the upper wall  202  and lower wall  206  can define the chamber  110 , and media  114  can be disposed on the inner surface  120  of the chamber  110 . The foregoing, however, is but an example. In other embodiments, the first electrode  204  can be part of the lower wall  206  and one or both of the electrode activation substrate  208  and/or second electrode  210  can be part of the upper wall  202 . As another example, the first electrode  204  can be part of the same wall  202  or  206  as the electrode activation substrate  208  and the second electrode  210 . For example, the electrode activation substrate  208  can comprise the first electrode  204  and/or the second electrode  210 . Moreover, the light source  214  can alternatively be located below the housing  102 , and/or the imaging device  220  and the light source  214  can alternatively be located on the same side of the housing  102 . 
     As mentioned, in some embodiments of the invention, part or all of each pen  112  can be “virtual,” which as used herein, means that part or all of the pen  112  comprises DEP forces from activated DEP electrodes at electrode regions of the interior surface  120  of the chamber  110  (as discussed above) rather than physical barriers. 
       FIG. 3  (which shows a partial, top cross-sectional view of part of the housing  102 ) illustrates an example of the device  100  of  FIGS. 1A-1C  in which the pens  112  (which are designated  302  in  FIG. 3 ) are virtual pens  302  according to some embodiments of the invention. The virtual pens  302  in  FIG. 3  can be created in the chamber  110  by the electrode mechanism  108  configured, for example, as the OET device of  FIG. 2 . That is, the virtual pens  302  can comprise a pattern of activated DEP electrodes at the inner surface  120  of the chamber  110 . Although one micro-object  116  is shown in each pen  302 , there can alternatively be more than one micro-object  116  in each pen. 
     As shown in  FIG. 3 , a flow  314  of media  114  through the chamber  110  can be provided in a flow path  126 . As illustrated in  FIG. 3 , each pen  302  can isolate the micro-object(s)  116  in the pen  302  from the micro-objects  116  in the other pens  302 . The flow  314  of media  114 , however, can be a common flow  314  provided to some or all of the pens  302  and thus the micro-objects  116  in the pens  302 . Configured as shown in  FIG. 3 , each pen  302  can thus isolate the micro-object(s)  116  inside the pen  302  from micro-objects  116  outside of the pen  302  including micro-objects  116  in other pens  302  and thus prevent a micro-object  116  from outside of a particular pen  302  from mixing with the micro-object(s) inside that particular pen  302  while allowing a common flow  314  of media  114  to flow into (by convection flow) and out of multiple pens  116  and thus, for example, supply nutrients and carry away waste from micro-objects  116  in multiple pens  116 . 
     The virtual pens  302  can comprise light enclosures in the light pattern  216  projected by the light source  214  into the housing  102  of the micro-fluidic device  100  as shown in  FIG. 2 . The power source  212  of the OET of  FIG. 2  can be configured with a frequency that causes the light enclosure that defines each pen  302  to repel a micro-object  116  so that each pen  302  holds a micro-object  116  inside the pen  302 . Moreover, one or more of the virtual pens  302  can be moved, expanded or contracted, turned off, or the like by changing the light pattern  216  projected into the housing  102 . 
     As shown in  FIG. 3 , the OET device depicted in  FIG. 2  can also create a light trap  304  (e.g., cage) that traps a micro-object  116  to select and move the micro-object  116 . The light trap  304  can be, for example, a light cage that traps the micro-object  116 . The frequency of the power source  212  in  FIG. 2  can be such that the light trap  304  repels the selected micro-object  116 . The micro-object  116  can thus be moved in the chamber  110  by moving the light trap  304  on the electrode activation substrate  208 . The detector  220  can capture images of the micro-objects  116  in the channel  110  (e.g., a flow path  126 ), which can be an example of a common space. Specific, desired individual ones of the micro-objects  116  can thus be identified and then selected with the selector  118  (e.g., configured as the OET device of  FIG. 2 ), for example, with light traps  302 ,  412  as discussed below with respect to  FIGS. 3 and 4 . The detector  220  and selector  118  (e.g., configured as the OET device of  FIG. 2 ) can thus be examples of a means for deterministically selecting or placing one or more of the micro-objects  116 . 
     Although illustrated as squares in  FIG. 3 , the pens  302  can alternatively be other shapes. For example, the pens  302  can be circles, ovals, rectangles, triangles, or the like. Moreover, the pens  302  need not be fully enclosed. For example, any of the pens  302  can have an opening  308  as illustrated by pen  302   a  in  FIG. 3 . Although illustrated as a circle, the light trap  304  can be other shapes such as square, oval, rectangular, triangular, or the like. In addition, the pens  302  can be difference sizes and can be disposed in different orientations. 
       FIG. 4  (which shows a partial, top cross-sectional view of part of the housing  102 ) illustrates another example of a configuration of the device  100  of  FIGS. 1A-1C . In the configuration illustrated in  FIG. 4 , the pens  112  (which are designated  402  in  FIG. 4 ) can be entirely physical or both physical and virtual. For example, as shown, each pen  402  can comprise a physical barrier  404  (e.g., as part of the housing  102 ), which can define or be part of an enclosure  406  with an opening  408  that is in fluidic communication (e.g., contact) with a flow  314  of media  114  through the chamber  110 . 
     Generally as discussed with respect to  FIG. 3 , a flow  314  of media  114  through the chamber  110  can be provided in a flow path  126 . Each pen  402  can isolate the micro-object(s)  116  in the pen  402  from the micro-objects  116  in the other pens  302 . For example, each pen  302  can prevent any micro-object  116  outside the pen  302  from mixing with any of the micro-objects inside the pen  302 . The flow  314  of media  114 , however, can be a common flow  314  provided to all of the pens  402  and thus all of the micro-objects  116  in the pens  402 . The pens  402 , however, can be structured so that the first medium  122  from the flow  314  does not flow directly into any of the pens  402 , but the structure of the pens  402  can allow diffusive mixing of the first medium  122  from the flow  314  and the second medium  124  inside the pens  402 . 
     For example, the barrier  404  of each pen  402  can be shaped and oriented to impede direct flow of the first medium  122  from the flow  314  in the flow path  126  into the pen  402 . For example, each pen  402  can be shaped and oriented such that a portion of the physical barrier  404  directly faces the direction of the flow  314  but no opening (e.g., the opening  408 ) directly faces the direction of the flow  314 . In the example illustrated in  FIG. 3 , each of the pens  402  thus impede direct flow of the first medium  122  from the flow  314  in the flow path  126  into the pen  402 . 
     As another example, the barrier  404  can be shaped and oriented to prevent convection flow of the first medium  122  from the flow  314  in the flow path  126  into the pen  402 . Each pen  402  can, however, be shaped and oriented to allow substantially only diffusion mixing of the first medium  122  from the flow  314  in the flow path  126  and the second medium  424  inside the pen  402 . For example, each pen  402  can comprise an opening shaped and oriented to allow such diffusive mixing. 
     In some embodiments, however, the pens  402  can be oriented with the opening  408  pointed in any direction with respect to the flow  314  of media  114 . As also shown, any of the pens  402  can comprise both a physical barrier and a virtual portion. For example, in some embodiments, a virtual door  410  comprising adjacent activated DEP electrodes on the inner surface  120  of the chamber  110  can be created and/or removed at the opening  408  of one or more of the physical barriers  404  to make the pen  402  selectively fully enclosed generally as shown in  FIG. 4 . The virtual door  410  can correspond to light in the light pattern  214  projected onto the electrode activation substrate  208 . (See  FIG. 2 .) 
     As also illustrated in  FIG. 4 , one or more of the pens  402  can comprise more than one such virtual door  410 . For example, as shown, pen  402   a  comprises more than one opening  408   a ,  408   b  into the pen  402   a , and there can be a virtual door  410   a ,  410   b  at each such opening  408   a ,  408   b . In operation, a micro-object  116  can be moved into the pen  402   a  through the first opening  408   a  while the first virtual door  410   a  is turned off, and the micro-object  116  can later be moved out of the pen  402   a  through the second opening  408   b  while the second virtual door  410   b  is turned off. 
     A light trap  412  (which can be similar to or the same as light trap  304 ) can be created on the surface  120  of the chamber  110  by the electrode mechanism  108  configured as the OET device of  FIG. 2 . The light trap  412  can be created that traps a micro-object  116  to select the micro-object  116 . The frequency of the power source  212  in  FIG. 2  can be such that the light trap  412  repels the selected micro-object  116 . The micro-object  116  can thus be moved in the chamber  110  by moving the light trap  412  on the photoconductive layer  308 . For example, a micro-object  116  can be selected and moved into and/or output of a pen  402  by forming a light trap  412  that traps the micro-object  116  and then moving the light trap  412  on the inner surface  102 . 
     Although illustrated as partial squares in  FIG. 4 , the pens  402  can alternatively be other shapes. For example, the pens  402  can be partial circles, ovals, rectangles, triangles, or the like. The light trap  412  can similarly have other shapes than the circle shown. 
     Like pens  302  and  402 , the pens  112  can, in some embodiments, also be configured to impede direct flow (e.g., convection flow) of the first medium  122  from a common flow in a flow path  126  into the pens  112  while allowing substantially only diffusive mixing of the first medium  122  from the common flow  314  in a flow path  126  and the second medium inside a pen  112 . 
     The housing  102 , however, need not be configured with a single common space for media  114 . Rather, the housing  102  can comprise one or more interconnected chambers, channels, or the like for containing media  114  and through which media  114  can flow.  FIGS. 5A-7  illustrate examples. 
     As shown in  FIGS. 5A and 5B , the housing  102  of the device  100  (see  FIGS. 1A-1C ) can comprise a base (e.g., a substrate)  502  on which is disposed one or more micro-fluidic structures  500 . The base  502  can comprise, for example, the lower wall  206  as discussed above with respect to  FIG. 2 , and all or part of the top surfaces of the micro-fluidic structure  500  can comprise the upper wall  202  including any variation discussed above. 
     As shown, the micro-fluidic structure  500  can comprise a channel  504  and pens  506 , each of which can comprise an enclosure  510  and an opening  508  to the channel  504 . As shown, the pens  506  and the channel  504  can be the same or a different height from the base  502 . The channel  504  and pens  506  can correspond to the chamber  110  of  FIGS. 1A-1C and 2 , and the surface  522  of the base  502  can correspond to the inner surface  120  of the chamber  110  of  FIGS. 1A-1C and 2 . Thus, in embodiments of the invention in which the housing  102  comprises the base  502  and the OET device of  FIG. 2 , DEP electrodes can be activated and deactivated in accordance with the light pattern  216  at the surface  522  of the base  502  rather than the inner surface  120  of the chamber  110 . 
     One or more micro-objects  116  can be deterministically selected and moved (e.g., using the detector  220  and selector  118  as discussed above) from the channel  504  (which can be an example of a common space and/or a flow path) through the opening  508  into the enclosure  510  of a pen  506 . The micro-object(s)  116  can then be held for a period of time in the pen  506 . The opening  508  and enclosure  510  of each pen can be sized and configured and the rate of the flow  520  of media  114  in the channel  504  can be such that the flow  520  creates little to no appreciable convection inside the enclosure  510 . Once placed in a pen  506 , micro-object(s)  116  thus tend to stay in the pen  506  until actively removed from the pen  506 . Diffusion through the opening  508  between media  114  in the channel  504  and the enclosure  510  can provide for inflow into the enclosure  510  from the channel  504  of nutrients for the micro-object(s)  116  in a pen  506  and outflow from the enclosure  510  into the channel  504  of waste from the micro-object(s)  116 . 
     The pens  506  can be structured so that a first medium  122  in the flow  520  in the channel  504  does not flow directly into any of the pens  506 , but the structure of the pens  506  allows diffusive mixing of the first medium  122  from the flow  520  through the opening  508  in the pen  506  with a second medium  124  inside the pen  506  generally as discussed above. 
     The channel  504  and the pens  506  can be physical structures as shown in  FIGS. 5A and 5B . For example, the micro-fluidic structure  500  can comprise a flexible material (e.g. rubber, plastic, an elastomer, polydimethylsioxane (“PDMS”), or the like), which can also be gas permeable in some embodiments. Alternatively, the micro-fluidic structure  500  can comprise other materials including rigid materials. Although one channel  504  and three pens  506  are shown, the micro-fluidic structure  500  can comprise more than one channel  504  and more or fewer than three pens  506 . As shown in  FIG. 5B , a virtual door  512  can optionally be created and removed closing and opening the opening  508  of each of the pens  506 . Such virtual doors  512  can be created by activating DEP electrodes at the surface  522  of the base  502  generally as discussed above with regard to the inner surface  120 . 
     Although the channel  504  and pens  506  are illustrated in  FIGS. 5A and 5B  as physical, the channel  504  and pens  506  can alternatively be virtual. For example, all or part of the channel  504  and/or the pens  506  can be created by activating DEP electrodes at the surface  522  of the base  502  generally as discussed above. 
     In the example shown in  FIGS. 6A and 6B , the housing  102  of the device  100  (see  FIGS. 1A-1C ) can comprise the base  502  of  FIGS. 5A and 5B  and a micro-fluidic structure  602  disposed on the surface  522  of the base  502 . As can be seen in  FIG. 6B , the micro-fluidic structure  602  can comprise a pen structure  612 , which can comprise pens  606 . Each such pen  606  can comprise an enclosure  610  in which a micro-object  116  can be placed and held for a time period. As also shown in  FIG. 6B , the micro-fluidic structure  602  can define channels  604 , and the opening  608  of each pen  606  can be in fluidic communication (e.g., contact) with media  114  in one of the channels  604 . 
     One or more micro-objects  116  can be deterministically selected (as discussed above) and moved from one of the channels  604  (which can be an example of a common space and/or a flow path) through the opening  608  into the enclosure  610  of a pen  606 . The micro-object(s)  116  can then be held in a pen  606  for a period of time. Thereafter, the micro-object(s)  116  can be moved from the enclosure  610  through the opening  608  into the channel  604 . Flows  620  of media  114  in the channels  604  can move micro-objects  116  in the channels  604 . 
     Because the openings  608  of the pens  606  are in fluidic communication with a channel  604 , the flows  620  of media  114  in the channels  604  can provide nutrients to the micro-objects  116  in the pens  606  and allow for the outflow of waste from the micro-objects  116  during the period of time that the micro-objects  116  are held in the pens  606 . The flows  620  in the channels  604  can thus constitute a common flow of media  114  to the pens  606 , which like pens  506 , can otherwise physically separate and isolate micro-objects  116 . 
     The pens  606  can be structured so that a first medium  122  in a flow  620  in a channel  604  does not flow directly into any of the pens  606 , but the structure of the pens  606  allows diffusive mixing of the first medium  122  from a flow  620  through an opening  608  in the pen  606  with a second medium  124  in a pen  606 . For example, a pen  606  can be physical (rather than virtual) and the opening  608  of the pen  606  can be oriented in any direction so long as no part of the opening  608  faces directly into a flow  620 . A pen  606  can thus impede direct flow of the first medium  122  into the pen  606 . 
     The pens  606  can be physical structures as shown in  FIG. 6B . For example, the micro-fluidic structure  600  can comprise any of the materials discussed above with respect to the micro-fluidic structure  500  of  FIGS. 5A and 5B . Although two channels  604  and twelve pens  606  are shown in  FIG. 6B , the micro-fluidic structure  602  can comprise more or less than two channels  604  and more or fewer than twelve pens  606 . Although not shown, a virtual door like door  512  of  FIG. 5B  can optionally be created at the openings  608  of one or more of the pens  606 . 
     Although the micro-fluidic structure  602  including the pen structure  612  are shown in  FIGS. 6A and 6B  as physical, all of part of the structure  602  can alternatively be virtual and thus created by activating DEP electrodes at the surface  522  of the base  502  as discussed above with respect to the inner surface  120 . For example, all or part of the pen structure  612  can be virtual rather than physical. 
       FIG. 7  is similar to  FIG. 6B  except that a channel  704  (which can be an example of a flow path  126 ) is disposed between pen structures  712  as shown. Otherwise, each pen  706  can be similar to each pen  606 . For example, each pen  706  can comprise an enclosure  710  in which a micro-object  116  can be placed and held. As also shown in  FIG. 7 , the opening  708  of each pen  706  can be in fluidic communication (e.g., contact) with media  114  in the channel  704 . One or more micro-objects  116  can be deterministically selected (as discussed above) and moved from the channel  704  (which can be an example of a common space) through the opening  708  into the enclosure  710  of a pen  706 , where the micro-object(s)  116  can be held for a period of time. Thereafter, the micro-object(s)  116  can be moved from the enclosure  710  through the opening  708  into the channel  704 . The flow  720  of media  114  in the channels  704  can move micro-objects  116  in the channel  704 . Alternatively or in addition, the micro-objects  116  can be moved by DEP forces, centrifugal forces, and/or the like. 
     Because the openings  708  of the pens  706  are in fluidic communication with the channel  704 , the flow  720  of media  114  in the channel can also provide nutrients to the micro-objects  116  in the pens  706  and provide for the outflow of waste from the micro-objects  116  during the period of time that the micro-objects  116  are held in the pens  706 . The flow  720  in the channel  704  can thus constitute a common flow of media  114  to all of the pens  706 . 
     The pens  706  can be structured so that a first medium  122  in the flow  720  in the channel  704  does not flow directly into any of the pens  706 , but the structure of the pens  706  allows diffusive mixing of the first medium  122  in the channel  704  through an opening  708  in the pen  706  with second medium  124  in a pen  706 . For example, a pen  706  can be physical and can be oriented so that no opening to the pen  706  faces directly into the flow  720 . 
     Although one channel  704  and twelve pens  706  are shown in  FIG. 7 , there can be more or fewer. Although not shown, a virtual door like door  512  of  FIG. 5B  can optionally be created at the openings  708  of one or more of the pens  706 . Although the pen structures  712  are shown in  FIG. 7  as physical, all or part of the pen structures  702  can alternatively be virtual and thus created by activating DEP electrodes at the surface  522  of the base  502  as discussed above with regard to inner surface  120 . 
     The shape and configuration of the pens  506 ,  606 ,  706  (or any pen disclosed herein) illustrated in  FIGS. 5A-7  are examples only, and those pens  506 ,  606 ,  706  (or any pen disclosed herein) can take other shapes and/or configurations. For example, any of pens  506 ,  606 ,  706  (or any pen disclosed herein) can be circular, oval, triangular, or the like rather than square or rectangular. As other examples, any of the pens  506 ,  606 ,  706  (or any pen disclosed herein) can be replaced by the pen  806 ,  826 ,  906 ,  926  illustrated in  FIGS. 8A-10 . 
     As shown in  FIG. 8A , a pen  806  can comprise an opening  812  (e.g., corresponding to openings  506 ,  606 ,  706 ) that is smaller than the full width of the enclosure  810  (e.g., corresponding to enclosures  510 ,  610 ,  710 ). As also shown in  FIG. 8A , a pen  806  can comprise one or more secondary openings  814  (one is shown but there can be more). The opening  812  can be larger than a micro-object  116  (not shown in  FIG. 8A ), and the secondary opening  814  can be smaller than a micro-object  116 . The secondary opening  814  can allow, for example, media  114  (not shown in  FIG. 8A ) to flow into or out of the pen  806 . For example, media  114  can flow into the pen  806  through the opening  812  and out of the pen  806  through the secondary opening  814 . As also shown in  FIG. 8A , the walls of a pen need not be the same thickness. 
     As shown in  FIG. 8B , a pen  826  can comprise an inner wall  834  that extends from an opening  832  (e.g., corresponding to openings  508 ,  608 ,  708 ,  812 ) to create an inner containment space  836  within the enclosure  840  (e.g., corresponding to enclosures  510 ,  610 ,  710 ). 
     As illustrated in  FIG. 9 , a pen  906  can comprise one or more additional pens  916  (one is shown but there can be more). For example, one or more inner pens  916  (one is shown but there can be more) comprising an opening  922  and an enclosure  920  can be disposed inside the enclosure  910  of an outer pen  906 , which can comprise opening  912 . One or more micro-objects  116  (not shown in  FIG. 9 ) can be disposed in the enclosure of each inner pen  916  and the outer pen  906 . 
     As shown in  FIG. 10 , a pen  926  (comprising an opening  932  and enclosure  930 ) can comprise multiple holding spaces  936  (although three are shown, there can be more or fewer) separated by interior walls  934 . One or more micro-objects  116  (not shown in  FIG. 10 ) can be disposed in each holding space  936 . For example, a different type of micro-object  116  can be disposed in each holding space  936 . 
     Any of the pens disclosed herein can be configured to be like or to have any of the characteristics of the pens  806 ,  826 ,  906 ,  926  illustrated in  FIGS. 8A-10 . 
     Regardless of the configuration of the pens, micro-objects  116  can be deterministically selected and moved from the flows  520 ,  620 ,  720  in the channels  504 ,  604 ,  704  into pens  506 ,  606 ,  706  in  FIGS. 5A-7  (including the variations of the pens  506 ,  606 ,  706  illustrated in  FIGS. 8A-10 ) by any of a variety of mechanisms.  FIGS. 11A-12B  illustrate examples in which the OET device of  FIG. 2  is used to do so. In  FIGS. 11A-12B , the channel  1104  can be any of the channels  504 ,  604 ,  704 ; the pen  1106  can be any of the pens  506 ,  606 ,  706 ; and the flow  1120  of media  114  can be any of the flows  520 ,  620 ,  720  in  FIGS. 5A-7 . 
     As shown in  FIG. 11A , a micro-object  116  can be deterministically selected in the flow  1120  in the channel  1104  by creating a light trap  1108  (e.g., like light trap  304 ) that traps the micro-object  116 , which can trap the micro-object  116  in the trap  1108 . As shown in  FIG. 11B , the light trap  1108  can then be moved from the channel  1104  into the pen  1106 , where the micro-object  116  can be released from the light trap  1108 . The light trap  1108  can be like and can be created and moved on the surface  522  of the base  502  by the OET device of  FIG. 2  in the same way as light traps  304 ,  412  are created and moved on the inner surface  120  as discussed above. 
     As shown in  FIG. 12A , a micro-object  116  can be deterministically selected in the flow  1120  in the channel  1104  by creating a virtual barrier  1208  in the path of the micro-object  116  in the channel  1104 . As illustrated in  FIG. 12B , the virtual barrier  1208  can deflect the micro-object  116  into the pen  1106 . The virtual barrier  1208  can be created by activating DEP electrodes on the surface  522  of the base  502  using the OET device of  FIG. 2  generally as discussed above. Once the selected micro-object  116  is deflected into the pen  1106 , the virtual barrier  1208  can be removed from the channel  1104 . 
     As mentioned above, micro-objects  116  can be contained in any of the pens disclosed herein for a period of time after which the micro-objects  116  can be removed from the pens. In some embodiments, micro-objects  116  can be removed from pens in any of the ways illustrated in  FIGS. 11A-12B . 
     For example, a light trap  1108  can be formed that traps a micro-object  116  in a pen  1106  and the light trap  1108  can be moved out of the pen  1106  into the channel  1104 , which is the reverse of the process shown in  FIGS. 11A and 11B . Once in the channel  1104 , the light trap  1108  can be turned off, releasing the micro-object  116  into the flow  1120  of media  114  in the channel  1104 . 
     As another example, a virtual barrier similar to the barrier  1208  shown in  FIGS. 12A and 12B  can be formed in a pen  1106  to nudge a micro-object  116  out of the pen  1106  into the flow  1120  of media  114  in the channel  1104 . The foregoing is the reverse of the process shown in  FIGS. 12A and 12B . 
     As yet another example, any of the physical pens disclosed herein can be configured like the outputting mechanisms  800  disclosed in the aforementioned U.S. patent application Ser. No. 13/856,781 (attorney docket no. BL1-US). In such a configuration, the pens can be configured like the expressing mechanism  804  in the foregoing patent application, and a striking mechanism (not shown) like the striking mechanism  802  in the foregoing patent can be provided to express the micro-objects  116  from the pens. 
       FIGS. 13A and 13B  illustrate a micro-fluidic device  1300  that can be an example of the device  100  of  FIGS. 1A-1C  in which the base  502  and a micro-fluidic structure  1302  are examples of the housing  102 , the chamber  1308  is an example of the chamber  110 , the inlet  1314  is an example of the inlet  104 , the outlet  1316  is an example of the outlet  106 , and the pens  1306  are examples of the pens  112 . (Compare to  FIGS. 1A-1C .) 
     As shown in  FIGS. 13A and 13B , the device  1300  can comprise a micro-fluidic structure  1302  disposed on the base  502  (which is described above with respect to  FIGS. 5A and 5B ). As can be seen in  FIG. 13B , the micro-fluidic structure  1302  and base  502  can define a chamber  1308  for media  114  and micro-objects  116 . Media  114  with micro-objects  116  can be input into the chamber  1308  through an inlet  1314  and output from the chamber  1308  through an outlet  1316 . A flow  1320  of media  114  can thus be provided in the chamber  1308  from the inlet  1314  to the outlet  1316 . The inlet  1314  and outlet  1316  can be the same as or similar to the inlet  104  and outlet  106  of  FIGS. 1A-1C  as discussed above. The channels  1304  are examples of common spaces and/or flow paths for media  114 . 
     As also shown in  FIG. 13B , a gas exchanger  1310  and an array  1312  of pens  1306  and channels  1304  can be disposed in the chamber  1308  between the inlet  1314  and the outlet  1316  and thus in the flow  1320  of media  114 . The flow  1320  of media  114  can thus pass from the inlet  1314  through the gas exchanger  1310 , through the channels  1304  of the pen array  1312 , and out the outlet  1316 . Alternatively, the inlet  1314  can be located between the gas exchanger  1310  and the pens  1304 , and the gas exchanger  1310  can thus be located upstream from the inlet  1314 . 
     The channels  1304  and pens  1306  can be like any of the channels and pens discussed herein. For example, the channels  1304  can be like any of channels  504 ,  604 ,  704 ,  1104 ,  1204  including any variation of those channels discussed above, and the pens  1306  can be like any of pens  112 ,  302 ,  402 ,  506 ,  606 ,  706 ,  806 ,  906 ,  1106 ,  1206  including any variation of those pens discussed above. 
     Openings of the pens  1306  can be in fluidic communication (e.g., contact) with one of the channels  1304 . As micro-objects  116  (not shown in  FIGS. 13A and 13B ) move with the flow  1320  of media  114 , ones of the micro-objects  116  can be selected in a channel  1304  and moved into a pen  1306 . A micro-object  116  can be deterministically selected in a channel  1304  and moved into a pen  1306  using any technique or mechanism discussed above (e.g., with light traps like light traps  304 ,  412 ,  1108 ; with a virtual barrier like barrier  1208 ; or the like). The flow  1320  of media  114  can also be a common flow that carries nutrients to and provides for the outflow of waste from micro-objects  116  in the pens  1306 , which can otherwise isolate micro-objects  116  from each other. Moreover, each of the pens  1306  can be structured so that media  114  (e.g., the first medium  122  shown in  FIGS. 1B and 1C ) in a flow  1320  in a channel  1304  does not flow directly into any of the pens  1306 , but the structure of each pen  130  can allow diffusive mixing of media  114  from a flow  1320  in a channel  1304  and media  114  (e.g., the second medium  124  shown in  FIGS. 1B and 1C ) in a pen  1306  generally as discussed above. 
     The configuration of the pen array  1312  in  FIG. 13B  is but an example.  FIGS. 14 and 15  illustrate examples of alternative configurations. 
     As shown in  FIG. 14 , a pen array  1400  can comprise rows of pens  1402 , and openings of the pens  1402  can be in fluidic communication (e.g., contact) with a single channel  1404 . The pen array  1400  and channel  1404  can replace the pen array  1312  and channels  1304  in  FIG. 13B , and the flow  1320  of media  114  in  FIG. 13B  can be through the channel  1404 . 
     The pen array  1500  and channels  1504  in  FIG. 15  can also replace the pen array  1312  and channels  1304  in  FIG. 13B . As shown in  FIG. 15 , the pen array  1500  can comprise rows of pens  1502  with openings in direct fluidic communication with channels  1504   c . A plurality of first branching channels  1504   b  can connect an input channel  1504   a  to the channels  1504   c  that flow directly past the pens  1502 . Other (second) branching channels  1504   d  can connect the channels  1504   c  to an output channel  1504   e . The flow  1320  of media  114  in  FIG. 13B  can be into the first channel  1504   a , through branching channels  1504   b  to the channels  1504   c  in direct fluidic communication with the pens  1502 , through other branching channels  1504   d  to the second channel  1504   e.    
     The channels  1404 ,  1504  in  FIGS. 14 and 15  can be like channels  1304  as discussed above. The pens  1402 ,  1502  can likewise be like pens  1306  as discussed above. The channels  1404 ,  1504  can be examples of common spaces and/or flow paths. Each pen  1402 ,  1502  can be structured so that media  114  (e.g., the first medium  122  in  FIGS. 1B and 1C ) in a flow in a channel  1404 ,  1504  does not flow directly into the pen  1402 ,  1502 , but the structure of each pen  1402 ,  1502  can allow diffusive mixing of media from a flow in a channel  1404 ,  1504  and media (e.g., the second medium  124  in  FIGS. 1B and 1C ) in a pen  1402 ,  1502  generally as discussed above. 
       FIG. 16  illustrates an example of a process  1600  for processing biological micro-objects in pens. The process  1600  can be performed using any of the micro-fluidic devices discussed above or similar devices. For example, the process  1600  can be performed using the micro-fluidic devices  100  and  1300  including any variation of those devices discussed above (e.g., as illustrated in  FIGS. 2-12B, 14, and 15 ). 
     As shown in  FIG. 16 , at step  1602 , the process  1600  can load biological micro-objects into a micro-fluidic device. For example, the process  1600  can introduce into the chamber  110  of the device  100  of  FIGS. 1A-1C  through the inlet  104  micro-objects  116  in media  114 . As another example, the process  1600  can introduce into the chamber  1308  of the device  1300  of  FIGS. 13A and 13B  micro-objects  116  in media  114  through the inlet  1314 . 
     At step  1604 , the process can select individual ones of the biological micro-objects loaded at step  1602 . For example, the process  1600  can select a sub-set of less than all of the micro-objects  116  in media  114  that have a particular characteristic. The micro-objects  116  can be monitored, for example, using the imaging device  220  of  FIG. 2 . At step  1604 , one micro-object  116  having a particular desired characteristic can be deterministically selected and loaded into one pen such that step  1604  results in one and only one micro-object  116  in each of a plurality of the pens. Alternatively, more than one micro-object  116  can be loaded into a pen. 
     At step  1606 , the process  1600  can place the micro-objects  116  selected at step  1604  into pens of the micro-fluidic device. For example, at step  1606 , the process  1600  can place selected micro-objects  116  into the pens  112 ,  302 ,  402 ,  506 ,  606 ,  706 ,  806 ,  906 ,  1106 ,  1206 ,  1306 ,  1402 ,  1502  using any of the techniques discussed above. As noted above and illustrated throughout the drawings, the foregoing pens can physically separate micro-objects  116  one from another. That is, each pen can physically separate the micro-object  116  or micro-objects  116  in the pen from all other micro-objects  116  in the micro-fluidic device  100 ,  1300 . After placing selected micro-objects  116  in the pens at step  1606 , the process  1600  can keep the micro-objects  116  in the pens for a time period. 
     At step  1608 , the process  1600  can provide a flow of liquid media  114  to the pens. Step  1608  can be accomplished by providing any of the flows  314 ,  314 ,  520 ,  620 ,  720 ,  1120 ,  1320  in the chambers  110 ,  1308  or channels  504 ,  604 ,  704 ,  1104  as discussed. It is noted that, at step  1606 , individual micro-objects  116  can be physically isolated from each other by being placed in physically separated pens, but at step  1608 , those micro-objects  116  in the pens can be provided with the same flow of media  114 . As noted above, the pens  112 ,  302 ,  402 ,  506 ,  606 ,  706 ,  806 ,  906 ,  1106 ,  1206 ,  1306 ,  1402 ,  1502  can be structured to impede direct flow of media  114  (e.g., the first medium  122  shown in  FIGS. 1B and 1C ) from the flows  314 ,  314 ,  520 ,  620 ,  720 ,  1120 ,  1320  in the chambers  110 ,  1308  or channels  504 ,  604 ,  704 ,  1104  into the pens  112 ,  302 ,  402 ,  506 ,  606 ,  706 ,  806 ,  906 ,  1106 ,  1206 ,  1306 ,  1402 ,  1502  while allowing diffusive mixing of media  114  (e.g., the first medium  122  shown in  FIGS. 1B and 1C ) from the flows  314 ,  314 ,  520 ,  620 ,  720 ,  1120 ,  1320  and media  114  (e.g., the second medium  124  shown in  FIGS. 1B and 1C ) inside the pens. 
     As mentioned, the micro-objects  116  placed into the pens at step  1606  can be kept in the pens for a time period during which step  1608  can provide the micro-objects  116  with the flow of media  114 , which through the diffusive mixing discussed above can provide the micro-objects  116  in the pens with nutrients and provide for the outflow of waste from the micro-objects  116 . At step  1610 , the process  1600  can monitor one or more biological activities of the micro-objects  116  in the pens. Examples of such biological activities can include clone production, secretion of certain biological substances, or the like. The monitoring at step  1610  can be continuous during the time period, periodically during the time period, at the end of the time period, or the like. The monitoring at step  1610  can be performed in any manner suitable for analyzing biological activities of the micro-objects  116 . For example, the monitoring at step  1610  can be performed using the imaging system  220  of  FIG. 2 , with sensors (not shown) in or adjacent the pens, or the like. 
     At step  1612 , the process  1600  can select the micro-objects  116  in the pens that meet a predetermined criteria, threshold, or condition associated with the biological activity or state monitored at step  1610 . The micro-objects  116  selected at step  1612  can be removed from the pens for further processing or use. For example, the selected micro-objects  116  can be removed from the pens using any technique or process discussed above. As another example, one or more micro-objects  116  can be removed from a pen by piercing the housing with a needle-like aspirator (not shown), and removing the micro-objects  116  with the aspirator. A specific, controlled number of micro-objects  116  can be removed, for example, by selecting and removing that number of micro-objects  116  or, if the micro-objects  116  are biological cells, removing all of the cells when a colony of cloned cells reaches the desired number. 
     At step  1614 , the process  1600  can discard the micro-objects  116  not selected at step  1612 , which are the micro-objects  116  that do not meet the predetermined criteria, threshold, or condition associated with the biological activity or state monitored at step  1610 . 
       FIG. 17  illustrates an example process  1700  for growing colonies of cloned cells from a single parent cell according to some embodiments of the invention. The process  1700  can be an example of the process  1600  of  FIG. 16 . For example, the process  1700  can start after steps  1602  and  1604  of  FIG. 16  are performed; steps  1702 - 1706  can be performed during steps  1606  and  1608 ; step  1708  can be an example of step  1610 ; step  1710  can be an example of step  1614 ; and step  1712  can be an example of step  1612 . 
     For ease of illustration and discussion the process  1700  is discussed below as performed with the device  100  configured with the OET device of  FIG. 2  for creating and manipulating the virtual pens  302  of  FIG. 3 . The process  1700 , however, can be performed with other configurations of the device  100  or the device  1300  in which the pens are virtual pens. 
     As shown in  FIG. 17 , at step  1702 , the process  1700  can process cells in pens. Such processing can include fusing two cells into one cell, transfecting a cell by injecting a biological vector into a cell, or the like.  FIGS. 18A-18C  illustrate an example. 
     As shown in  FIG. 18A , two different types of micro-objects  116  and  1804  can be placed in media  114  in the chamber  110 . The OET device of  FIG. 2  can generate light traps  1806 ,  1808  (e.g., like light trap  304 ) to select one of the first cell type  116  and one of the second cell type  1804 . The light traps  1806  and  1808  can then be moved into contact such that the first cell type  116  and the second cell type are in contact as shown in  FIG. 18B . Such paired cells  1810  can then be subjected to one or more treatments (e.g., including in the flow  314  a fusing chemical (e.g., polyethylene glycol (PEG), the Sendai virus, piercing the membranes of one of the cells  116 ,  1804 , electric fields, pressure, or the like)) that fuse the paired cells  1810  together to form a fused cell  1812  as shown in  FIG. 18C . That is, each fused cell  1812  can comprise one of the first cell types  116  and one of the second cell types  1804  fused together. The light traps  1806  and  1808  can be like and can be created and manipulated like the light trap  304 ,  412  as discussed above including any variation thereof. 
     As also shown in  FIG. 18C , individual fused cells  1812  can be placed in virtual pens  1814 ,  1816 ,  1818 ,  1820 . Although four pens  1814 ,  1816 ,  1818 ,  1820  are shown, there can be more or fewer. The virtual pens  1814 ,  1816 ,  1818 ,  1820  can be the same as or similar to the pens  302  of  FIG. 3  as described above. 
     Alternatively, element  1804  in  FIG. 18A  can be a biological vector to be transfected into a micro-object  116 . In such a case, each cell  1812  in  FIG. 18C  can be one of the micro-objects  116  transfected with a vector  1804 . 
     As yet another alternative, the cells  1812  in  FIG. 18C  (whether fused cells or transfected cells) can be processed in another device and then placed into the pens  1814 ,  1816 ,  1818 ,  1820 . In such an alternative, step  1702  is not included in the process of  FIG. 17 . As a still further alternative, cells  1812  can be simple cells rather than fused or transfected cells. 
     Referring again to  FIG. 17 , at step  1704 , clones can be grown in each pen  1814 ,  1816 ,  1818 ,  1820  from the cell  1812  in the pen. This can be facilitated by including a growth medium in the flow  314  through the chamber  114 . At step  1706 , the pens  1814 ,  1816 ,  1818 ,  1820  can be expanded as the clones grow in each pen.  FIG. 19  illustrates an example. As shown in  FIG. 19 , as the number of cells  1812  in each pen  1814 ,  1816 ,  1818 ,  1820  increases, the size of the pens  1814 ′,  1816 ′,  1818 ′,  1820 ′ can be expanded to accommodate the growing clone populations in each pen. 
     At step  1708  of  FIG. 17 , each pen  1814 ,  1816 ,  1818 ,  1820  can be examined and clone growth in the pen can be analyzed. For example, a fluorescent label (e.g., a biological fluorescent compound that fluoresces when stimulated or otherwise) that binds to the clones can be included in the flow  314  through the chamber  110 . The level that each pen  1814 ,  1816 ,  1810 ,  1820  fluoresces can then be analyzed to determine clone growth in each pen. 
     At step  1710 , the clones in the pens  1814 ,  1816 ,  1818 ,  1820  in which the clones  1812  are growing at less than a minimum amount (or are otherwise undesirable) can be discarded.  FIG. 20  illustrates an example. For purposes of the example illustrated in  FIG. 20 , it is assumed that at step  1710  it was determined that the clones  1812  in pens  1814 ′,  1820 ′ of  FIG. 19  grew less than a minimum threshold amount and are to be discarded. As shown in  FIG. 20 , the pens  1814 ′,  1820 ′ can be turned off, freeing the clones  1812  in those pens. The pens  1814 ′,  1820 ′ can be turned off simply by removing from the light pattern  216  being directed into the housing  102   FIG. 2  the light that corresponds to pens  1814 ′,  1820 ′. The now freed clones  1812  that were in pens  1814 ′,  1820 ′ can be flushed out of the chamber  110  (e.g., by flow  314 ) and discarded. 
     As shown in  FIG. 17 , the steps  1704  through  1710  can be repeated to continue growing clones  1812  in the pens  1816 ′,  1818 ′. Alternatively, at step  1712 , individual ones of the clones  1812  from pens  1814 ′,  1820 ′ can be selected and placed as daughter clones in new pens, and the steps  1704  through  1710  can be repeated to grow, test, and discard slow growers in the new pens.  FIG. 21  shows an example in which individual daughter clones  1812  from the pens  1816 ′,  1818 ′ in  FIG. 20  are selected and each placed in a new pen  2102 . The new pens  2102  can be created and manipulated in the same way that pens  1814 ,  1816 ,  1818 ,  1820  are created and manipulated as discussed above. Individual daughter clones  1812  can be selected and moved generally as discussed above (e.g., with light traps like light trap  304 ,  412  of  FIG. 4 ). 
       FIG. 22  illustrates a process  2200  that is a variation of the process  1700  of  FIG. 17 . 
     As shown in  FIG. 22 , one or more cells can be held in and secrete into the pens. For example, as shown in  FIG. 18C , a cell  1812  can be disposed in each of the pens  1814 ,  1816 ,  1818 ,  1820 . Alternatively, there can be more than one cell  1812  in each pen  1814 ,  1816 ,  1818 ,  1820 . The cells  1812  can be fused or transfected cells as discussed above with respect to  FIGS. 18A-18C . Alternatively, cells  1812  can be simple cells rather than fused or transfected cells. 
     At step  2204  of  FIG. 22 , each pen  1814 ,  1816 ,  1818 ,  1820  can be examined and the productivity of the cells  1812  in the pen can be analyzed. For example, one or more cells  1812  can be removed from each pen  1814 ,  1816 ,  1818 ,  1820  and observed, tested, or the like to determine the secretion productivity of the removed cells  1812 . 
     At step  2206 , the pens  1814 ,  1816 ,  1818 ,  1820  in which the cells  1812  are secreting at less than a threshold level can be discarded. This can be accomplished generally as shown in  FIG. 20  and discussed above. That is, pens  1814 ,  1816 ,  1818 ,  1820  that contain low producing cells  1812  can be turned off and the low performing cells  1812  washed away generally in accordance with the discussion of  FIG. 20  above. 
     Referring again to  FIG. 22 , the steps  2202  through  2206  can be repeated to continue to have the cells  1812  in the remaining pens secrete, to test the secretion productivity of the cells in each pen, and discard cells  1812  in low producing pens. Alternatively, at step  2208 , individual ones of the high producing cells  1812  can be selected and placed as daughter cells in new pens (e.g., generally in accordance with the example shown in  FIG. 21 ), and the steps  2202  through  2206  can be repeated to have the daughter cells secrete in their new pens, test the secretion productivity of the daughter cells in each pen, and discard daughter cells in low secreting pens. 
     Although specific embodiments and applications of the invention have been described in this specification, these embodiments and applications are exemplary only, and many variations are possible.