Patent Publication Number: US-11376591-B2

Title: Light sequencing and patterns for dielectrophoretic transport

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
     This application is a non-provisional application claiming the benefit under 35 U.S.C. 119(e) of U.S. Provisional Application No. 62/323,436, filed on Apr. 15, 2016; and of U.S. Provisional Application No. 62/428,992, filed on Dec. 1, 2016, each of which disclosures is herein incorporated by reference in its entirety. 
    
    
     BACKGROUND 
     Optically-actuated microfluidic devices allow researchers to use spatially-modulated light to manipulate micro-objects such as biological cells. The present disclosure relates to systems and methods for providing sequences of light to move and direct a plurality of micro-objects. 
     SUMMARY 
     In one aspect, a method for re-positioning a plurality of micro-objects in a microfluidic device is provided, the method including: projecting a plurality of light bars on a portion of the microfluidic device, where each light bar has an initial position within the portion of the microfluidic device and the plurality of micro-objects are positioned within the portion of the microfluidic device; and, moving each of the plurality of light bars of the plurality along a common trajectory towards an end position, wherein each of the light bars provides sufficient force to move one or more of the plurality of micro-objects. 
     In another aspect, a method for transporting one or more micro-objects in a microfluidic device, the method including: identifying one or more micro-objects disposed within an enclosure of the microfluidic device, wherein the enclosure includes a flow region and a substrate including a dielectrophoresis configuration; generating a light cage having a size configured to surround the identified one or more micro-objects, and further wherein a shape of the light cage includes an angled leading edge; and, transporting the one or more micro-objects from a first location to a second location within the enclosure of the microfluidic device. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG. 1A  illustrates an example of a system for use with a microfluidic device and associated control equipment according to some embodiments of the disclosure. 
         FIGS. 1B and 1C  illustrate a microfluidic device according to some embodiments of the disclosure. 
         FIGS. 2A and 2B  illustrate isolation pens according to some embodiments of the disclosure. 
         FIG. 2C  illustrates a detailed sequestration pen according to some embodiments of the disclosure. 
         FIGS. 2D-F  illustrate sequestration pens according to some other embodiments of the disclosure. 
         FIG. 2G  illustrates a microfluidic device according to an embodiment of the disclosure. 
         FIG. 2H  illustrates a coated surface of the microfluidic device according to an embodiment of the disclosure. 
         FIG. 3A  illustrates a specific example of a system for use with a microfluidic device and associated control equipment according to some embodiments of the disclosure. 
         FIG. 3B  illustrates an imaging device according to some embodiments of the disclosure. 
         FIG. 3C  illustrates the communications between an imaging module and a light modulating subsystem to project patterns of light according to some embodiments of the disclosure. 
         FIGS. 4A-4D  provide a schematic illustration of a conveyor light sequence used to move micro-objects from a sequestration pen to a channel according to a specific embodiment of the disclosure. 
         FIGS. 5A-5B  provide a schematic illustration of a conveyor light sequence used to move micro-objects from a channel to a sequestration pen according to a specific embodiment of the disclosure. 
         FIGS. 6A-6B  provide a schematic illustration of a conveyor light sequence used in conjunction with a barrier light bar according to a specific embodiment of the disclosure. 
         FIG. 7  provides a schematic illustration of a conveyor light sequence used with static light bars according to a specific embodiment of the disclosure. 
         FIG. 8  provides a schematic illustration of a conveyor light sequence used in conjunction with moving light bars using to direct micro-objects on the conveyor light sequence according to a specific embodiment of the disclosure. 
         FIG. 9  provides a schematic illustration of the use of multiple, staggered conveyor light sequences according to a specific embodiment of the disclosure. 
         FIG. 10  provides a schematic illustration of a conveyor light sequence used in conjunction with lights sequences to separate cells within a channel according to a specific embodiment of the disclosure. 
         FIG. 11  provides a schematic illustration of a conveyor light sequence used with an oscillatory flow according to a specific embodiment of the disclosure. 
         FIG. 12  is a flowchart depicting steps performed to determine whether to use a conveyor light sequence according to a specific embodiment of the disclosure. 
         FIGS. 13A-13C  provide a schematic illustration of a staged conveyor light sequence according to a specific embodiment of the disclosure. 
         FIG. 14A-14C  depict the use of a conveyor light sequence to move a plurality of micro-objects into sequestration pens in parallel according to a specific embodiment of the disclosure. 
         FIGS. 15A and 15B  depict a conveyor light sequence used to export select cells from sequestration pens according to a specific embodiment of the disclosure. 
         FIGS. 16A-16C  depict a conveyor light sequence used to move cells from an upper region of a channel to a lower region of the channel proximal to sequestration pens according to a specific embodiment of the disclosure. 
         FIGS. 17A-17D  depict the use of a conveyor light sequence in conjunction with light sequences used to separate cells as they are exported by the conveyor light sequence into a channel according to a specific embodiment of the disclosure. 
         FIGS. 18A-18F  depicts a conveyor light sequence that has a non-linear trajectory according to a specific embodiment of the disclosure. 
         FIGS. 19A-19E  depict the use of a conveyor light sequences with light sequences that are used to deterministically separate cells once they enter the channel. 
         FIGS. 20A-B  are graphical representations of two embodiments of light cages for selective transit of micro-object using a light cage having an angled leading edge. 
     
    
    
     DETAILED DESCRIPTION 
     This specification describes exemplary embodiments and applications of the disclosure. The disclosure, 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. In addition, as the terms “on,” “attached to,” “connected to,” “coupled to,” or similar words are used herein, one element (e.g., a material, a layer, a substrate, etc.) can be “on,” “attached to,” “connected to,” or “coupled to” another element regardless of whether the one element is directly on, attached to, connected to, or coupled to the other element or there are one or more intervening elements between the one element and the other element. Also, unless the context dictates otherwise, 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. Section divisions in the specification are for ease of review only and do not limit any combination of elements discussed. 
     Where dimensions of microfluidic features are described as having a width or an area, the dimension typically is described relative to an x-axial and/or y-axial dimension, both of which lie within a plane that is parallel to the substrate and/or cover of the microfluidic device. The height of a microfluidic feature may be described relative to a z-axial direction, which is perpendicular to a plane that is parallel to the substrate and/or cover of the microfluidic device. In some instances, a cross sectional area of a microfluidic feature, such as a channel or a passageway, may be in reference to a x-axial/z-axial, a y-axial/z-axial, or an x-axially-axial area. 
     As used herein, “substantially” means sufficient to work for the intended purpose. The term “substantially” thus allows for minor, insignificant variations from an absolute or perfect state, dimension, measurement, result, or the like such as would be expected by a person of ordinary skill in the field but that do not appreciably affect overall performance. When used with respect to numerical values or parameters or characteristics that can be expressed as numerical values, “substantially” means within ten percent. 
     The term “ones” means more than one. 
     As used herein, the term “plurality” can be 2, 3, 4, 5, 6, 7, 8, 9, 10, or more. 
     As used herein, the term “disposed” encompasses within its meaning “located.” 
     As used herein, a “microfluidic device” or “microfluidic apparatus” is a device that includes one or more discrete microfluidic circuits configured to hold a fluid, each microfluidic circuit comprised of fluidically interconnected circuit elements, including but not limited to region(s), flow path(s), channel(s), chamber(s), and/or pen(s), and at least one port configured to allow the fluid (and, optionally, micro-objects suspended in the fluid) to flow into and/or out of the microfluidic device. Typically, a microfluidic circuit of a microfluidic device will include a flow region, which may include a microfluidic channel, and at least one chamber, and will hold a volume of fluid of less than about 1 mL, e.g., less than about 750, 500, 250, 200, 150, 100, 75, 50, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, or 2 μL. In certain embodiments, the microfluidic circuit holds about 1-2, 1-3, 1-4, 1-5, 2-5, 2-8, 2-10, 2-12, 2-15, 2-20, 5-20, 5-30, 5-40, 5-50, 10-50, 10-75, 10-100, 20-100, 20-150, 20-200, 50-200, 50-250, or 50-300 μL. The microfluidic circuit may be configured to have a first end fluidically connected with a first port (e.g., an inlet) in the microfluidic device and a second end fluidically connected with a second port (e.g., an outlet) in the microfluidic device. 
     As used herein, a “nanofluidic device” or “nanofluidic apparatus” is a type of microfluidic device having a microfluidic circuit that contains at least one circuit element configured to hold a volume of fluid of less than about 1 μL, e.g., less than about 750, 500, 250, 200, 150, 100, 75, 50, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 nL or less. A nanofluidic device may comprise a plurality of circuit elements (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 75, 100, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 6000, 7000, 8000, 9000, 10,000, or more). In certain embodiments, one or more (e.g., all) of the at least one circuit elements is configured to hold a volume of fluid of about 100 pL to 1 nL, 100 pL to 2 nL, 100 pL to 5 nL, 250 pL to 2 nL, 250 pL to 5 nL, 250 pL to 10 nL, 500 pL to 5 nL, 500 pL to 10 nL, 500 pL to 15 nL, 750 pL to 10 nL, 750 pL to 15 nL, 750 pL to 20 nL, 1 to 10 nL, 1 to 15 nL, 1 to 20 nL, 1 to 25 nL, or 1 to 50 nL. In other embodiments, one or more (e.g., all) of the at least one circuit elements are configured to hold a volume of fluid of about 20 nL to 200 nL, 100 to 200 nL, 100 to 300 nL, 100 to 400 nL, 100 to 500 nL, 200 to 300 nL, 200 to 400 nL, 200 to 500 nL, 200 to 600 nL, 200 to 700 nL, 250 to 400 nL, 250 to 500 nL, 250 to 600 nL, or 250 to 750 nL. 
     A microfluidic device or a nanofluidic device may be referred to herein as a “microfluidic chip” or a “chip”; or “nanofluidic chip” or “chip”. 
     A “microfluidic channel” or “flow channel” as used herein refers to flow region of a microfluidic device having a length that is significantly longer than both the horizontal and vertical dimensions. For example, the flow channel can be at least 5 times the length of either the horizontal or vertical dimension, e.g., at least 10 times the length, at least 25 times the length, at least 100 times the length, at least 200 times the length, at least 500 times the length, at least 1,000 times the length, at least 5,000 times the length, or longer. In some embodiments, the length of a flow channel is in the range of from about 100,000 microns to about 500,000 microns, including any range therebetween. In some embodiments, the horizontal dimension is in the range of from about 100 microns to about 1000 microns (e.g., about 150 to about 500 microns) and the vertical dimension is in the range of from about 25 microns to about 200 microns, e.g., from about 40 to about 150 microns. It is noted that a flow channel may have a variety of different spatial configurations in a microfluidic device, and thus is not restricted to a perfectly linear element. For example, a flow channel may be, or include one or more sections having, the following configurations: curve, bend, spiral, incline, decline, fork (e.g., multiple different flow paths), and any combination thereof. In addition, a flow channel may have different cross-sectional areas along its path, widening and constricting to provide a desired fluid flow therein. The flow channel may include valves, and the valves may be of any type known in the art of microfluidics. Examples of microfluidic channels that include valves are disclosed in U.S. Pat. Nos. 6,408,878 and 9,227,200, each of which is herein incorporated by reference in its entirety. 
     As used herein, the term “obstruction” refers generally to a bump or similar type of structure that is sufficiently large so as to partially (but not completely) impede movement of target micro-objects between two different regions or circuit elements in a microfluidic device. The two different regions/circuit elements can be, for example, the connection region and the isolation region of a microfluidic sequestration pen. 
     As used herein, the term “constriction” refers generally to a narrowing of a width of a circuit element (or an interface between two circuit elements) in a microfluidic device. The constriction can be located, for example, at the interface between the isolation region and the connection region of a microfluidic sequestration pen of the instant disclosure. 
     As used herein, the term “transparent” refers to a material which allows visible light to pass through without substantially altering the light as is passes through. 
     As used herein, the term “micro-object” refers generally to any microscopic object that may be isolated and/or manipulated in accordance with the present disclosure. Non-limiting examples of micro-objects include: inanimate micro-objects such as microparticles; microbeads (e.g., polystyrene beads, Luminex™ beads, or the like); magnetic beads; microrods; microwires; quantum dots, and the like; biological micro-objects such as cells; biological organelles; vesicles, or complexes; synthetic vesicles; liposomes (e.g., synthetic or derived from membrane preparations); lipid nanorafts, and the like; or a combination of inanimate micro-objects and biological micro-objects (e.g., microbeads attached to cells, liposome-coated micro-beads, liposome-coated magnetic beads, or the like). Beads may include moieties/molecules covalently or non-covalently attached, such as fluorescent labels, proteins, carbohydrates, antigens, small molecule signaling moieties, or other chemical/biological species capable of use in an assay. Lipid nanorafts have been described, for example, in Ritchie et al. (2009) “Reconstitution of Membrane Proteins in Phospholipid Bilayer Nanodiscs,” Methods Enzymol., 464:211-231. 
     As used herein, the term “cell” is used interchangeably with the term “biological cell.” Non-limiting examples of biological cells include eukaryotic cells, plant cells, animal cells, such as mammalian cells, reptilian cells, avian cells, fish cells, or the like, prokaryotic cells, bacterial cells, fungal cells, protozoan cells, or the like, cells dissociated from a tissue, such as muscle, cartilage, fat, skin, liver, lung, neural tissue, and the like, immunological cells, such as T cells, B cells, natural killer cells, macrophages, and the like, embryos (e.g., zygotes), oocytes, ova, sperm cells, hybridomas, cultured cells, cells from a cell line, cancer cells, infected cells, transfected and/or transformed cells, reporter cells, and the like. A mammalian cell can be, for example, from a human, a mouse, a rat, a horse, a goat, a sheep, a cow, a primate, or the like. 
     A colony of biological cells is “clonal” if all of the living cells in the colony that are capable of reproducing are daughter cells derived from a single parent cell. In certain embodiments, all the daughter cells in a clonal colony are derived from the single parent cell by no more than 10 divisions. In other embodiments, all the daughter cells in a clonal colony are derived from the single parent cell by no more than 14 divisions. In other embodiments, all the daughter cells in a clonal colony are derived from the single parent cell by no more than 17 divisions. In other embodiments, all the daughter cells in a clonal colony are derived from the single parent cell by no more than 20 divisions. The term “clonal cells” refers to cells of the same clonal colony. 
     As used herein, a “colony” of biological cells refers to 2 or more cells (e.g. about 2 to about 20, about 4 to about 40, about 6 to about 60, about 8 to about 80, about 10 to about 100, about 20 to about 200, about 40 to about 400, about 60 to about 600, about 80 to about 800, about 100 to about 1000, or greater than 1000 cells). 
     As used herein, the term “maintaining (a) cell(s)” refers to providing an environment comprising both fluidic and gaseous components and, optionally a surface, that provides the conditions necessary to keep the cells viable and/or expanding. 
     As used herein, the term “expanding” when referring to cells, refers to increasing in cell number. 
     A “component” of a fluidic medium is any chemical or biochemical molecule present in the medium, including solvent molecules, ions, small molecules, antibiotics, nucleotides and nucleosides, nucleic acids, amino acids, peptides, proteins, sugars, carbohydrates, lipids, fatty acids, cholesterol, metabolites, or the like. 
     As used herein, “flowable polymer” is a polymer monomer or macromer that is soluble or dispersible within a fluidic medium (e.g., a pre-polymer solution). The flowable polymer may be input into a microfluidic flow region and flow with other components of a fluidic medium therein. 
     As used herein, “photoinitiated polymer” refers to a polymer (or a monomeric molecule that can be used to generate the polymer) that upon exposure to light, is capable of crosslinking covalently, forming specific covalent bonds, changing regiochemistry around a rigidified chemical motif, or forming ion pairs which cause a change in physical state, and thereby forming a polymer network. In some instances, a photoinitiated polymer may include a polymer segment bound to one or more chemical moieties capable of crosslinking covalently, forming specific covalent bonds, changing regiochemistry around a rigidified chemical motif, or forming ion pairs which cause a change in physical state. In some instances, a photoinitiated polymer may require a photoactivatable radical initiator to initiate formation of the polymer network (e.g., via polymerization of the polymer). 
     As used herein in reference to a fluidic medium, “diffuse” and “diffusion” refer to thermodynamic movement of a component of the fluidic medium down a concentration gradient. 
     The phrase “flow of a medium” means bulk movement of a fluidic medium primarily due to any mechanism other than diffusion. For example, flow of a medium can involve movement of the fluidic medium from one point to another point due to a pressure differential between the points. Such flow can include a continuous, pulsed, periodic, random, intermittent, or reciprocating flow of the liquid, or any combination thereof. When one fluidic medium flows into another fluidic medium, turbulence and mixing of the media can result. 
     The phrase “substantially no flow” refers to a rate of flow of a fluidic medium that, averaged over time, is less than the rate of diffusion of components of a material (e.g., an analyte of interest) into or within the fluidic medium. The rate of diffusion of components of such a material can depend on, for example, temperature, the size of the components, and the strength of interactions between the components and the fluidic medium. 
     As used herein in reference to different regions within a microfluidic device, the phrase “fluidically connected” means that, when the different regions are substantially filled with fluid, such as fluidic media, the fluid in each of the regions is connected so as to form a single body of fluid. This does not mean that the fluids (or fluidic media) in the different regions are necessarily identical in composition. Rather, the fluids in different fluidically connected regions of a microfluidic device can have different compositions (e.g., different concentrations of solutes, such as proteins, carbohydrates, ions, or other molecules) which are in flux as solutes move down their respective concentration gradients and/or fluids flow through the microfluidic device. 
     As used herein, a “flow path” refers to one or more fluidically connected circuit elements (e.g. channel(s), region(s), chamber(s) and the like) that define, and are subject to, the trajectory of a flow of medium. A flow path is thus an example of a swept region of a microfluidic device. Other circuit elements (e.g., unswept regions) may be fluidically connected with the circuit elements that comprise the flow path without being subject to the flow of medium in the flow path. 
     As used herein, “isolating a micro-object” confines a micro-object to a defined area within the microfluidic device. 
     A microfluidic (or nanofluidic) device can comprise “swept” regions and “unswept” regions. As used herein, a “swept” region is comprised of one or more fluidically interconnected circuit elements of a microfluidic circuit, each of which experiences a flow of medium when fluid is flowing through the microfluidic circuit. The circuit elements of a swept region can include, for example, regions, channels, and all or parts of chambers. As used herein, an “unswept” region is comprised of one or more fluidically interconnected circuit element of a microfluidic circuit, each of which experiences substantially no flux of fluid when fluid is flowing through the microfluidic circuit. An unswept region can be fluidically connected to a swept region, provided the fluidic connections are structured to enable diffusion but substantially no flow of media between the swept region and the unswept region. The microfluidic device can thus be structured to substantially isolate an unswept region from a flow of medium in a swept region, while enabling substantially only diffusive fluidic communication between the swept region and the unswept region. For example, a flow channel of a microfluidic device is an example of a swept region while an isolation region (described in further detail below) of a microfluidic device is an example of an unswept region. 
     The capability of biological micro-objects (e.g., biological cells) to produce specific biological materials (e.g., proteins, such as antibodies) can be assayed in such a microfluidic device. In a specific embodiment of an assay, sample material comprising biological micro-objects (e.g., cells) to be assayed for production of an analyte of interest can be loaded into a swept region of the microfluidic device. Ones of the biological micro-objects (e.g., mammalian cells, such as human cells) can be selected for particular characteristics and disposed in unswept regions. The remaining sample material can then be flowed out of the swept region and an assay material flowed into the swept region. Because the selected biological micro-objects are in unswept regions, the selected biological micro-objects are not substantially affected by the flowing out of the remaining sample material or the flowing in of the assay material. The selected biological micro-objects can be allowed to produce the analyte of interest, which can diffuse from the unswept regions into the swept region, where the analyte of interest can react with the assay material to produce localized detectable reactions, each of which can be correlated to a particular unswept region. Any unswept region associated with a detected reaction can be analyzed to determine which, if any, of the biological micro-objects in the unswept region are sufficient producers of the analyte of interest. 
     Microfluidic devices and systems for operating and observing such devices.  FIG. 1A  illustrates an example of a microfluidic device  100  and a system  150  which can be used for maintaining, isolating, assaying or culturing biological micro-objects. A perspective view of the microfluidic device  100  is shown having a partial cut-away of its cover  110  to provide a partial view into the microfluidic device  100 . The microfluidic device  100  generally comprises a microfluidic circuit  120  comprising a flow path  106  through which a fluidic medium  180  can flow, optionally carrying one or more micro-objects (not shown) into and/or through the microfluidic circuit  120 . Although a single microfluidic circuit  120  is illustrated in  FIG. 1A , suitable microfluidic devices can include a plurality (e.g., 2 or 3) of such microfluidic circuits. Regardless, the microfluidic device  100  can be configured to be a nanofluidic device. As illustrated in  FIG. 1A , the microfluidic circuit  120  may include a plurality of microfluidic sequestration pens  124 ,  126 ,  128 , and  130 , where each sequestration pens may have one or more openings in fluidic communication with flow path  106 . In some embodiments of the device of  FIG. 1A , the sequestration pens may have only a single opening in fluidic communication with the flow path  106 . As discussed further below, the microfluidic sequestration pens comprise various features and structures that have been optimized for retaining micro-objects in the microfluidic device, such as microfluidic device  100 , even when a medium  180  is flowing through the flow path  106 . Before turning to the foregoing, however, a brief description of microfluidic device  100  and system  150  is provided. 
     As generally illustrated in  FIG. 1A , the microfluidic circuit  120  is defined by an enclosure  102 . Although the enclosure  102  can be physically structured in different configurations, in the example shown in  FIG. 1A  the enclosure  102  is depicted as comprising a support structure  104  (e.g., a base), a microfluidic circuit structure  108 , and a cover  110 . The support structure  104 , microfluidic circuit structure  108 , and cover  110  can be attached to each other. For example, the microfluidic circuit structure  108  can be disposed on an inner surface  109  of the support structure  104 , and the cover  110  can be disposed over the microfluidic circuit structure  108 . Together with the support structure  104  and cover  110 , the microfluidic circuit structure  108  can define the elements of the microfluidic circuit  120 . 
     The support structure  104  can be at the bottom and the cover  110  at the top of the microfluidic circuit  120  as illustrated in  FIG. 1A . Alternatively, the support structure  104  and the cover  110  can be configured in other orientations. For example, the support structure  104  can be at the top and the cover  110  at the bottom of the microfluidic circuit  120 . Regardless, there can be one or more ports  107  each comprising a passage into or out of the enclosure  102 . Examples of a passage include a valve, a gate, a pass-through hole, or the like. As illustrated, port  107  is a pass-through hole created by a gap in the microfluidic circuit structure  108 . However, the port  107  can be situated in other components of the enclosure  102 , such as the cover  110 . Only one port  107  is illustrated in  FIG. 1A  but the microfluidic circuit  120  can have two or more ports  107 . For example, there can be a first port  107  that functions as an inlet for fluid entering the microfluidic circuit  120 , and there can be a second port  107  that functions as an outlet for fluid exiting the microfluidic circuit  120 . Whether a port  107  function as an inlet or an outlet can depend upon the direction that fluid flows through flow path  106 . 
     The support structure  104  can comprise one or more electrodes (not shown) and a substrate or a plurality of interconnected substrates. For example, the support structure  104  can comprise one or more semiconductor substrates, each of which is electrically connected to an electrode (e.g., all or a subset of the semiconductor substrates can be electrically connected to a single electrode). The support structure  104  can further comprise a printed circuit board assembly (“PCBA”). For example, the semiconductor substrate(s) can be mounted on a PCBA. 
     The microfluidic circuit structure  108  can define circuit elements of the microfluidic circuit  120 . Such circuit elements can comprise spaces or regions that can be fluidly interconnected when microfluidic circuit  120  is filled with fluid, such as flow regions (which may include or be one or more flow channels), chambers, pens, traps, and the like. In the microfluidic circuit  120  illustrated in  FIG. 1A , the microfluidic circuit structure  108  comprises a frame  114  and a microfluidic circuit material  116 . The frame  114  can partially or completely enclose the microfluidic circuit material  116 . The frame  114  can be, for example, a relatively rigid structure substantially surrounding the microfluidic circuit material  116 . For example, the frame  114  can comprise a metal material. 
     The microfluidic circuit material  116  can be patterned with cavities or the like to define circuit elements and interconnections of the microfluidic circuit  120 . The microfluidic circuit material  116  can comprise a flexible material, such as a flexible polymer (e.g. rubber, plastic, elastomer, silicone, polydimethylsiloxane (“PDMS”), or the like), which can be gas permeable. Other examples of materials that can compose microfluidic circuit material  116  include molded glass, an etchable material such as silicone (e.g. photo-patternable silicone or “PPS”), photo-resist (e.g., SU8), or the like. In some embodiments, such materials—and thus the microfluidic circuit material  116 —can be rigid and/or substantially impermeable to gas. Regardless, microfluidic circuit material  116  can be disposed on the support structure  104  and inside the frame  114 . 
     The cover  110  can be an integral part of the frame  114  and/or the microfluidic circuit material  116 . Alternatively, the cover  110  can be a structurally distinct element, as illustrated in  FIG. 1A . The cover  110  can comprise the same or different materials than the frame  114  and/or the microfluidic circuit material  116 . Similarly, the support structure  104  can be a separate structure from the frame  114  or microfluidic circuit material  116  as illustrated, or an integral part of the frame  114  or microfluidic circuit material  116 . Likewise, the frame  114  and microfluidic circuit material  116  can be separate structures as shown in  FIG. 1A  or integral portions of the same structure. 
     In some embodiments, the cover  110  can comprise a rigid material. The rigid material may be glass or a material with similar properties. In some embodiments, the cover  110  can comprise a deformable material. The deformable material can be a polymer, such as PDMS. In some embodiments, the cover  110  can comprise both rigid and deformable materials. For example, one or more portions of cover  110  (e.g., one or more portions positioned over sequestration pens  124 ,  126 ,  128 ,  130 ) can comprise a deformable material that interfaces with rigid materials of the cover  110 . In some embodiments, the cover  110  can further include one or more electrodes. The one or more electrodes can comprise a conductive oxide, such as indium-tin-oxide (ITO), which may be coated on glass or a similarly insulating material. Alternatively, the one or more electrodes can be flexible electrodes, such as single-walled nanotubes, multi-walled nanotubes, nanowires, clusters of electrically conductive nanoparticles, or combinations thereof, embedded in a deformable material, such as a polymer (e.g., PDMS). Flexible electrodes that can be used in microfluidic devices have been described, for example, in U.S. 2012/0325665 (Chiou et al.), the contents of which are incorporated herein by reference. In some embodiments, the cover  110  can be modified (e.g., by conditioning all or part of a surface that faces inward toward the microfluidic circuit  120 ) to support cell adhesion, viability and/or growth. The modification may include a coating of a synthetic or natural polymer. In some embodiments, the cover  110  and/or the support structure  104  can be transparent to light. The cover  110  may also include at least one material that is gas permeable (e.g., PDMS or PPS). 
       FIG. 1A  also shows a system  150  for operating and controlling microfluidic devices, such as microfluidic device  100 . System  150  includes an electrical power source  192 , an imaging device  194  (incorporated within imaging module  164 , where device  194  is not illustrated in  FIG. 1A , per se), and a tilting device  190  (part of tilting module  166 , where device  190  is not illustrated in  FIG. 1A ). 
     The electrical power source  192  can provide electric power to the microfluidic device  100  and/or tilting device  190 , providing biasing voltages or currents as needed. The electrical power source  192  can, for example, comprise one or more alternating current (AC) and/or direct current (DC) voltage or current sources. The imaging device  194  (part of imaging module  164 , discussed below) can comprise a device, such as a digital camera, for capturing images inside microfluidic circuit  120 . In some instances, the imaging device  194  further comprises a detector having a fast frame rate and/or high sensitivity (e.g. for low light applications). The imaging device  194  can also include a mechanism for directing stimulating radiation and/or light beams into the microfluidic circuit  120  and collecting radiation and/or light beams reflected or emitted from the microfluidic circuit  120  (or micro-objects contained therein). The emitted light beams may be in the visible spectrum and may, e.g., include fluorescent emissions. The reflected light beams may include reflected emissions originating from an LED or a wide spectrum lamp, such as a mercury lamp (e.g. a high pressure mercury lamp) or a Xenon arc lamp. As discussed with respect to  FIG. 3B , the imaging device  194  may further include a microscope (or an optical train), which may or may not include an eyepiece. 
     System  150  further comprises a tilting device  190  (part of tilting module  166 , discussed below) configured to rotate a microfluidic device  100  about one or more axes of rotation. In some embodiments, the tilting device  190  is configured to support and/or hold the enclosure  102  comprising the microfluidic circuit  120  about at least one axis such that the microfluidic device  100  (and thus the microfluidic circuit  120 ) can be held in a level orientation (i.e. at 0° relative to x- and y-axes), a vertical orientation (i.e. at 90° relative to the x-axis and/or the y-axis), or any orientation therebetween. The orientation of the microfluidic device  100  (and the microfluidic circuit  120 ) relative to an axis is referred to herein as the “tilt” of the microfluidic device  100  (and the microfluidic circuit  120 ). For example, the tilting device  190  can tilt the microfluidic device  100  at 0.1°, 0.2°, 0.3°, 0.4°, 0.5°, 0.6°, 0.7°, 0.8°, 0.9°, 1°, 2°, 3°, 4°, 5°, 10°, 15°, 20°, 25°, 30°, 35°, 40°, 45°, 50°, 55°, 60°, 65°, 70°, 75°, 80°, 90° relative to the x-axis or any degree therebetween. The level orientation (and thus the x- and y-axes) is defined as normal to a vertical axis defined by the force of gravity. The tilting device can also tilt the microfluidic device  100  (and the microfluidic circuit  120 ) to any degree greater than 90° relative to the x-axis and/or y-axis, or tilt the microfluidic device  100  (and the microfluidic circuit  120 ) 180° relative to the x-axis or the y-axis in order to fully invert the microfluidic device  100  (and the microfluidic circuit  120 ). Similarly, in some embodiments, the tilting device  190  tilts the microfluidic device  100  (and the microfluidic circuit  120 ) about an axis of rotation defined by flow path  106  or some other portion of microfluidic circuit  120 . 
     In some instances, the microfluidic device  100  is tilted into a vertical orientation such that the flow path  106  is positioned above or below one or more sequestration pens. The term “above” as used herein denotes that the flow path  106  is positioned higher than the one or more sequestration pens on a vertical axis defined by the force of gravity (i.e. an object in a sequestration pen above a flow path  106  would have a higher gravitational potential energy than an object in the flow path). The term “below” as used herein denotes that the flow path  106  is positioned lower than the one or more sequestration pens on a vertical axis defined by the force of gravity (i.e. an object in a sequestration pen below a flow path  106  would have a lower gravitational potential energy than an object in the flow path). 
     In some instances, the tilting device  190  tilts the microfluidic device  100  about an axis that is parallel to the flow path  106 . Moreover, the microfluidic device  100  can be tilted to an angle of less than 90° such that the flow path  106  is located above or below one or more sequestration pens without being located directly above or below the sequestration pens. In other instances, the tilting device  190  tilts the microfluidic device  100  about an axis perpendicular to the flow path  106 . In still other instances, the tilting device  190  tilts the microfluidic device  100  about an axis that is neither parallel nor perpendicular to the flow path  106 . 
     System  150  can further include a media source  178 . The media source  178  (e.g., a container, reservoir, or the like) can comprise multiple sections or containers, each for holding a different fluidic medium  180 . Thus, the media source  178  can be a device that is outside of and separate from the microfluidic device  100 , as illustrated in  FIG. 1A . Alternatively, the media source  178  can be located in whole or in part inside the enclosure  102  of the microfluidic device  100 . For example, the media source  178  can comprise reservoirs that are part of the microfluidic device  100 . 
       FIG. 1A  also illustrates simplified block diagram depictions of examples of control and monitoring equipment  152  that constitute part of system  150  and can be utilized in conjunction with a microfluidic device  100 . As shown, examples of such control and monitoring equipment  152  include a master controller  154  comprising a media module  160  for controlling the media source  178 , a motive module  162  for controlling movement and/or selection of micro-objects (not shown) and/or medium (e.g., droplets of medium) in the microfluidic circuit  120 , an imaging module  164  for controlling an imaging device  194  (e.g., a camera, microscope, light source or any combination thereof) for capturing images (e.g., digital images), and a tilting module  166  for controlling a tilting device  190 . The control equipment  152  can also include other modules  168  for controlling, monitoring, or performing other functions with respect to the microfluidic device  100 . As shown, the equipment  152  can further include a display device  170  and an input/output device  172 . 
     The master controller  154  can comprise a control module  156  and a digital memory  158 . The control module  156  can comprise, for example, a digital processor configured to operate in accordance with machine executable instructions (e.g., software, firmware, source code, or the like) stored as non-transitory data or signals in the memory  158 . Alternatively, or in addition, the control module  156  can comprise hardwired digital circuitry and/or analog circuitry. The media module  160 , motive module  162 , imaging module  164 , tilting module  166 , and/or other modules  168  can be similarly configured. Thus, functions, processes acts, actions, or steps of a process discussed herein as being performed with respect to the microfluidic device  100  or any other microfluidic apparatus can be performed by any one or more of the master controller  154 , media module  160 , motive module  162 , imaging module  164 , tilting module  166 , and/or other modules  168  configured as discussed above. Similarly, the master controller  154 , media module  160 , motive module  162 , imaging module  164 , tilting module  166 , and/or other modules  168  may be communicatively coupled to transmit and receive data used in any function, process, act, action or step discussed herein. 
     The media module  160  controls the media source  178 . For example, the media module  160  can control the media source  178  to input a selected fluidic medium  180  into the enclosure  102  (e.g., through an inlet port  107 ). The media module  160  can also control removal of media from the enclosure  102  (e.g., through an outlet port (not shown)). One or more media can thus be selectively input into and removed from the microfluidic circuit  120 . The media module  160  can also control the flow of fluidic medium  180  in the flow path  106  inside the microfluidic circuit  120 . For example, in some embodiments media module  160  stops the flow of media  180  in the flow path  106  and through the enclosure  102  prior to the tilting module  166  causing the tilting device  190  to tilt the microfluidic device  100  to a desired angle of incline. 
     The motive module  162  can be configured to control selection, trapping, and movement of micro-objects (not shown) in the microfluidic circuit  120 . As discussed below with respect to  FIGS. 1B and 1C , the enclosure  102  can comprise a dielectrophoresis (DEP), optoelectronic tweezers (OET) and/or opto-electrowetting (OEW) configuration (not shown in  FIG. 1A ), and the motive module  162  can control the activation of electrodes and/or transistors (e.g., phototransistors) to select and move micro-objects (not shown) and/or droplets of medium (not shown) in the flow path  106  and/or sequestration pens  124 ,  126 ,  128 ,  130 . 
     The imaging module  164  can control the imaging device  194 . For example, the imaging module  164  can receive and process image data from the imaging device  194 . Image data from the imaging device  194  can comprise any type of information captured by the imaging device  194  (e.g., the presence or absence of micro-objects, droplets of medium, accumulation of label, such as fluorescent label, etc.). Using the information captured by the imaging device  194 , the imaging module  164  can further calculate the position of objects (e.g., micro-objects, droplets of medium) and/or the rate of motion of such objects within the microfluidic device  100 . 
     The tilting module  166  can control the tilting motions of tilting device  190 . Alternatively, or in addition, the tilting module  166  can control the tilting rate and timing to optimize transfer of micro-objects to the one or more sequestration pens via gravitational forces. The tilting module  166  is communicatively coupled with the imaging module  164  to receive data describing the motion of micro-objects and/or droplets of medium in the microfluidic circuit  120 . Using this data, the tilting module  166  may adjust the tilt of the microfluidic circuit  120  in order to adjust the rate at which micro-objects and/or droplets of medium move in the microfluidic circuit  120 . The tilting module  166  may also use this data to iteratively adjust the position of a micro-object and/or droplet of medium in the microfluidic circuit  120 . 
     In the example shown in  FIG. 1A , the microfluidic circuit  120  is illustrated as comprising a microfluidic channel  122  and sequestration pens  124 ,  126 ,  128 ,  130 . Each pen comprises an opening to channel  122 , but otherwise is enclosed such that the pens can substantially isolate micro-objects inside the pen from fluidic medium  180  and/or micro-objects in the flow path  106  of channel  122  or in other pens. The walls of the sequestration pen extend from the inner surface  109  of the base to the inside surface of the cover  110  to provide enclosure. The opening of the pen to the microfluidic channel  122  is oriented at an angle to the flow  106  of fluidic medium  180  such that flow  106  is not directed into the pens. The flow may be tangential or orthogonal to the plane of the opening of the pen. In some instances, pens  124 ,  126 ,  128 ,  130  are configured to physically corral one or more micro-objects within the microfluidic circuit  120 . Sequestration pens in accordance with the present disclosure can comprise various shapes, surfaces and features that are optimized for use with DEP, OET, OEW, fluid flow, and/or gravitational forces, as will be discussed and shown in detail below. 
     The microfluidic circuit  120  may comprise any number of microfluidic sequestration pens. Although five sequestration pens are shown, microfluidic circuit  120  may have fewer or more sequestration pens. As shown, microfluidic sequestration pens  124 ,  126 ,  128 , and  130  of microfluidic circuit  120  each comprise differing features and shapes which may provide one or more benefits useful for maintaining, isolating, assaying or culturing biological micro-objects. In some embodiments, the microfluidic circuit  120  comprises a plurality of identical microfluidic sequestration pens. 
     In the embodiment illustrated in  FIG. 1A , a single channel  122  and flow path  106  is shown. However, other embodiments may contain multiple channels  122 , each configured to comprise a flow path  106 . The microfluidic circuit  120  further comprises an inlet valve or port  107  in fluid communication with the flow path  106  and fluidic medium  180 , whereby fluidic medium  180  can access channel  122  via the inlet port  107 . In some instances, the flow path  106  comprises a single path. In some instances, the single path is arranged in a zigzag pattern whereby the flow path  106  travels across the microfluidic device  100  two or more times in alternating directions. 
     In some instances, microfluidic circuit  120  comprises a plurality of parallel channels  122  and flow paths  106 , wherein the fluidic medium  180  within each flow path  106  flows in the same direction. In some instances, the fluidic medium within each flow path  106  flows in at least one of a forward or reverse direction. In some instances, a plurality of sequestration pens is configured (e.g., relative to a channel  122 ) such that the sequestration pens can be loaded with target micro-objects in parallel. 
     In some embodiments, microfluidic circuit  120  further comprises one or more micro-object traps  132 . The traps  132  are generally formed in a wall forming the boundary of a channel  122 , and may be positioned opposite an opening of one or more of the microfluidic sequestration pens  124 ,  126 ,  128 ,  130 . In some embodiments, the traps  132  are configured to receive or capture a single micro-object from the flow path  106 . In some embodiments, the traps  132  are configured to receive or capture a plurality of micro-objects from the flow path  106 . In some instances, the traps  132  comprise a volume approximately equal to the volume of a single target micro-object. 
     The traps  132  may further comprise an opening which is configured to assist the flow of targeted micro-objects into the traps  132 . In some instances, the traps  132  comprise an opening having a height and width that is approximately equal to the dimensions of a single target micro-object, whereby larger micro-objects are prevented from entering into the micro-object trap. The traps  132  may further comprise other features configured to assist in retention of targeted micro-objects within the trap  132 . In some instances, the trap  132  is aligned with and situated on the opposite side of a channel  122  relative to the opening of a microfluidic sequestration pen, such that upon tilting the microfluidic device  100  about an axis parallel to the microfluidic channel  122 , the trapped micro-object exits the trap  132  at a trajectory that causes the micro-object to fall into the opening of the sequestration pen. In some instances, the trap  132  comprises a side passage  134  that is smaller than the target micro-object in order to facilitate flow through the trap  132  and thereby increase the likelihood of capturing a micro-object in the trap  132 . 
     In some embodiments, dielectrophoretic (DEP) forces are applied across the fluidic medium  180  (e.g., in the flow path and/or in the sequestration pens) via one or more electrodes (not shown) to manipulate, transport, separate and sort micro-objects located therein. For example, in some embodiments, DEP forces are applied to one or more portions of microfluidic circuit  120  in order to transfer a single micro-object from the flow path  106  into a desired microfluidic sequestration pen. In some embodiments, DEP forces are used to prevent a micro-object within a sequestration pen (e.g., sequestration pen  124 ,  126 ,  128 , or  130 ) from being displaced therefrom. Further, in some embodiments, DEP forces are used to selectively remove a micro-object from a sequestration pen that was previously collected in accordance with the embodiments of the current disclosure. In some embodiments, the DEP forces comprise optoelectronic tweezer (OET) forces. 
     In other embodiments, optoelectrowetting (OEW) forces are applied to one or more positions in the support structure  104  (and/or the cover  110 ) of the microfluidic device  100  (e.g., positions helping to define the flow path and/or the sequestration pens) via one or more electrodes (not shown) to manipulate, transport, separate and sort droplets located in the microfluidic circuit  120 . For example, in some embodiments, OEW forces are applied to one or more positions in the support structure  104  (and/or the cover  110 ) in order to transfer a single droplet from the flow path  106  into a desired microfluidic sequestration pen. In some embodiments, OEW forces are used to prevent a droplet within a sequestration pen (e.g., sequestration pen  124 ,  126 ,  128 , or  130 ) from being displaced therefrom. Further, in some embodiments, OEW forces are used to selectively remove a droplet from a sequestration pen that was previously collected in accordance with the embodiments of the current disclosure. 
     In some embodiments, DEP and/or OEW forces are combined with other forces, such as flow and/or gravitational force, so as to manipulate, transport, separate and sort micro-objects and/or droplets within the microfluidic circuit  120 . For example, the enclosure  102  can be tilted (e.g., by tilting device  190 ) to position the flow path  106  and micro-objects located therein above the microfluidic sequestration pens, and the force of gravity can transport the micro-objects and/or droplets into the pens. In some embodiments, the DEP and/or OEW forces can be applied prior to the other forces. In other embodiments, the DEP and/or OEW forces can be applied after the other forces. In still other instances, the DEP and/or OEW forces can be applied at the same time as the other forces or in an alternating manner with the other forces. 
       FIGS. 1B, 1C, and 2A-2H  illustrates various embodiments of microfluidic devices that can be used in the practice of the embodiments of the present disclosure.  FIG. 1B  depicts an embodiment in which the microfluidic device  200  is configured as an optically-actuated electrokinetic device. A variety of optically-actuated electrokinetic devices are known in the art, including devices having an optoelectronic tweezer (OET) configuration and devices having an opto-electrowetting (OEW) configuration. Examples of suitable OET configurations are illustrated in the following U.S. patent documents, each of which is incorporated herein by reference in its entirety: U.S. Pat. No. RE 44,711 (Wu et al.) (originally issued as U.S. Pat. No. 7,612,355); and U.S. Pat. No. 7,956,339 (Ohta et al.). Examples of OEW configurations are illustrated in U.S. Pat. No. 6,958,132 (Chiou et al.) and U.S. Patent Application Publication No. 2012/0024708 (Chiou et al.), both of which are incorporated by reference herein in their entirety. Yet another example of an optically-actuated electrokinetic device includes a combined OET/OEW configuration, examples of which are shown in U.S. Patent Publication Nos. 20150306598 (Khandros et al.) and 20150306599 (Khandros et al.) and their corresponding PCT Publications WO2015/164846 and WO2015/164847, all of which are incorporated herein by reference in their entirety. 
     Examples of microfluidic devices having pens in which biological micro-objects can be placed, cultured, and/or monitored have been described, for example, in US 2014/0116881 (application Ser. No. 14/060,117, filed Oct. 22, 2013), US 2015/0151298 (application Ser. No. 14/520,568, filed Oct. 22, 2014), and US 2015/0165436 (application Ser. No. 14/521,447, filed Oct. 22, 2014), each of which is incorporated herein by reference in its entirety. US application Ser. Nos. 14/520,568 and 14/521,447 also describe exemplary methods of analyzing secretions of cells cultured in a microfluidic device. Each of the foregoing applications further describes microfluidic devices configured to produce dielectrophoretic (DEP) forces, such as optoelectronic tweezers (OET) or configured to provide opto-electro wetting (OEW). For example, the optoelectronic tweezers device illustrated in FIG. 2 of US 2014/0116881 is an example of a device that can be utilized in embodiments of the present disclosure to select and move an individual biological micro-object or a group of biological micro-objects. 
     Microfluidic device motive configurations. As described above, the control and monitoring equipment of the system can comprise a motive module for selecting and moving objects, such as micro-objects or droplets, in the microfluidic circuit of a microfluidic device. The microfluidic device can have a variety of motive configurations, depending upon the type of object being moved and other considerations. For example, a dielectrophoresis (DEP) configuration can be utilized to select and move micro-objects in the microfluidic circuit. Thus, the support structure  104  and/or cover  110  of the microfluidic device  100  can comprise a DEP configuration for selectively inducing DEP forces on micro-objects in a fluidic medium  180  in the microfluidic circuit  120  and thereby select, capture, and/or move individual micro-objects or groups of micro-objects. Alternatively, the support structure  104  and/or cover  110  of the microfluidic device  100  can comprise an electrowetting (EW) configuration for selectively inducing EW forces on droplets in a fluidic medium  180  in the microfluidic circuit  120  and thereby select, capture, and/or move individual droplets or groups of droplets. 
     One example of a microfluidic device  200  comprising a DEP configuration is illustrated in  FIGS. 1B and 1C . While for purposes of simplicity  FIGS. 1B and 1C  show a side cross-sectional view and a top cross-sectional view, respectively, of a portion of an enclosure  102  of the microfluidic device  200  having a region/chamber  202 , it should be understood that the region/chamber  202  may be part of a fluidic circuit element having a more detailed structure, such as a growth chamber, a sequestration pen, a flow region, or a flow channel. Furthermore, the microfluidic device  200  may include other fluidic circuit elements. For example, the microfluidic device  200  can include a plurality of growth chambers or sequestration pens and/or one or more flow regions or flow channels, such as those described herein with respect to microfluidic device  100 . A DEP configuration may be incorporated into any such fluidic circuit elements of the microfluidic device  200 , or select portions thereof. It should be further appreciated that any of the above or below described microfluidic device components and system components may be incorporated in and/or used in combination with the microfluidic device  200 . For example, system  150  including control and monitoring equipment  152 , described above, may be used with microfluidic device  200 , including one or more of the media module  160 , motive module  162 , imaging module  164 , tilting module  166 , and other modules  168 . 
     As seen in  FIG. 1B , the microfluidic device  200  includes a support structure  104  having a bottom electrode  204  and an electrode activation substrate  206  overlying the bottom electrode  204 , and a cover  110  having a top electrode  210 , with the top electrode  210  spaced apart from the bottom electrode  204 . The top electrode  210  and the electrode activation substrate  206  define opposing surfaces of the region/chamber  202 . A medium  180  contained in the region/chamber  202  thus provides a resistive connection between the top electrode  210  and the electrode activation substrate  206 . A power source  212  configured to be connected to the bottom electrode  204  and the top electrode  210  and create a biasing voltage between the electrodes, as required for the generation of DEP forces in the region/chamber  202 , is also shown. The power source  212  can be, for example, an alternating current (AC) power source. 
     In certain embodiments, the microfluidic device  200  illustrated in  FIGS. 1B and 1C  can have an optically-actuated DEP configuration. Accordingly, changing patterns of light  218  from the light source  216 , which may be controlled by the motive module  162 , can selectively activate and deactivate changing patterns of DEP electrodes at regions  214  of the inner surface  208  of the electrode activation substrate  206 . (Hereinafter the regions  214  of a microfluidic device having a DEP configuration are referred to as “DEP electrode regions.”) As illustrated in  FIG. 1C , a light pattern  218  directed onto the inner surface  208  of the electrode activation substrate  206  can illuminate select DEP electrode regions  214   a  (shown in white) in a pattern, such as a square. The non-illuminated DEP electrode regions  214  (cross-hatched) are hereinafter referred to as “dark” DEP electrode regions  214 . The relative electrical impedance through the DEP electrode activation substrate  206  (i.e., from the bottom electrode  204  up to the inner surface  208  of the electrode activation substrate  206  which interfaces with the medium  180  in the flow region  106 ) is greater than the relative electrical impedance through the medium  180  in the region/chamber  202  (i.e., from the inner surface  208  of the electrode activation substrate  206  to the top electrode  210  of the cover  110 ) at each dark DEP electrode region  214 . An illuminated DEP electrode region  214   a , however, exhibits a reduced relative impedance through the electrode activation substrate  206  that is less than the relative impedance through the medium  180  in the region/chamber  202  at each illuminated DEP electrode region  214   a.    
     With the power source  212  activated, the foregoing DEP configuration creates an electric field gradient in the fluidic medium  180  between illuminated DEP electrode regions  214   a  and adjacent dark DEP electrode regions  214 , which in turn creates local DEP forces that attract or repel nearby micro-objects (not shown) in the fluidic medium  180 . DEP electrodes that attract or repel micro-objects in the fluidic medium  180  can thus be selectively activated and deactivated at many different such DEP electrode regions  214  at the inner surface  208  of the region/chamber  202  by changing light patterns  218  projected from a light source  216  into the microfluidic device  200 . Whether the DEP forces attract or repel nearby micro-objects can depend on such parameters as the frequency of the power source  212  and the dielectric properties of the medium  180  and/or micro-objects (not shown). 
     The square pattern  220  of illuminated DEP electrode regions  214   a  illustrated in  FIG. 1C  is an example only. Any pattern of the DEP electrode regions  214  can be illuminated (and thereby activated) by the pattern of light  218  projected into the microfluidic device  200 , and the pattern of illuminated/activated DEP electrode regions  214  can be repeatedly changed by changing or moving the light pattern  218 . 
     In some embodiments, the electrode activation substrate  206  can comprise or consist of a photoconductive material. In such embodiments, the inner surface  208  of the electrode activation substrate  206  can be featureless. For example, the electrode activation substrate  206  can comprise or consist of a layer of hydrogenated amorphous silicon (a-Si:H). The a-Si:H can comprise, for example, about 8% to 40% hydrogen (calculated as 100*the number of hydrogen atoms/the total number of hydrogen and silicon atoms). The layer of a-Si:H can have a thickness of about 500 nm to about 2.0 μm. In such embodiments, the DEP electrode regions  214  can be created anywhere and in any pattern on the inner surface  208  of the electrode activation substrate  206 , in accordance with the light pattern  218 . The number and pattern of the DEP electrode regions  214  thus need not be fixed, but can correspond to the light pattern  218 . Examples of microfluidic devices having a DEP configuration comprising a photoconductive layer such as discussed above have been described, for example, in U.S. Pat. No. RE 44,711 (Wu et al.) (originally issued as U.S. Pat. No. 7,612,355), the entire contents of which are incorporated herein by reference. 
     In other embodiments, the electrode activation substrate  206  can comprise a substrate comprising a plurality of doped layers, electrically insulating layers (or regions), and electrically conductive layers that form semiconductor integrated circuits, such as is known in semiconductor fields. For example, the electrode activation substrate  206  can comprise a plurality of phototransistors, including, for example, lateral bipolar phototransistors, each phototransistor corresponding to a DEP electrode region  214 . Alternatively, the electrode activation substrate  206  can comprise electrodes (e.g., conductive metal electrodes) controlled by phototransistor switches, with each such electrode corresponding to a DEP electrode region  214 . The electrode activation substrate  206  can include a pattern of such phototransistors or phototransistor-controlled electrodes. The pattern, for example, can be an array of substantially square phototransistors or phototransistor-controlled electrodes arranged in rows and columns, such as shown in  FIG. 2B . Alternatively, the pattern can be an array of substantially hexagonal phototransistors or phototransistor-controlled electrodes that form a hexagonal lattice. Regardless of the pattern, electric circuit elements can form electrical connections between the DEP electrode regions  214  at the inner surface  208  of the electrode activation substrate  206  and the bottom electrode  210 , and those electrical connections (i.e., phototransistors or electrodes) can be selectively activated and deactivated by the light pattern  218 . When not activated, each electrical connection can have high impedance such that the relative impedance through the electrode activation substrate  206  (i.e., from the bottom electrode  204  to the inner surface  208  of the electrode activation substrate  206  which interfaces with the medium  180  in the region/chamber  202 ) is greater than the relative impedance through the medium  180  (i.e., from the inner surface  208  of the electrode activation substrate  206  to the top electrode  210  of the cover  110 ) at the corresponding DEP electrode region  214 . When activated by light in the light pattern  218 , however, the relative impedance through the electrode activation substrate  206  is less than the relative impedance through the medium  180  at each illuminated DEP electrode region  214 , thereby activating the DEP electrode at the corresponding DEP electrode region  214  as discussed above. DEP electrodes that attract or repel micro-objects (not shown) in the medium  180  can thus be selectively activated and deactivated at many different DEP electrode regions  214  at the inner surface  208  of the electrode activation substrate  206  in the region/chamber  202  in a manner determined by the light pattern  218 . 
     Examples of microfluidic devices having electrode activation substrates that comprise phototransistors have been described, for example, in U.S. Pat. No. 7,956,339 (Ohta et al.) (see, e.g., device  300  illustrated in  FIGS. 21 and 22 , and descriptions thereof), the entire contents of which are incorporated herein by reference. Examples of microfluidic devices having electrode activation substrates that comprise electrodes controlled by phototransistor switches have been described, for example, in U.S. Patent Publication No. 2014/0124370 (Short et al.) (see, e.g., devices  200 ,  400 ,  500 ,  600 , and  900  illustrated throughout the drawings, and descriptions thereof), the entire contents of which are incorporated herein by reference. 
     In some embodiments of a DEP configured microfluidic device, the top electrode  210  is part of a first wall (or cover  110 ) of the enclosure  102 , and the electrode activation substrate  206  and bottom electrode  204  are part of a second wall (or support structure  104 ) of the enclosure  102 . The region/chamber  202  can be between the first wall and the second wall. In other embodiments, the electrode  210  is part of the second wall (or support structure  104 ) and one or both of the electrode activation substrate  206  and/or the electrode  210  are part of the first wall (or cover  110 ). Moreover, the light source  216  can alternatively be used to illuminate the enclosure  102  from below. 
     With the microfluidic device  200  of  FIGS. 1B-1C  having a DEP configuration, the motive module  162  can select a micro-object (not shown) in the medium  180  in the region/chamber  202  by projecting a light pattern  218  into the microfluidic device  200  to activate a first set of one or more DEP electrodes at DEP electrode regions  214   a  of the inner surface  208  of the electrode activation substrate  206  in a pattern (e.g., square pattern  220 ) that surrounds and captures the micro-object. The motive module  162  can then move the in situ-generated captured micro-object by moving the light pattern  218  relative to the microfluidic device  200  to activate a second set of one or more DEP electrodes at DEP electrode regions  214 . Alternatively, the microfluidic device  200  can be moved relative to the light pattern  218 . 
     In other embodiments, the microfluidic device  200  can have a DEP configuration that does not rely upon light activation of DEP electrodes at the inner surface  208  of the electrode activation substrate  206 . For example, the electrode activation substrate  206  can comprise selectively addressable and energizable electrodes positioned opposite to a surface including at least one electrode (e.g., cover  110 ). Switches (e.g., transistor switches in a semiconductor substrate) may be selectively opened and closed to activate or inactivate DEP electrodes at DEP electrode regions  214 , thereby creating a net DEP force on a micro-object (not shown) in region/chamber  202  in the vicinity of the activated DEP electrodes. Depending on such characteristics as the frequency of the power source  212  and the dielectric properties of the medium (not shown) and/or micro-objects in the region/chamber  202 , the DEP force can attract or repel a nearby micro-object. By selectively activating and deactivating a set of DEP electrodes (e.g., at a set of DEP electrodes regions  214  that forms a square pattern  220 ), one or more micro-objects in region/chamber  202  can be trapped and moved within the region/chamber  202 . The motive module  162  in  FIG. 1A  can control such switches and thus activate and deactivate individual ones of the DEP electrodes to select, trap, and move particular micro-objects (not shown) around the region/chamber  202 . Microfluidic devices having a DEP configuration that includes selectively addressable and energizable electrodes are known in the art and have been described, for example, in U.S. Pat. No. 6,294,063 (Becker et al.) and U.S. Pat. No. 6,942,776 (Medoro), the entire contents of which are incorporated herein by reference. 
     As yet another example, the microfluidic device  200  can have an electrowetting (EW) configuration, which can be in place of the DEP configuration or can be located in a portion of the microfluidic device  200  that is separate from the portion which has the DEP configuration. The EW configuration can be an opto-electrowetting configuration or an electrowetting on dielectric (EWOD) configuration, both of which are known in the art. In some EW configurations, the support structure  104  has an electrode activation substrate  206  sandwiched between a dielectric layer (not shown) and the bottom electrode  204 . The dielectric layer can comprise a hydrophobic material and/or can be coated with a hydrophobic material, as described below. For microfluidic devices  200  that have an EW configuration, the inner surface  208  of the support structure  104  is the inner surface of the dielectric layer or its hydrophobic coating. 
     The dielectric layer (not shown) can comprise one or more oxide layers, and can have a thickness of about 50 nm to about 250 nm (e.g., about 125 nm to about 175 nm). In certain embodiments, the dielectric layer may comprise a layer of oxide, such as a metal oxide (e.g., aluminum oxide or hafnium oxide). In certain embodiments, the dielectric layer can comprise a dielectric material other than a metal oxide, such as silicon oxide or a nitride. Regardless of the exact composition and thickness, the dielectric layer can have an impedance of about 10 kOhms to about 50 kOhms. 
     In some embodiments, the surface of the dielectric layer that faces inward toward region/chamber  202  is coated with a hydrophobic material. The hydrophobic material can comprise, for example, fluorinated carbon molecules. Examples of fluorinated carbon molecules include perfluoro-polymers such as polytetrafluoroethylene (e.g., TEFLON®) or poly(2,3-difluoromethylenyl-perfluorotetrahydrofuran) (e.g., CYTOP™). Molecules that make up the hydrophobic material can be covalently bonded to the surface of the dielectric layer. For example, molecules of the hydrophobic material can be covalently bound to the surface of the dielectric layer by means of a linker such as a siloxane group, a phosphonic acid group, or a thiol group. Thus, in some embodiments, the hydrophobic material can comprise alkyl-terminated siloxane, alkyl-termination phosphonic acid, or alkyl-terminated thiol. The alkyl group can be long-chain hydrocarbons (e.g., having a chain of at least 10 carbons, or at least 16, 18, 20, 22, or more carbons). Alternatively, fluorinated (or perfluorinated) carbon chains can be used in place of the alkyl groups. Thus, for example, the hydrophobic material can comprise fluoroalkyl-terminated siloxane, fluoroalkyl-terminated phosphonic acid, or fluoroalkyl-terminated thiol. In some embodiments, the hydrophobic coating has a thickness of about 10 nm to about 50 nm. In other embodiments, the hydrophobic coating has a thickness of less than 10 nm (e.g., less than 5 nm, or about 1.5 to 3.0 nm). 
     In some embodiments, the cover  110  of a microfluidic device  200  having an electrowetting configuration is coated with a hydrophobic material (not shown) as well. The hydrophobic material can be the same hydrophobic material used to coat the dielectric layer of the support structure  104 , and the hydrophobic coating can have a thickness that is substantially the same as the thickness of the hydrophobic coating on the dielectric layer of the support structure  104 . Moreover, the cover  110  can comprise an electrode activation substrate  206  sandwiched between a dielectric layer and the top electrode  210 , in the manner of the support structure  104 . The electrode activation substrate  206  and the dielectric layer of the cover  110  can have the same composition and/or dimensions as the electrode activation substrate  206  and the dielectric layer of the support structure  104 . Thus, the microfluidic device  200  can have two electrowetting surfaces. 
     In some embodiments, the electrode activation substrate  206  can comprise a photoconductive material, such as described above. Accordingly, in certain embodiments, the electrode activation substrate  206  can comprise or consist of a layer of hydrogenated amorphous silicon (a-Si:H). The a-Si:H can comprise, for example, about 8% to 40% hydrogen (calculated as 100*the number of hydrogen atoms/the total number of hydrogen and silicon atoms). The layer of a-Si:H can have a thickness of about 500 nm to about 2.0 μm. Alternatively, the electrode activation substrate  206  can comprise electrodes (e.g., conductive metal electrodes) controlled by phototransistor switches, as described above. Microfluidic devices having an opto-electrowetting configuration are known in the art and/or can be constructed with electrode activation substrates known in the art. For example, U.S. Pat. No. 6,958,132 (Chiou et al.), the entire contents of which are incorporated herein by reference, discloses opto-electrowetting configurations having a photoconductive material such as a-Si:H, while U.S. Patent Publication No. 2014/0124370 (Short et al.), referenced above, discloses electrode activation substrates having electrodes controlled by phototransistor switches. 
     The microfluidic device  200  thus can have an opto-electrowetting configuration, and light patterns  218  can be used to activate photoconductive EW regions or photoresponsive EW electrodes in the electrode activation substrate  206 . Such activated EW regions or EW electrodes of the electrode activation substrate  206  can generate an electrowetting force at the inner surface  208  of the support structure  104  (i.e., the inner surface of the overlaying dielectric layer or its hydrophobic coating). By changing the light patterns  218  (or moving microfluidic device  200  relative to the light source  216 ) incident on the electrode activation substrate  206 , droplets (e.g., containing an aqueous medium, solution, or solvent) contacting the inner surface  208  of the support structure  104  can be moved through an immiscible fluid (e.g., an oil medium) present in the region/chamber  202 . 
     In other embodiments, microfluidic devices  200  can have an EWOD configuration, and the electrode activation substrate  206  can comprise selectively addressable and energizable electrodes that do not rely upon light for activation. The electrode activation substrate  206  thus can include a pattern of such electrowetting (EW) electrodes. The pattern, for example, can be an array of substantially square EW electrodes arranged in rows and columns, such as shown in  FIG. 2B . Alternatively, the pattern can be an array of substantially hexagonal EW electrodes that form a hexagonal lattice. Regardless of the pattern, the EW electrodes can be selectively activated (or deactivated) by electrical switches (e.g., transistor switches in a semiconductor substrate). By selectively activating and deactivating EW electrodes in the electrode activation substrate  206 , droplets (not shown) contacting the inner surface  208  of the overlaying dielectric layer or its hydrophobic coating can be moved within the region/chamber  202 . The motive module  162  in  FIG. 1A  can control such switches and thus activate and deactivate individual EW electrodes to select and move particular droplets around region/chamber  202 . Microfluidic devices having a EWOD configuration with selectively addressable and energizable electrodes are known in the art and have been described, for example, in U.S. Pat. No. 8,685,344 (Sundarsan et al.), the entire contents of which are incorporated herein by reference. 
     Regardless of the configuration of the microfluidic device  200 , a power source  212  can be used to provide a potential (e.g., an AC voltage potential) that powers the electrical circuits of the microfluidic device  200 . The power source  212  can be the same as, or a component of, the power source  192  referenced in  FIG. 1 . Power source  212  can be configured to provide an AC voltage and/or current to the top electrode  210  and the bottom electrode  204 . For an AC voltage, the power source  212  can provide a frequency range and an average or peak power (e.g., voltage or current) range sufficient to generate net DEP forces (or electrowetting forces) strong enough to trap and move individual micro-objects (not shown) in the region/chamber  202 , as discussed above, and/or to change the wetting properties of the inner surface  208  of the support structure  104  (i.e., the dielectric layer and/or the hydrophobic coating on the dielectric layer) in the region/chamber  202 , as also discussed above. Such frequency ranges and average or peak power ranges are known in the art. See, e.g., U.S. Pat. No. 6,958,132 (Chiou et al.), US Patent No. RE 44,711 (Wu et al.) (originally issued as U.S. Pat. No. 7,612,355), and US Patent Application Publication Nos. US2014/0124370 (Short et al.), US 2015/0306598 (Khandros et al.), and US 2015/0306599 (Khandros et al.). 
     Sequestration pens. Non-limiting examples of generic sequestration pens  224 ,  226 , and  228  are shown within the microfluidic device  230  depicted in  FIGS. 2A-2C . Each sequestration pen  224 ,  226 , and  228  can comprise an isolation structure  232  defining an isolation region  240  and a connection region  236  fluidically connecting the isolation region  240  to a channel  122 . The connection region  236  can comprise a proximal opening  234  to the microfluidic channel  122  and a distal opening  238  to the isolation region  240 . The connection region  236  can be configured so that the maximum penetration depth of a flow of a fluidic medium (not shown) flowing from the microfluidic channel  122  into the sequestration pen  224 ,  226 ,  228  does not extend into the isolation region  240 . Thus, due to the connection region  236 , a micro-object (not shown) or other material (not shown) disposed in an isolation region  240  of a sequestration pen  224 ,  226 ,  228  can thus be isolated from, and not substantially affected by, a flow of medium  180  in the microfluidic channel  122 . 
     The sequestration pens  224 ,  226 , and  228  of  FIGS. 2A-2C  each have a single opening which opens directly to the microfluidic channel  122 . The opening of the sequestration pen opens laterally from the microfluidic channel  122 . The electrode activation substrate  206  underlays both the microfluidic channel  122  and the sequestration pens  224 ,  226 , and  228 . The upper surface of the electrode activation substrate  206  within the enclosure of a sequestration pen, forming the floor of the sequestration pen, is disposed at the same level or substantially the same level of the upper surface the of electrode activation substrate  206  within the microfluidic channel  122  (or flow region if a channel is not present), forming the floor of the flow channel (or flow region, respectively) of the microfluidic device. The electrode activation substrate  206  may be featureless or may have an irregular or patterned surface that varies from its highest elevation to its lowest depression by less than about 3 microns, 2.5 microns, 2 microns, 1.5 microns, 1 micron, 0.9 microns, 0.5 microns, 0.4 microns, 0.2 microns, 0.1 microns or less. The variation of elevation in the upper surface of the substrate across both the microfluidic channel  122  (or flow region) and sequestration pens may be less than about 3%, 2%, 1%, 0.9%, 0.8%, 0.5%, 0.3% or 0.1% of the height of the walls of the sequestration pen or walls of the microfluidic device. While described in detail for the microfluidic device  200 , this also applies to any of the microfluidic devices  100 ,  230 ,  250 ,  280 ,  290 ,  320 ,  400 ,  450 ,  500 ,  700  described herein. 
     The microfluidic channel  122  can thus be an example of a swept region, and the isolation regions  240  of the sequestration pens  224 ,  226 ,  228  can be examples of unswept regions. As noted, the microfluidic channel  122  and sequestration pens  224 ,  226 ,  228  can be configured to contain one or more fluidic media  180 . In the example shown in  FIGS. 2A-2B , the ports  222  are connected to the microfluidic channel  122  and allow a fluidic medium  180  to be introduced into or removed from the microfluidic device  230 . Prior to introduction of the fluidic medium  180 , the microfluidic device may be primed with a gas such as carbon dioxide gas. Once the microfluidic device  230  contains the fluidic medium  180 , the flow  242  of fluidic medium  180  in the microfluidic channel  122  can be selectively generated and stopped. For example, as shown, the ports  222  can be disposed at different locations (e.g., opposite ends) of the microfluidic channel  122 , and a flow  242  of medium can be created from one port  222  functioning as an inlet to another port  222  functioning as an outlet. 
       FIG. 2C  illustrates a detailed view of an example of a sequestration pen  224  according to the present disclosure. Examples of micro-objects  246  are also shown. 
     As is known, a flow  242  of fluidic medium  180  in a microfluidic channel  122  past a proximal opening  234  of sequestration pen  224  can cause a secondary flow  244  of the medium  180  into and/or out of the sequestration pen  224 . To isolate micro-objects  246  in the isolation region  240  of a sequestration pen  224  from the secondary flow  244 , the length L con  of the connection region  236  of the sequestration pen  224  (i.e., from the proximal opening  234  to the distal opening  238 ) should be greater than the penetration depth D p  of the secondary flow  244  into the connection region  236 . The penetration depth D p  of the secondary flow  244  depends upon the velocity of the fluidic medium  180  flowing in the microfluidic channel  122  and various parameters relating to the configuration of the microfluidic channel  122  and the proximal opening  234  of the connection region  236  to the microfluidic channel  122 . For a given microfluidic device, the configurations of the microfluidic channel  122  and the opening  234  will be fixed, whereas the rate of flow  242  of fluidic medium  180  in the microfluidic channel  122  will be variable. Accordingly, for each sequestration pen  224 , a maximal velocity V max  for the flow  242  of fluidic medium  180  in channel  122  can be identified that ensures that the penetration depth D p  of the secondary flow  244  does not exceed the length L con  of the connection region  236 . As long as the rate of the flow  242  of fluidic medium  180  in the microfluidic channel  122  does not exceed the maximum velocity V max , the resulting secondary flow  244  can be limited to the microfluidic channel  122  and the connection region  236  and kept out of the isolation region  240 . The flow  242  of medium  180  in the microfluidic channel  122  will thus not draw micro-objects  246  out of the isolation region  240 . Rather, micro-objects  246  located in the isolation region  240  will stay in the isolation region  240  regardless of the flow  242  of fluidic medium  180  in the microfluidic channel  122 . 
     Moreover, as long as the rate of flow  242  of medium  180  in the microfluidic channel  122  does not exceed V max , the flow  242  of fluidic medium  180  in the microfluidic channel  122  will not move miscellaneous particles (e.g., microparticles and/or nanoparticles) from the microfluidic channel  122  into the isolation region  240  of a sequestration pen  224 . Having the length L con  of the connection region  236  be greater than the maximum penetration depth D p  of the secondary flow  244  can thus prevent contamination of one sequestration pen  224  with miscellaneous particles from the microfluidic channel  122  or another sequestration pen (e.g., sequestration pens  226 ,  228  in  FIG. 2D ). 
     Because the microfluidic channel  122  and the connection regions  236  of the sequestration pens  224 ,  226 ,  228  can be affected by the flow  242  of medium  180  in the microfluidic channel  122 , the microfluidic channel  122  and connection regions  236  can be deemed swept (or flow) regions of the microfluidic device  230 . The isolation regions  240  of the sequestration pens  224 ,  226 ,  228 , on the other hand, can be deemed unswept (or non-flow) regions. For example, components (not shown) in a first fluidic medium  180  in the microfluidic channel  122  can mix with a second fluidic medium  248  in the isolation region  240  substantially only by diffusion of components of the first medium  180  from the microfluidic channel  122  through the connection region  236  and into the second fluidic medium  248  in the isolation region  240 . Similarly, components (not shown) of the second medium  248  in the isolation region  240  can mix with the first medium  180  in the microfluidic channel  122  substantially only by diffusion of components of the second medium  248  from the isolation region  240  through the connection region  236  and into the first medium  180  in the microfluidic channel  122 . In some embodiments, the extent of fluidic medium exchange between the isolation region of a sequestration pen and the flow region by diffusion is greater than about 90%, 91%, 92%, 93%, 94% 95%, 96%, 97%, 98%, or greater than about 99% of fluidic exchange. The first medium  180  can be the same medium or a different medium than the second medium  248 . Moreover, the first medium  180  and the second medium  248  can start out being the same, then become different (e.g., through conditioning of the second medium  248  by one or more cells in the isolation region  240 , or by changing the medium  180  flowing through the microfluidic channel  122 ). 
     The maximum penetration depth D p  of the secondary flow  244  caused by the flow  242  of fluidic medium  180  in the microfluidic channel  122  can depend on a number of parameters, as mentioned above. Examples of such parameters include: the shape of the microfluidic channel  122  (e.g., the microfluidic channel can direct medium into the connection region  236 , divert medium away from the connection region  236 , or direct medium in a direction substantially perpendicular to the proximal opening  234  of the connection region  236  to the microfluidic channel  122 ); a width W ch  (or cross-sectional area) of the microfluidic channel  122  at the proximal opening  234 ; and a width W con  (or cross-sectional area) of the connection region  236  at the proximal opening  234 ; the velocity V of the flow  242  of fluidic medium  180  in the microfluidic channel  122 ; the viscosity of the first medium  180  and/or the second medium  248 , or the like. 
     In some embodiments, the dimensions of the microfluidic channel  122  and sequestration pens  224 ,  226 ,  228  can be oriented as follows with respect to the vector of the flow  242  of fluidic medium  180  in the microfluidic channel  122 : the microfluidic channel width W ch  (or cross-sectional area of the microfluidic channel  122 ) can be substantially perpendicular to the flow  242  of medium  180 ; the width W con  (or cross-sectional area) of the connection region  236  at opening  234  can be substantially parallel to the flow  242  of medium  180  in the microfluidic channel  122 ; and/or the length L con  of the connection region can be substantially perpendicular to the flow  242  of medium  180  in the microfluidic channel  122 . The foregoing are examples only, and the relative position of the microfluidic channel  122  and sequestration pens  224 ,  226 ,  228  can be in other orientations with respect to each other. 
     As illustrated in  FIG. 2C , the width W con  of the connection region  236  can be uniform from the proximal opening  234  to the distal opening  238 . The width W con  of the connection region  236  at the distal opening  238  can thus be in any of the ranges identified herein for the width W con  of the connection region  236  at the proximal opening  234 . Alternatively, the width W con  of the connection region  236  at the distal opening  238  can be larger than the width W con  of the connection region  236  at the proximal opening  234 . 
     As illustrated in  FIG. 2C , the width of the isolation region  240  at the distal opening  238  can be substantially the same as the width W con  of the connection region  236  at the proximal opening  234 . The width of the isolation region  240  the distal opening  238  can thus be in any of the ranges identified herein for the width W con  of the connection region  236  at the proximal opening  234 . Alternatively, the width of the isolation region  240  at the distal opening  238  can be larger or smaller than the width W con  of the connection region  236  at the proximal opening  234 . Moreover, the distal opening  238  may be smaller than the proximal opening  234  and the width W con  of the connection region  236  may be narrowed between the proximal opening  234  and distal opening  238 . For example, the connection region  236  may be narrowed between the proximal opening and the distal opening, using a variety of different geometries (e g chamfering the connection region, beveling the connection region). Further, any part or subpart of the connection region  236  may be narrowed (e.g. a portion of the connection region adjacent to the proximal opening  234 ). 
       FIGS. 2D-2F  depict another exemplary embodiment of a microfluidic device  250  containing a microfluidic circuit  262  and flow channels  264 , which are variations of the respective microfluidic device  100 , circuit  132  and channel  134  of  FIG. 1A . The microfluidic device  250  also has a plurality of sequestration pens  266  that are additional variations of the above-described sequestration pens  124 ,  126 ,  128 ,  130 ,  224 ,  226  or  228 . In particular, it should be appreciated that the sequestration pens  266  of device  250  shown in  FIGS. 2D-2F  can replace any of the above-described sequestration pens  124 ,  126 ,  128 ,  130 ,  224 ,  226  or  228  in devices  100 ,  200 ,  230 ,  280 ,  290 ,  300 . Likewise, the microfluidic device  250  is another variant of the microfluidic device  100 , and may also have the same or a different DEP configuration as the above-described microfluidic device  100 ,  200 ,  230 ,  280 ,  290 ,  300  as well as any of the other microfluidic system components described herein. 
     The microfluidic device  250  of  FIGS. 2D-2F  comprises a support structure (not visible in  FIGS. 2D-2F , but can be the same or generally similar to the support structure  104  of device  100  depicted in  FIG. 1A ), a microfluidic circuit structure  256 , and a cover (not visible in  FIGS. 2D-2F , but can be the same or generally similar to the cover  122  of device  100  depicted in  FIG. 1A ). The microfluidic circuit structure  256  includes a frame  252  and microfluidic circuit material  260 , which can be the same as or generally similar to the frame  114  and microfluidic circuit material  116  of device  100  shown in  FIG. 1A . As shown in  FIG. 2D , the microfluidic circuit  262  defined by the microfluidic circuit material  260  can comprise multiple channels  264  (two are shown but there can be more) to which multiple sequestration pens  266  are fluidically connected. 
     Each sequestration pen  266  can comprise an isolation structure  272 , an isolation region  270  within the isolation structure  272 , and a connection region  268 . From a proximal opening  274  at the microfluidic channel  264  to a distal opening  276  at the isolation structure  272 , the connection region  268  fluidically connects the microfluidic channel  264  to the isolation region  270 . Generally, in accordance with the above discussion of  FIGS. 2B and 2C , a flow  278  of a first fluidic medium  254  in a channel  264  can create secondary flows  282  of the first medium  254  from the microfluidic channel  264  into and/or out of the respective connection regions  268  of the sequestration pens  266 . 
     As illustrated in  FIG. 2E , the connection region  268  of each sequestration pen  266  generally includes the area extending between the proximal opening  274  to a channel  264  and the distal opening  276  to an isolation structure  272 . The length L con  of the connection region  268  can be greater than the maximum penetration depth D p  of secondary flow  282 , in which case the secondary flow  282  will extend into the connection region  268  without being redirected toward the isolation region  270  (as shown in  FIG. 2D ). Alternatively, at illustrated in  FIG. 2F , the connection region  268  can have a length L con  that is less than the maximum penetration depth D p , in which case the secondary flow  282  will extend through the connection region  268  and be redirected toward the isolation region  270 . In this latter situation, the sum of lengths L c1  and L c2  of connection region  268  is greater than the maximum penetration depth D p , so that secondary flow  282  will not extend into isolation region  270 . Whether length L con  of connection region  268  is greater than the penetration depth D p , or the sum of lengths L c1  and L c2  of connection region  268  is greater than the penetration depth D p , a flow  278  of a first medium  254  in channel  264  that does not exceed a maximum velocity V max  will produce a secondary flow having a penetration depth D p , and micro-objects (not shown but can be the same or generally similar to the micro-objects  246  shown in  FIG. 2C ) in the isolation region  270  of a sequestration pen  266  will not be drawn out of the isolation region  270  by a flow  278  of first medium  254  in channel  264 . Nor will the flow  278  in channel  264  draw miscellaneous materials (not shown) from channel  264  into the isolation region  270  of a sequestration pen  266 . As such, diffusion is the only mechanism by which components in a first medium  254  in the microfluidic channel  264  can move from the microfluidic channel  264  into a second medium  258  in an isolation region  270  of a sequestration pen  266 . Likewise, diffusion is the only mechanism by which components in a second medium  258  in an isolation region  270  of a sequestration pen  266  can move from the isolation region  270  to a first medium  254  in the microfluidic channel  264 . The first medium  254  can be the same medium as the second medium  258 , or the first medium  254  can be a different medium than the second medium  258 . Alternatively, the first medium  254  and the second medium  258  can start out being the same, then become different, e.g., through conditioning of the second medium by one or more cells in the isolation region  270 , or by changing the medium flowing through the microfluidic channel  264 . 
     As illustrated in  FIG. 2E , the width W ch  of the microfluidic channels  264  (i.e., taken transverse to the direction of a fluid medium flow through the microfluidic channel indicated by arrows  278  in  FIG. 2D ) in the microfluidic channel  264  can be substantially perpendicular to a width W con1  of the proximal opening  274  and thus substantially parallel to a width W con2  of the distal opening  276 . The width W con1  of the proximal opening  274  and the width W con2  of the distal opening  276 , however, need not be substantially perpendicular to each other. For example, an angle between an axis (not shown) on which the width W con1  of the proximal opening  274  is oriented and another axis on which the width W con2  of the distal opening  276  is oriented can be other than perpendicular and thus other than 90°. Examples of alternatively oriented angles include angles in any of the following ranges: from about 30° to about 90°, from about 45° to about 90°, from about 60° to about 90°, or the like. 
     In various embodiments of sequestration pens (e.g.  124 ,  126 ,  128 ,  130 ,  224 ,  226 ,  228 , or  266 ), the isolation region (e.g.  240  or  270 ) is configured to contain a plurality of micro-objects. In other embodiments, the isolation region can be configured to contain only one, two, three, four, five, or a similar relatively small number of micro-objects. Accordingly, the volume of an isolation region can be, for example, at least 1×10 6 , 2×10 6 , 4×10 6 , 6×10 6  cubic microns, or more. 
     In various embodiments of sequestration pens, the width W ch  of the microfluidic channel (e.g.,  122 ) at a proximal opening (e.g.  234 ) can be within any of the following ranges: about 50-1000 microns, 50-500 microns, 50-400 microns, 50-300 microns, 50-250 microns, 50-200 microns, 50-150 microns, 50-100 microns, 70-500 microns, 70-400 microns, 70-300 microns, 70-250 microns, 70-200 microns, 70-150 microns, 90-400 microns, 90-300 microns, 90-250 microns, 90-200 microns, 90-150 microns, 100-300 microns, 100-250 microns, 100-200 microns, 100-150 microns, and 100-120 microns. In some other embodiments, the width W ch  of the microfluidic channel (e.g.,  122 ) at a proximal opening (e.g.  234 ) can be about 200-800 microns, 200-700 microns, or 200-600 microns. The foregoing are examples only, and the width W ch  of the microfluidic channel  122  can be in other ranges (e.g., a range defined by any of the endpoints listed above). Moreover, the W ch  of the microfluidic channel  122  can be selected to be in any of these ranges in regions of the microfluidic channel other than at a proximal opening of a sequestration pen. 
     In some embodiments, a sequestration pen has a height of about 30 to about 200 microns, or about 50 to about 150 microns. In some embodiments, the sequestration pen has a cross-sectional area of about 1×10 4 -3×10 6  square microns, 2×10 4 -2×10 6  square microns, 4×10 4 -1×10 6  square microns, 2×10 4 -5×10 5  square microns, 2×10 4 -1×10 5  square microns or about 2×10 5 -2×10 6  square microns. 
     In various embodiments of sequestration pens, the height H ch  of the microfluidic channel (e.g.,  122 ) at a proximal opening (e.g.,  234 ) can be within any of the following ranges: 20-100 microns, 20-90 microns, 20-80 microns, 20-70 microns, 20-60 microns, 20-50 microns, 30-100 microns, 30-90 microns, 30-80 microns, 30-70 microns, 30-60 microns, 30-50 microns, 40-100 microns, 40-90 microns, 40-80 microns, 40-70 microns, 40-60 microns, or 40-50 microns. The foregoing are examples only, and the height H ch  of the microfluidic channel (e.g.,  122 ) can be in other ranges (e.g., a range defined by any of the endpoints listed above). The height H ch  of the microfluidic channel  122  can be selected to be in any of these ranges in regions of the microfluidic channel other than at a proximal opening of a sequestration pen. 
     In various embodiments of sequestration pens a cross-sectional area of the microfluidic channel (e.g.,  122 ) at a proximal opening (e.g.,  234 ) can be within any of the following ranges: 500-50,000 square microns, 500-40,000 square microns, 500-30,000 square microns, 500-25,000 square microns, 500-20,000 square microns, 500-15,000 square microns, 500-10,000 square microns, 500-7,500 square microns, 500-5,000 square microns, 1,000-25,000 square microns, 1,000-20,000 square microns, 1,000-15,000 square microns, 1,000-10,000 square microns, 1,000-7,500 square microns, 1,000-5,000 square microns, 2,000-20,000 square microns, 2,000-15,000 square microns, 2,000-10,000 square microns, 2,000-7,500 square microns 2,000-6,000 square microns, 3,000-20,000 square microns, 3,000-15,000 square microns, 3,000-10,000 square microns, 3,000-7,500 square microns, or 3,000 to 6,000 square microns. The foregoing are examples only, and the cross-sectional area of the microfluidic channel (e.g.,  122 ) at a proximal opening (e.g.,  234 ) can be in other ranges (e.g., a range defined by any of the endpoints listed above). 
     In various embodiments of sequestration pens, the length L con  of the connection region (e.g.,  236 ) can be in any of the following ranges: about 1-600 microns, 5-550 microns, 10-500 microns, 15-400 microns, 20-300 microns, 20-500 microns, 40-400 microns, 60-100 microns, 80-200 microns, or about 100-150 microns. The foregoing are examples only, and length L con  of a connection region (e.g.,  236 ) can be in a different range than the foregoing examples (e.g., a range defined by any of the endpoints listed above). 
     In various embodiments of sequestration pens the width W con  of a connection region (e.g.,  236 ) at a proximal opening (e.g.,  234 ) can be in any of the following ranges: 20-500 microns, 20-400 microns, 20-300 microns, 20-200 microns, 20-150 microns, 20-100 microns, 20-80 microns, 20-60 microns, 30-400 microns, 30-300 microns, 30-200 microns, 30-150 microns, 30-100 microns, 30-80 microns, 30-60 microns, 40-300 microns, 40-200 microns, 40-150 microns, 40-100 microns, 40-80 microns, 40-60 microns, 50-250 microns, 50-200 microns, 50-150 microns, 50-100 microns, 50-80 microns, 60-200 microns, 60-150 microns, 60-100 microns, 60-80 microns, 70-150 microns, 70-100 microns, and 80-100 microns. The foregoing are examples only, and the width W ch  of a connection region (e.g.,  236 ) at a proximal opening (e.g.,  234 ) can be different than the foregoing examples (e.g., a range defined by any of the endpoints listed above). 
     In various embodiments of sequestration pens, the width W con  of a connection region (e.g.,  236 ) at a proximal opening (e.g.,  234 ) can be at least as large as the largest dimension of a micro-object (e.g., biological cell which may be a T cell, B cell, or an ovum or embryo) that the sequestration pen is intended for. The foregoing are examples only, and the width W con  of a connection region (e.g.,  236 ) at a proximal opening (e.g.,  234 ) can be different than the foregoing examples (e.g., a range defined by any of the endpoints listed above). 
     In various embodiments of sequestration pens, the width W pr  of a proximal opening of a connection region may be at least as large as the largest dimension of a micro-object (e.g., a biological micro-object such as a cell) that the sequestration pen is intended for. For example, the width W pr  may be about 50 microns, about 60 microns, about 100 microns, about 200 microns, about 300 microns or may be about 50-300 microns, about 50-200 microns, about 50-100 microns, about 75-150 microns, about 75-100 microns, or about 200-300 microns 
     In various embodiments of sequestration pens, a ratio of the length L con  of a connection region (e.g.,  236 ) to a width W con  of the connection region (e.g.,  236 ) at the proximal opening  234  can be greater than or equal to any of the following ratios: 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, or more. The foregoing are examples only, and the ratio of the length L con  of a connection region  236  to a width W con  of the connection region  236  at the proximal opening  234  can be different than the foregoing examples. 
     In various embodiments of microfluidic devices  100 ,  200 ,  23 ,  250 ,  280 ,  290 ,  300 , V max  can be set around 0.2, 0.5, 0.7, 1.0, 1.3, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.7, 7.0, 7.5, 8.0, 8.5, 9.0, 10, 11, 12, 13, 14, or 15 microliters/sec. 
     In various embodiments of microfluidic devices having sequestration pens, the volume of an isolation region (e.g.,  240 ) of a sequestration pen can be, for example, at least 5×10 5 , 8×10 5 , 1×10 6 , 2×10 6 , 4×10 6 , 6×10 6 , 8×10 6 , 1×10 7 , 5×10 7 , 1×10 8 , 5×10 8 , or 8×10 8  cubic microns, or more. In various embodiments of microfluidic devices having sequestration pens, the volume of a sequestration pen may be about 5×10 5 , 6×10 5 , 8×10 5 , 1×10 6 , 2×10 6 , 4×10 6 , 8×10 6 , 1×10 7 , 3×10 7 , 5×10 7 , or about 8×10 7  cubic microns, or more. In some other embodiments, the volume of a sequestration pen may be about 1 nanoliter to about 50 nanoliters, 2 nanoliters to about 25 nanoliters, 2 nanoliters to about 20 nanoliters, about 2 nanoliters to about 15 nanoliters, or about 2 nanoliters to about 10 nanoliters. 
     In various embodiment, the microfluidic device has sequestration pens configured as in any of the embodiments discussed herein where the microfluidic device has about 5 to about 10 sequestration pens, about 10 to about 50 sequestration pens, about 100 to about 500 sequestration pens; about 200 to about 1000 sequestration pens, about 500 to about 1500 sequestration pens, about 1000 to about 2000 sequestration pens, about 1000 to about 3500, or about 2500 to about 5000 sequestration pens. The sequestration pens need not all be the same size and may include a variety of configurations (e.g., different widths, different features within the sequestration pen). 
     Sequestration pens  424 ,  524 ,  526 ,  624 ,  626 ,  724 ,  824 ,  826 ,  828 ,  924 ,  1024 ,  1124 ,  1126 ,  1127 ,  1324  described below may have dimensions and properties like that of any of the sequestration pens  124 ,  126 ,  128 ,  130 ,  224 ,  226 ,  228 , or  266  as described above, in any combination. 
       FIG. 2G  illustrates a microfluidic device  280  according to one embodiment. The microfluidic device  280  illustrated in  FIG. 2G  is a stylized diagram of a microfluidic device  100 . In practice the microfluidic device  280  and its constituent circuit elements (e.g. channels  122  and sequestration pens  128 ) would have the dimensions discussed herein. The microfluidic circuit  120  illustrated in  FIG. 2G  has two ports  107 , four distinct channels  122  and four distinct flow paths  106 . The microfluidic device  280  further comprises a plurality of sequestration pens opening off of each channel  122 . In the microfluidic device illustrated in  FIG. 2G , the sequestration pens have a geometry similar to the pens illustrated in  FIG. 2C  and thus, have both connection regions and isolation regions. Accordingly, the microfluidic circuit  120  includes both swept regions (e g channels  122  and portions of the connection regions  236  within the maximum penetration depth D p  of the secondary flow  244 ) and non-swept regions (e.g. isolation regions  240  and portions of the connection regions  236  not within the maximum penetration depth D p  of the secondary flow  244 ). 
       FIGS. 3A through 3B  shows various embodiments of system  150  which can be used to operate and observe microfluidic devices (e.g.  100 ,  200 ,  230 ,  250 ,  280 ,  290 ,  300 ) according to the present disclosure. As illustrated in  FIG. 3A , the system  150  can include a structure (“nest”)  300  configured to hold a microfluidic device  100  (not shown), or any other microfluidic device described herein. The nest  300  can include a socket  302  capable of interfacing with the microfluidic device  320  (e.g., an optically-actuated electrokinetic device  100 ) and providing electrical connections from power source  192  to microfluidic device  320 . The nest  300  can further include an integrated electrical signal generation subsystem  304 . The electrical signal generation subsystem  304  can be configured to supply a biasing voltage to socket  302  such that the biasing voltage is applied across a pair of electrodes in the microfluidic device  320  when it is being held by socket  302 . Thus, the electrical signal generation subsystem  304  can be part of power source  192 . The ability to apply a biasing voltage to microfluidic device  320  does not mean that a biasing voltage will be applied at all times when the microfluidic device  320  is held by the socket  302 . Rather, in most cases, the biasing voltage will be applied intermittently, e.g., only as needed to facilitate the generation of electrokinetic forces, such as dielectrophoresis or electro-wetting, in the microfluidic device  320 . 
     As illustrated in  FIG. 3A , the nest  300  can include a printed circuit board assembly (PCBA)  322 . The electrical signal generation subsystem  304  can be mounted on and electrically integrated into the PCBA  322 . The exemplary support includes socket  302  mounted on PCBA  322 , as well. 
     Typically, the electrical signal generation subsystem  304  will include a waveform generator (not shown). The electrical signal generation subsystem  304  can further include an oscilloscope (not shown) and/or a waveform amplification circuit (not shown) configured to amplify a waveform received from the waveform generator. The oscilloscope, if present, can be configured to measure the waveform supplied to the microfluidic device  320  held by the socket  302 . In certain embodiments, the oscilloscope measures the waveform at a location proximal to the microfluidic device  320  (and distal to the waveform generator), thus ensuring greater accuracy in measuring the waveform actually applied to the device. Data obtained from the oscilloscope measurement can be, for example, provided as feedback to the waveform generator, and the waveform generator can be configured to adjust its output based on such feedback. An example of a suitable combined waveform generator and oscilloscope is the Red Pitaya™. 
     In certain embodiments, the nest  300  further comprises a controller  308 , such as a microprocessor used to sense and/or control the electrical signal generation subsystem  304 . Examples of suitable microprocessors include the Arduino™ microprocessors, such as the Arduino Nano™. The controller  308  may be used to perform functions and analysis or may communicate with an external master controller  154  (shown in  FIG. 1A ) to perform functions and analysis. In the embodiment illustrated in  FIG. 3A  the controller  308  communicates with a master controller  154  through an interface  310  (e.g., a plug or connector). 
     In some embodiments, the nest  300  can comprise an electrical signal generation subsystem  304  comprising a Red Pitaya™ waveform generator/oscilloscope unit (“Red Pitaya unit”) and a waveform amplification circuit that amplifies the waveform generated by the Red Pitaya unit and passes the amplified voltage to the microfluidic device  100 . In some embodiments, the Red Pitaya unit is configured to measure the amplified voltage at the microfluidic device  320  and then adjust its own output voltage as needed such that the measured voltage at the microfluidic device  320  is the desired value. In some embodiments, the waveform amplification circuit can have a +6.5V to −6.5V power supply generated by a pair of DC-DC converters mounted on the PCBA  322 , resulting in a signal of up to 13 Vpp at the microfluidic device  100 . 
     As illustrated in  FIG. 3A , the support structure  300  (e.g., nest) can further include a thermal control subsystem  306 . The thermal control subsystem  306  can be configured to regulate the temperature of microfluidic device  320  held by the support structure  300 . For example, the thermal control subsystem  306  can include a Peltier thermoelectric device (not shown) and a cooling unit (not shown). The Peltier thermoelectric device can have a first surface configured to interface with at least one surface of the microfluidic device  320 . The cooling unit can be, for example, a cooling block (not shown), such as a liquid-cooled aluminum block. A second surface of the Peltier thermoelectric device (e.g., a surface opposite the first surface) can be configured to interface with a surface of such a cooling block. The cooling block can be connected to a fluidic path  314  configured to circulate cooled fluid through the cooling block. In the embodiment illustrated in  FIG. 3A , the support structure  300  comprises an inlet  316  and an outlet  318  to receive cooled fluid from an external reservoir (not shown), introduce the cooled fluid into the fluidic path  314  and through the cooling block, and then return the cooled fluid to the external reservoir. In some embodiments, the Peltier thermoelectric device, the cooling unit, and/or the fluidic path  314  can be mounted on a casing  312  of the support structure  300 . In some embodiments, the thermal control subsystem  306  is configured to regulate the temperature of the Peltier thermoelectric device so as to achieve a target temperature for the microfluidic device  320 . Temperature regulation of the Peltier thermoelectric device can be achieved, for example, by a thermoelectric power supply, such as a Pololu™ thermoelectric power supply (Pololu Robotics and Electronics Corp.). The thermal control subsystem  306  can include a feedback circuit, such as a temperature value provided by an analog circuit. Alternatively, the feedback circuit can be provided by a digital circuit. 
     In some embodiments, the nest  300  can include a thermal control subsystem  306  with a feedback circuit that is an analog voltage divider circuit (not shown) which includes a resistor (e.g., with resistance 1 kOhm+/−0.1%, temperature coefficient+/−0.02 ppm/C0) and a NTC thermistor (e.g., with nominal resistance 1 kOhm+/−0.01%). In some instances, the thermal control subsystem  306  measures the voltage from the feedback circuit and then uses the calculated temperature value as input to an on-board PID control loop algorithm. Output from the PID control loop algorithm can drive, for example, both a directional and a pulse-width-modulated signal pin on a Pololu™ motor drive (not shown) to actuate the thermoelectric power supply, thereby controlling the Peltier thermoelectric device. 
     The nest  300  can include a serial port  324  which allows the microprocessor of the controller  308  to communicate with an external master controller  154  via the interface  310  (not shown). In addition, the microprocessor of the controller  308  can communicate (e.g., via a Plink tool (not shown)) with the electrical signal generation subsystem  304  and thermal control subsystem  306 . Thus, via the combination of the controller  308 , the interface  310 , and the serial port  324 , the electrical signal generation subsystem  304  and the thermal control subsystem  306  can communicate with the external master controller  154 . In this manner, the master controller  154  can, among other things, assist the electrical signal generation subsystem  304  by performing scaling calculations for output voltage adjustments. A Graphical User Interface (GUI) (not shown) provided via a display device  170  coupled to the external master controller  154 , can be configured to plot temperature and waveform data obtained from the thermal control subsystem  306  and the electrical signal generation subsystem  304 , respectively. Alternatively, or in addition, the GUI can allow for updates to the controller  308 , the thermal control subsystem  306 , and the electrical signal generation subsystem  304 . 
     As discussed above, system  150  can include an imaging device  194 . In some embodiments, the imaging device  194  comprises a light modulating subsystem  330  (See  FIG. 3B ). The light modulating subsystem  330  can include a digital mirror device (DMD) or a microshutter array system (MSA), either of which can be configured to receive light from a light source  332  and transmits a subset of the received light into an optical train of microscope  350 . Alternatively, the light modulating subsystem  330  can include a device that produces its own light (and thus dispenses with the need for a light source  332 ), such as an organic light emitting diode display (OLED), a liquid crystal on silicon (LCOS) device, a ferroelectric liquid crystal on silicon device (FLCOS), or a transmissive liquid crystal display (LCD). The light modulating subsystem  330  can be, for example, a projector. Thus, the light modulating subsystem  330  can be capable of emitting both structured and unstructured light. In certain embodiments, imaging module  164  and/or motive module  162  of system  150  can control the light modulating subsystem  330 . 
     In certain embodiments, the imaging device  194  further comprises a microscope  350 . In such embodiments, the nest  300  and light modulating subsystem  330  can be individually configured to be mounted on the microscope  350 . The microscope  350  can be, for example, a standard research-grade light microscope or fluorescence microscope. Thus, the nest  300  can be configured to be mounted on the stage  344  of the microscope  350  and/or the light modulating subsystem  330  can be configured to mount on a port of microscope  350 . In other embodiments, the nest  300  and the light modulating subsystem  330  described herein can be integral components of microscope  350 . 
     In certain embodiments, the microscope  350  can further include one or more detectors  348 . In some embodiments, the detector  348  is controlled by the imaging module  164 . The detector  348  can include an eye piece, a charge-coupled device (CCD), a camera (e.g., a digital camera), or any combination thereof. If at least two detectors  348  are present, one detector can be, for example, a fast-frame-rate camera while the other detector can be a high sensitivity camera. Furthermore, the microscope  350  can include an optical train configured to receive reflected and/or emitted light from the microfluidic device  320  and focus at least a portion of the reflected and/or emitted light on the one or more detectors  348 . The optical train of the microscope can also include different tube lenses (not shown) for the different detectors, such that the final magnification on each detector can be different. 
     In certain embodiments, imaging device  194  is configured to use at least two light sources. For example, a first light source  332  can be used to produce structured light (e.g., via the light modulating subsystem  330 ) and a second light source  334  can be used to provide unstructured light. The first light source  332  can produce structured light for optically-actuated electrokinesis and/or fluorescent excitation, and the second light source  334  can be used to provide bright field illumination. In these embodiments, the motive module  164  can be used to control the first light source  332  and the imaging module  164  can be used to control the second light source  334 . The optical train of the microscope  350  can be configured to (1) receive structured light from the light modulating subsystem  330  and focus the structured light on at least a first region in a microfluidic device, such as an optically-actuated electrokinetic device, when the device is being held by the nest  300 , and (2) receive reflected and/or emitted light from the microfluidic device and focus at least a portion of such reflected and/or emitted light onto detector  348 . The optical train can be further configured to receive unstructured light from a second light source and focus the unstructured light on at least a second region of the microfluidic device, when the device is held by the nest  300 . In certain embodiments, the first and second regions of the microfluidic device can be overlapping regions. For example, the first region can be a subset of the second region. In other embodiments, the second light source  334  may additionally or alternatively include a laser, which may have any suitable wavelength of light. The representation of the optical system shown in  FIG. 3B  is a schematic representation only, and the optical system may include additional filters, notch filters, lenses and the like. When the second light source  334  includes one or more light source(s) for brightfield and/or fluorescent excitation, as well as laser illumination the physical arrangement of the light source(s) may vary from that shown in  FIG. 3B , and the laser illumination may be introduced at any suitable physical location within the optical system. The schematic locations of light source  334  and light source  332 /light modulating subsystem  330  may be interchanged as well. 
     In  FIG. 3B , the first light source  332  is shown supplying light to a light modulating subsystem  330 , which provides structured light to the optical train of the microscope  350  of system  355  (not shown). The second light source  334  is shown providing unstructured light to the optical train via a beam splitter  336 . Structured light from the light modulating subsystem  330  and unstructured light from the second light source  334  travel from the beam splitter  336  through the optical train together to reach a second beam splitter (or dichroic filter  338 , depending on the light provided by the light modulating subsystem  330 ), where the light gets reflected down through the objective  336  to the sample plane  342 . Reflected and/or emitted light from the sample plane  342  then travels back up through the objective  340 , through the beam splitter and/or dichroic filter  338 , and to a dichroic filter  346 . Only a fraction of the light reaching dichroic filter  346  passes through and reaches the detector  348 . 
     In some embodiments, the second light source  334  emits blue light. With an appropriate dichroic filter  346 , blue light reflected from the sample plane  342  is able to pass through dichroic filter  346  and reach the detector  348 . In contrast, structured light coming from the light modulating subsystem  330  gets reflected from the sample plane  342 , but does not pass through the dichroic filter  346 . In this example, the dichroic filter  346  is filtering out visible light having a wavelength longer than 495 nm. Such filtering out of the light from the light modulating subsystem  330  would only be complete (as shown) if the light emitted from the light modulating subsystem did not include any wavelengths shorter than 495 nm. In practice, if the light coming from the light modulating subsystem  330  includes wavelengths shorter than 495 nm (e.g., blue wavelengths), then some of the light from the light modulating subsystem would pass through filter  346  to reach the detector  348 . In such an embodiment, the filter  346  acts to change the balance between the amount of light that reaches the detector  348  from the first light source  332  and the second light source  334 . This can be beneficial if the first light source  332  is significantly stronger than the second light source  334 . In other embodiments, the second light source  334  can emit red light, and the dichroic filter  346  can filter out visible light other than red light (e.g., visible light having a wavelength shorter than 650 nm). 
       FIG. 3C  illustrates communications between the motive module  162  and the light modulating subsystem  330  to project patterns of light on a microfluidic device according to a specific embodiment of the disclosure. As discussed above with respect to  FIG. 3B , the light modulating subsystem  330  may comprise an electrically-addressed spatial light modulator and/or an optically-addressed spatial light modulator. Electrically-addressed spatial light modulators comprise an array of individually-addressable spatial light modulators that are controlled by electrodes. In  FIG. 3C , the light modulating subsystem  330  is a Digital Mirror Device (DMD)  460  comprising an array of individually-addressable micro-mirrors  464  that are controlled by one or more electrodes. However, in other embodiments, the light modulating subsystem  330  can be a Liquid Crystal on Silicon (LCoS) device comprising an array of individually-addressable electrodes that correspond to pixels in a liquid crystal display. 
     In the embodiment illustrated in  FIG. 3C , the light modulating subsystem  330  uses a separate light source  440  to receive and modulate light. However, in other embodiments, the light modulating subsystem  330  comprises its own light source. 
     As illustrated in  FIG. 3C , the motive module  162  transmits information  450  specifying a specific light pattern (“pattern information”) to the light modulating subsystem  330 . In some embodiments, the pattern information  450  can comprise a bitmap (or similar pixel-based data structure), vector data, or any combination thereof. For purposes of illustration, the pattern information  450  in  FIG. 3C  is illustrated as a bitmap comprising an array of pixels  456  and including a square pattern  452  of pixels. Depending on the embodiment, the pattern information  450  can be binary (i.e. specify whether or not to project a pattern of light) or contain values indicating an intensity of light to project. In instances where the spatial light modulators are micro-mirrors  464 , the micro-mirrors  464  may create different intensities of light by rapidly switching the mirrors between an “on” and “off” state (i.e. “dithering” the micro-mirrors). 
     The light modulating subsystem  330  receives the pattern information  450  from the motive module  162  and uses the pattern information  450  to direct the projection of a light pattern  468  onto DEP electrode regions  474  on the microfluidic device  470 . In the embodiment illustrated in  FIG. 3C , a DMD  460  rotates a plurality  462  of individually-addressable micro-mirrors  464  corresponding to the square pattern information  450  into an “on state.” The square pattern of individual-addressable micro-mirrors  462  modulates the light from the light source  440  to project a light pattern  468  onto the microfluidic device  470  that illuminates a square pattern of DEP electrode regions  472  in the array of DEP electrode regions  474  in the microfluidic device  470 . 
     In some embodiments, there is a one-to-one correspondence between the array of individually-addressable spatial light modulating elements  464  that project light onto the microfluidic device  470  and the array of DEP electrode regions  474  in the microfluidic device  470 . In this way, each individually-addressable spatial light modulating element  464  can project light to generate light-actuated DEP force at a corresponding DEP electrode region  474 . In these embodiments, the motive module  162  can send pattern information  450  to the light modulating subsystem  330  that specifies the DEP electrode regions  474  to project light onto. For example, instead of sending bitmap and or vector data to the light modulating subsystem  330 , the motive module  162  can communicate directly with the individually-addressable spatial light modulators to control which of the DEP electrode regions  474  are illuminated on the microfluidic device  470 . Once illuminated the DEP electrode regions  474  may exert OET or OEW force on surrounding micro-objects. 
     As discussed above, in some embodiments, the spatial light modulating elements  464  can receive pattern information  450  specifying an intensity of light to project. In a specific embodiment, the pattern information  450  may specify a gradation of light to project over adjacent DEP electrode regions  474  in the microfluidic device. In some embodiments, the pattern information  450  may specify a gradation of light that decreases in intensity over adjacent DEP electrode regions  474 . For example, the pattern information  450  may specify that about 100% of the maximum light intensity is to be projected at a first DEP electrode region  474 , that 70% of the maximum light intensity is to be projected at a second DEP electrode region  474  adjacent to the first DEP electrode region  474 , and that 10% of the maximum light intensity is to be projected at a third DEP electrode region  474  adjacent to the second DEP electrode region  474 . Various combinations of light intensities may be used to project a gradation over various numbers of DEP electrode regions  474  (e.g. any decreasing combination of about 100%, about 90%, about 80%, about 70%, about 60%, about 50%, about 40%, about 30%, about 20%, and about 10%, and any values therebetween, of the maximum light intensity over any number of DEP electrode regions  474 ). Similarly, the pattern information  450  may specify a gradation of light that increases in intensity over any number of DEP electrode regions  474  or a gradation of light that both increases and decreases in intensity over any number of DEP electrode regions  474 . 
     Coating solutions and coating agents. Without intending to be limited by theory, maintenance of a biological micro-object (e.g., a biological cell) within a microfluidic device (e.g., a DEP-configured and/or EW-configured microfluidic device) may be facilitated (i.e., the biological micro-object exhibits increased viability, greater expansion and/or greater portability within the microfluidic device) when at least one or more inner surfaces of the microfluidic device have been conditioned or coated so as to present a layer of organic and/or hydrophilic molecules that provides the primary interface between the microfluidic device and biological micro-object(s) maintained therein. In some embodiments, one or more of the inner surfaces of the microfluidic device (e.g. the inner surface of the electrode activation substrate of a DEP-configured microfluidic device, the cover of the microfluidic device, and/or the surfaces of the circuit material) may be treated with or modified by a coating solution and/or coating agent to generate the desired layer of organic and/or hydrophilic molecules. 
     The coating may be applied before or after introduction of biological micro-object(s), or may be introduced concurrently with the biological micro-object(s). In some embodiments, the biological micro-object(s) may be imported into the microfluidic device in a fluidic medium that includes one or more coating agents. In other embodiments, the inner surface(s) of the microfluidic device (e.g., a DEP-configured microfluidic device) are treated or “primed” with a coating solution comprising a coating agent prior to introduction of the biological micro-object(s) into the microfluidic device. 
     In some embodiments, at least one surface of the microfluidic device includes a coating material that provides a layer of organic and/or hydrophilic molecules suitable for maintenance and/or expansion of biological micro-object(s) (e.g. provides a conditioned surface as described below). In some embodiments, substantially all the inner surfaces of the microfluidic device include the coating material. The coated inner surface(s) may include the surface of a flow region (e.g., channel), chamber, or sequestration pen, or a combination thereof. In some embodiments, each of a plurality of sequestration pens has at least one inner surface coated with coating materials. In other embodiments, each of a plurality of flow regions or channels has at least one inner surface coated with coating materials. In some embodiments, at least one inner surface of each of a plurality of sequestration pens and each of a plurality of channels is coated with coating materials. 
     Coating agent/Solution. Any convenient coating agent/coating solution can be used, including but not limited to: serum or serum factors, bovine serum albumin (BSA), polymers, detergents, enzymes, and any combination thereof. 
     Polymer-based coating materials. The at least one inner surface may include a coating material that comprises a polymer. The polymer may be covalently or non-covalently bound (or may be non-specifically adhered) to the at least one surface. The polymer may have a variety of structural motifs, such as found in block polymers (and copolymers), star polymers (star copolymers), and graft or comb polymers (graft copolymers), all of which may be suitable for the methods disclosed herein. 
     The polymer may include a polymer including alkylene ether moieties. A wide variety of alkylene ether containing polymers may be suitable for use in the microfluidic devices described herein. One non-limiting exemplary class of alkylene ether containing polymers are amphiphilic nonionic block copolymers which include blocks of polyethylene oxide (PEO) and polypropylene oxide (PPO) subunits in differing ratios and locations within the polymer chain. Pluronic® polymers (BASF) are block copolymers of this type and are known in the art to be suitable for use when in contact with living cells. The polymers may range in average molecular mass M w  from about 2000 Da to about 20 KDa. In some embodiments, the PEO-PPO block copolymer can have a hydrophilic-lipophilic balance (HLB) greater than about 10 (e.g. 12-18). Specific Pluronic® polymers useful for yielding a coated surface include Pluronic® L44, L64, P85, and F127 (including F127NF). Another class of alkylene ether containing polymers is polyethylene glycol (PEG M w &lt;100,000 Da) or alternatively polyethylene oxide (PEO, M w &gt;100,000). In some embodiments, a PEG may have an M w  of about 1000 Da, 5000 Da, 10,000 Da or 20,000 Da. 
     In other embodiments, the coating material may include a polymer containing carboxylic acid moieties. The carboxylic acid subunit may be an alkyl, alkenyl or aromatic moiety containing subunit. One non-limiting example is polylactic acid (PLA). In other embodiments, the coating material may include a polymer containing phosphate moieties, either at a terminus of the polymer backbone or pendant from the backbone of the polymer. In yet other embodiments, the coating material may include a polymer containing sulfonic acid moieties. The sulfonic acid subunit may be an alkyl, alkenyl or aromatic moiety containing subunit. One non-limiting example is polystyrene sulfonic acid (PSSA) or polyanethole sulfonic acid. In further embodiments, the coating material may include a polymer including amine moieties. The polyamino polymer may include a natural polyamine polymer or a synthetic polyamine polymer. Examples of natural polyamines include spermine, spermidine, and putrescine. 
     In other embodiments, the coating material may include a polymer containing saccharide moieties. In a non-limiting example, polysaccharides such as xanthan gum or dextran may be suitable to form a material which may reduce or prevent cell sticking in the microfluidic device. For example, a dextran polymer having a size about 3 kDa may be used to provide a coating material for a surface within a microfluidic device. 
     In other embodiments, the coating material may include a polymer containing nucleotide moieties, i.e. a nucleic acid, which may have ribonucleotide moieties or deoxyribonucleotide moieties, providing a polyelectrolyte surface. The nucleic acid may contain only natural nucleotide moieties or may contain unnatural nucleotide moieties which comprise nucleobase, ribose or phosphate moiety analogs such as 7-deazaadenine, pentose, methyl phosphonate or phosphorothioate moieties without limitation. 
     In yet other embodiments, the coating material may include a polymer containing amino acid moieties. The polymer containing amino acid moieties may include a natural amino acid containing polymer or an unnatural amino acid containing polymer, either of which may include a peptide, a polypeptide or a protein. In one non-limiting example, the protein may be bovine serum albumin (BSA) and/or serum (or a combination of multiple different sera) comprising albumin and/or one or more other similar proteins as coating agents. The serum can be from any convenient source, including but not limited to fetal calf serum, sheep serum, goat serum, horse serum, and the like. In certain embodiments, BSA in a coating solution is present in a concentration from about 1 mg/mL to about 100 mg/mL, including 5 mg/mL, 10 mg/mL, 20 mg/mL, 30 mg/mL, 40 mg/mL, 50 mg/mL, 60 mg/mL, 70 mg/mL, 80 mg/mL, 90 mg/mL, or more or anywhere in between. In certain embodiments, serum in a coating solution may be present in a concentration of about 20% (v/v) to about 50% v/v, including 25%, 30%, 35%, 40%, 45%, or more or anywhere in between. In some embodiments, BSA may be present as a coating agent in a coating solution at 5 mg/mL, whereas in other embodiments, BSA may be present as a coating agent in a coating solution at 70 mg/mL. In certain embodiments, serum is present as a coating agent in a coating solution at 30%. In some embodiments, an extracellular matrix (ECM) protein may be provided within the coating material for optimized cell adhesion to foster cell growth. A cell matrix protein, which may be included in a coating material, can include, but is not limited to, a collagen, an elastin, an RGD-containing peptide (e.g. a fibronectin), or a laminin. In yet other embodiments, growth factors, cytokines, hormones or other cell signaling species may be provided within the coating material of the microfluidic device. 
     In some embodiments, the coating material may include a polymer containing more than one of alkylene oxide moieties, carboxylic acid moieties, sulfonic acid moieties, phosphate moieties, saccharide moieties, nucleotide moieties, or amino acid moieties. In other embodiments, the polymer conditioned surface may include a mixture of more than one polymer each having alkylene oxide moieties, carboxylic acid moieties, sulfonic acid moieties, phosphate moieties, saccharide moieties, nucleotide moieties, and/or amino acid moieties, which may be independently or simultaneously incorporated into the coating material. 
     Covalently linked coating materials. In some embodiments, the at least one inner surface includes covalently linked molecules that provide a layer of organic and/or hydrophilic molecules suitable for maintenance/expansion of biological micro-object(s) within the microfluidic device, providing a conditioned surface for such cells. 
     The covalently linked molecules include a linking group, wherein the linking group is covalently linked to one or more surfaces of the microfluidic device, as described below. The linking group is also covalently linked to a moiety configured to provide a layer of organic and/or hydrophilic molecules suitable for maintenance/expansion of biological micro-object(s). 
     In some embodiments, the covalently linked moiety configured to provide a layer of organic and/or hydrophilic molecules suitable for maintenance/expansion of biological micro-object(s) may include alkyl or fluoroalkyl (which includes perfluoroalkyl) moieties; mono- or polysaccharides (which may include but is not limited to dextran); alcohols (including but not limited to propargyl alcohol); polyalcohols, including but not limited to polyvinyl alcohol; alkylene ethers, including but not limited to polyethylene glycol; polyelectrolytes (including but not limited to polyacrylic acid or polyvinyl phosphonic acid); amino groups (including derivatives thereof, such as, but not limited to alkylated amines, hydroxyalkylated amino group, guanidinium, and heterocylic groups containing an unaromatized nitrogen ring atom, such as, but not limited to morpholinyl or piperazinyl); carboxylic acids including but not limited to propiolic acid (which may provide a carboxylate anionic surface); phosphonic acids, including but not limited to ethynyl phosphonic acid (which may provide a phosphonate anionic surface); sulfonate anions; carboxybetaines; sulfobetaines; sulfamic acids; or amino acids. 
     In various embodiments, the covalently linked moiety configured to provide a layer of organic and/or hydrophilic molecules suitable for maintenance/expansion of biological micro-object(s) in the microfluidic device may include non-polymeric moieties such as an alkyl moiety, a substituted alkyl moiety, such as a fluoroalkyl moiety (including but not limited to a perfluoroalkyl moiety), amino acid moiety, alcohol moiety, amino moiety, carboxylic acid moiety, phosphonic acid moiety, sulfonic acid moiety, sulfamic acid moiety, or saccharide moiety. Alternatively, the covalently linked moiety may include polymeric moieties, which may be any of the moieties described above. 
     In some embodiments, the covalently linked alkyl moiety may comprises carbon atoms forming a linear chain (e.g., a linear chain of at least 10 carbons, or at least 14, 16, 18, 20, 22, or more carbons) and may be an unbranched alkyl moiety. In some embodiments, the alkyl group may include a substituted alkyl group (e.g., some of the carbons in the alkyl group can be fluorinated or perfluorinated). In some embodiments, the alkyl group may include a first segment, which may include a perfluoroalkyl group, joined to a second segment, which may include a non-substituted alkyl group, where the first and second segments may be joined directly or indirectly (e.g., by means of an ether linkage). The first segment of the alkyl group may be located distal to the linking group, and the second segment of the alkyl group may be located proximal to the linking group. 
     In other embodiments, the covalently linked moiety may include at least one amino acid, which may include more than one type of amino acid. Thus, the covalently linked moiety may include a peptide or a protein. In some embodiments, the covalently linked moiety may include an amino acid which may provide a zwitterionic surface to support cell growth, viability, portability, or any combination thereof. 
     In other embodiments, the covalently linked moiety may include at least one alkylene oxide moiety, and may include any alkylene oxide polymer as described above. One useful class of alkylene ether containing polymers is polyethylene glycol (PEG M w &lt;100,000 Da) or alternatively polyethylene oxide (PEO, M w &gt;100,000). In some embodiments, a PEG may have an M w  of about 1000 Da, 5000 Da, 10,000 Da or 20,000 Da. 
     The covalently linked moiety may include one or more saccharides. The covalently linked saccharides may be mono-, di-, or polysaccharides. The covalently linked saccharides may be modified to introduce a reactive pairing moiety which permits coupling or elaboration for attachment to the surface. Exemplary reactive pairing moieties may include aldehyde, alkyne or halo moieties. A polysaccharide may be modified in a random fashion, wherein each of the saccharide monomers may be modified or only a portion of the saccharide monomers within the polysaccharide are modified to provide a reactive pairing moiety that may be coupled directly or indirectly to a surface. One exemplar may include a dextran polysaccharide, which may be coupled indirectly to a surface via an unbranched linker. 
     The covalently linked moiety may include one or more amino groups. The amino group may be a substituted amine moiety, guanidine moiety, nitrogen-containing heterocyclic moiety or heteroaryl moiety. The amino containing moieties may have structures permitting pH modification of the environment within the microfluidic device, and optionally, within the sequestration pens and/or flow regions (e.g., channels). 
     The coating material providing a conditioned surface may comprise only one kind of covalently linked moiety or may include more than one different kind of covalently linked moiety. For example, the fluoroalkyl conditioned surfaces (including perfluoroalkyl) may have a plurality of covalently linked moieties which are all the same, e.g., having the same linking group and covalent attachment to the surface, the same overall length, and the same number of fluoromethylene units comprising the fluoroalkyl moiety. Alternatively, the coating material may have more than one kind of covalently linked moiety attached to the surface. For example, the coating material may include molecules having covalently linked alkyl or fluoroalkyl moieties having a specified number of methylene or fluoromethylene units and may further include a further set of molecules having charged moieties covalently attached to an alkyl or fluoroalkyl chain having a greater number of methylene or fluoromethylene units, which may provide capacity to present bulkier moieties at the coated surface. In this instance, the first set of molecules having different, less sterically demanding termini and fewer backbone atoms can help to functionalize the entire substrate surface and thereby prevent undesired adhesion or contact with the silicon/silicon oxide, hafnium oxide or alumina making up the substrate itself. In another example, the covalently linked moieties may provide a zwitterionic surface presenting alternating charges in a random fashion on the surface. 
     Conditioned surface properties. Aside from the composition of the conditioned surface, other factors such as physical thickness of the hydrophobic material can impact DEP force. Various factors can alter the physical thickness of the conditioned surface, such as the manner in which the conditioned surface is formed on the substrate (e.g. vapor deposition, liquid phase deposition, spin coating, flooding, and electrostatic coating). In some embodiments, the conditioned surface has a thickness in the range of about 1 nm to about 10 nm; about 1 nm to about 7 nm; about 1 nm to about 5 nm; or any individual value therebetween. In other embodiments, the conditioned surface formed by the covalently linked moieties may have a thickness of about 10 nm to about 50 nm. In various embodiments, the conditioned surface prepared as described herein has a thickness of less than 10 nm. In some embodiments, the covalently linked moieties of the conditioned surface may form a monolayer when covalently linked to the surface of the microfluidic device (e.g., a DEP configured substrate surface) and may have a thickness of less than 10 nm (e.g., less than 5 nm, or about 1.5 to 3.0 nm). These values are in contrast to that of a surface prepared by spin coating, for example, which may typically have a thickness in the range of about 30 nm. In some embodiments, the conditioned surface does not require a perfectly formed monolayer to be suitably functional for operation within a DEP-configured microfluidic device. 
     In various embodiments, the coating material providing a conditioned surface of the microfluidic device may provide desirable electrical properties. Without intending to be limited by theory, one factor that impacts robustness of a surface coated with a particular coating material is intrinsic charge trapping. Different coating materials may trap electrons, which can lead to breakdown of the coating material. Defects in the coating material may increase charge trapping and lead to further breakdown of the coating material. Similarly, different coating materials have different dielectric strengths (i.e. the minimum applied electric field that results in dielectric breakdown), which may impact charge trapping. In certain embodiments, the coating material can have an overall structure (e.g., a densely-packed monolayer structure) that reduces or limits that amount of charge trapping. 
     In addition to its electrical properties, the conditioned surface may also have properties that are beneficial in use with biological molecules. For example, a conditioned surface that contains fluorinated (or perfluorinated) carbon chains may provide a benefit relative to alkyl-terminated chains in reducing the amount of surface fouling. Surface fouling, as used herein, refers to the amount of indiscriminate material deposition on the surface of the microfluidic device, which may include permanent or semi-permanent deposition of biomaterials such as protein and its degradation products, nucleic acids and respective degradation products and the like. 
     Unitary or Multi-part conditioned surface. The covalently linked coating material may be formed by reaction of a molecule which already contains the moiety configured to provide a layer of organic and/or hydrophilic molecules suitable for maintenance/expansion of biological micro-object(s) in the microfluidic device, as is described below. Alternatively, the covalently linked coating material may be formed in a two-part sequence by coupling the moiety configured to provide a layer of organic and/or hydrophilic molecules suitable for maintenance/expansion of biological micro-object(s) to a surface modifying ligand that itself has been covalently linked to the surface. 
     Methods of preparing a covalently linked coating material. In some embodiments, a coating material that is covalently linked to the surface of a microfluidic device (e.g., including at least one surface of the sequestration pens and/or flow regions) has a structure of Formula 1 or Formula 2. When the coating material is introduced to the surface in one step, it has a structure of Formula 1, while when the coating material is introduced in a multiple step process, it has a structure of Formula 2. 
     
       
         
         
             
             
         
       
     
     The coating material may be linked covalently to oxides of the surface of a DEP-configured or EW-configured substrate. The DEP- or EW-configured substrate may comprise silicon, silicon oxide, alumina, or hafnium oxide. Oxides may be present as part of the native chemical structure of the substrate or may be introduced as discussed below. 
     The coating material may be attached to the oxides via a linking group (“LG”), which may be a siloxy or phosphonate ester group formed from the reaction of a siloxane or phosphonic acid group with the oxides. The moiety configured to provide a layer of organic and/or hydrophilic molecules suitable for maintenance/expansion of biological micro-object(s) in the microfluidic device can be any of the moieties described herein. The linking group LG may be directly or indirectly connected to the moiety configured to provide a layer of organic and/or hydrophilic molecules suitable for maintenance/expansion of biological micro-object(s) in the microfluidic device. When the linking group LG is directly connected to the moiety, optional linker (“L”) is not present and n is 0. When the linking group LG is indirectly connected to the moiety, linker L is present and n is 1. The linker L may have a linear portion where a backbone of the linear portion may include 1 to 200 non-hydrogen atoms selected from any combination of silicon, carbon, nitrogen, oxygen, sulfur and phosphorus atoms, subject to chemical bonding limitations as is known in the art. It may be interrupted with any combination of one or more moieties, which may be chosen from ether, amino, carbonyl, amido, or phosphonate groups, arylene, heteroarylene, or heterocyclic groups. In some embodiments, the backbone of the linker L may include 10 to 20 atoms. In other embodiments, the backbone of the linker L may include about 5 atoms to about 200 atoms; about 10 atoms to about 80 atoms; about 10 atoms to about 50 atoms; or about 10 atoms to about 40 atoms. In some embodiments, the backbone atoms are all carbon atoms. 
     In some embodiments, the moiety configured to provide a layer of organic and/or hydrophilic molecules suitable for maintenance/expansion of biological micro-object(s) may be added to the surface of the substrate in a multi-step process, and has a structure of Formula 2, as shown above. The moiety may be any of the moieties described above. 
     In some embodiments, the coupling group CG represents the resultant group from reaction of a reactive moiety R x  and a reactive pairing moiety R px  (i.e., a moiety configured to react with the reactive moiety R x ). For example, one typical coupling group CG may include a carboxamidyl group, which is the result of the reaction of an amino group with a derivative of a carboxylic acid, such as an activated ester, an acid chloride or the like. Other CG may include a triazolylene group, a carboxamidyl, thioamidyl, an oxime, a mercaptyl, a disulfide, an ether, or alkenyl group, or any other suitable group that may be formed upon reaction of a reactive moiety with its respective reactive pairing moiety. The coupling group CG may be located at the second end (i.e., the end proximal to the moiety configured to provide a layer of organic and/or hydrophilic molecules suitable for maintenance/expansion of biological micro-object(s) in the microfluidic device) of linker L, which may include any combination of elements as described above. In some other embodiments, the coupling group CG may interrupt the backbone of the linker L. When the coupling group CG is triazolylene, it may be the product resulting from a Click coupling reaction and may be further substituted (e.g., a dibenzocylcooctenyl fused triazolylene group). 
     In some embodiments, the coating material (or surface modifying ligand) is deposited on the inner surfaces of the microfluidic device using chemical vapor deposition. The vapor deposition process can be optionally improved, for example, by pre-cleaning the cover  110 , the microfluidic circuit material  116 , and/or the substrate (e.g., the inner surface  208  of the electrode activation substrate  206  of a DEP-configured substrate, or a dielectric layer of the support structure  104  of an EW-configured substrate), by exposure to a solvent bath, sonication or a combination thereof. Alternatively, or in addition, such pre-cleaning can include treating the cover  110 , the microfluidic circuit material  116 , and/or the substrate in an oxygen plasma cleaner, which can remove various impurities, while at the same time introducing an oxidized surface (e.g. oxides at the surface, which may be covalently modified as described herein). Alternatively, liquid-phase treatments, such as a mixture of hydrochloric acid and hydrogen peroxide or a mixture of sulfuric acid and hydrogen peroxide (e.g., piranha solution, which may have a ratio of sulfuric acid to hydrogen peroxide from about 3:1 to about 7:1) may be used in place of an oxygen plasma cleaner. 
     In some embodiments, vapor deposition is used to coat the inner surfaces of the microfluidic device  200  after the microfluidic device  200  has been assembled to form an enclosure  102  defining a microfluidic circuit  120 . Without intending to be limited by theory, depositing such a coating material on a fully-assembled microfluidic circuit  120  may be beneficial in preventing delamination caused by a weakened bond between the microfluidic circuit material  116  and the electrode activation substrate  206  dielectric layer and/or the cover  110 . In embodiments where a two-step process is employed the surface modifying ligand may be introduced via vapor deposition as described above, with subsequent introduction of the moiety configured provide a layer of organic and/or hydrophilic molecules suitable for maintenance/expansion of biological micro-object(s). The subsequent reaction may be performed by exposing the surface modified microfluidic device to a suitable coupling reagent in solution. 
       FIG. 2H  depicts a cross-sectional view of a microfluidic device  290  having an exemplary covalently linked coating material providing a conditioned surface. As illustrated, the coating materials  298  (shown schematically) can comprise a monolayer of densely-packed molecules covalently bound to both the inner surface  294  of a base  286 , which may be a DEP substrate, and the inner surface  292  of a cover  288  of the microfluidic device  290 . The coating material  298  can be disposed on substantially all inner surfaces  294 ,  292  proximal to, and facing inwards towards, the enclosure  284  of the microfluidic device  290 , including, in some embodiments and as discussed above, the surfaces of microfluidic circuit material (not shown) used to define circuit elements and/or structures within the microfluidic device  290 . In alternate embodiments, the coating material  298  can be disposed on only one or some of the inner surfaces of the microfluidic device  290 . 
     In the embodiment shown in  FIG. 2H , the coating material  298  can include a monolayer of organosiloxane molecules, each molecule covalently bonded to the inner surfaces  292 ,  294  of the microfluidic device  290  via a siloxy linker  296 . Any of the above-discussed coating materials  298  can be used (e.g. an alkyl-terminated, a fluoroalkyl terminated moiety, a PEG-terminated moiety, a dextran terminated moiety, or a terminal moiety containing positive or negative charges for the organosiloxy moieties), where the terminal moiety is disposed at its enclosure-facing terminus (i.e. the portion of the monolayer of the coating material  298  that is not bound to the inner surfaces  292 ,  294  and is proximal to the enclosure  284 ). 
     In other embodiments, the coating material  298  used to coat the inner surface(s)  292 ,  294  of the microfluidic device  290  can include anionic, cationic, or zwitterionic moieties, or any combination thereof. Without intending to be limited by theory, by presenting cationic moieties, anionic moieties, and/or zwitterionic moieties at the inner surfaces of the enclosure  284  of the microfluidic circuit  120 , the coating material  298  can form strong hydrogen bonds with water molecules such that the resulting water of hydration acts as a layer (or “shield”) that separates the biological micro-objects from interactions with non-biological molecules (e.g., the silicon and/or silicon oxide of the substrate). In addition, in embodiments in which the coating material  298  is used in conjunction with coating agents, the anions, cations, and/or zwitterions of the coating material  298  can form ionic bonds with the charged portions of non-covalent coating agents (e.g. proteins in solution) that are present in a medium  180  (e.g. a coating solution) in the enclosure  284 . 
     In still other embodiments, the coating material may comprise or be chemically modified to present a hydrophilic coating agent at its enclosure-facing terminus. In some embodiments, the coating material may include an alkylene ether containing polymer, such as PEG. In some embodiments, the coating material may include a polysaccharide, such as dextran. Like the charged moieties discussed above (e.g., anionic, cationic, and zwitterionic moieties), the hydrophilic coating agent can form strong hydrogen bonds with water molecules such that the resulting water of hydration acts as a layer (or “shield”) that separates the biological micro-objects from interactions with non-biological molecules (e.g., the silicon and/or silicon oxide of the substrate). 
     Further details of appropriate coating treatments and modifications may be found at U.S. application Ser. No. 15/135,707, filed on Apr. 22, 2016, and is incorporated by reference in its entirety. 
     Additional system components for maintenance of viability of cells within the sequestration pens of the microfluidic device. In order to promote growth and/or expansion of cell populations, environmental conditions conducive to maintaining functional cells may be provided by additional components of the system. For example, such additional components can provide nutrients, cell growth signaling species, pH modulation, gas exchange, temperature control, and removal of waste products from cells. 
     Light sequencing and patterns for transport of micro-objects. In some embodiments, the present disclosure is directed to the use of pattern information to project various sequences of light patterns (referred to herein in some embodiments as “light sequences”) to move, transport, and/or capture micro-objects. The term “move” as used herein with respect to light sequences refers to a light sequence that is in some embodiments sequentially projected at positions on any surface of the microfluidic device so that it appears to be moved along that surface of the microfluidic device. In some instances, certain light sequences (e.g. light sequences comprising a single light bar that is moved along the surface of the microfluidic device) provide insufficient force to move more than a single micro-object. However, due to the time necessary to move individual micro-objects, it may be necessary and more efficient to move a plurality of micro-objects in series or in parallel. In one aspect, the present disclosure is directed to light sequences that allow for the parallel manipulation of a plurality of micro-objects. In various embodiments, the present disclosure is directed to the use of a light sequence of plurality of moving light bars (referred to herein as a “conveyor light sequence”) to move one more micro-objects. 
     In various embodiments, the light bars (or a portion of the light bar) will have a substantially rectangular shape with a length that is greater than the width of the light bar. As discussed below, other portions of light bars may be curved, comprise indentations, irregularities, or have an otherwise non-planar or non-flat shape. In some embodiments, the light bars in the conveyor light sequence will be substantially parallel to each other. 
     Depending on the embodiments and the functionality required, the light bars in the conveyor light sequence may be of varying length and width to illuminate a varying number of DEP electrodes in an array of DEP electrodes, thus providing varying amounts of DEP force. For example, the light bars may illuminate a single row of DEP electrodes or several rows of DEP electrodes. Likewise, the light bars in the conveyor light sequence may vary in length to span different portions of the microfluidic device (e.g. circuit elements within the microfluidic device) depending on the functionality required. For example, in instances where the conveyor light sequence is used to move micro-objects from a sequestration pen to a channel in a microfluidic device, the light bars may have a length sufficient to span a portion (or all) of the length or width of the sequestration pen. Similarly, instances where the conveyor light sequence is used to move micro-objects from the channel in the microfluidic device to one or more sequestration pens, the light bars may span all or part of the channel. 
     Each conveyor light sequence has a starting position and an end position, as well as intermediate or temporary starting and ending positions. In some embodiments, lights bars in a conveyor light sequence are projected at a starting position and moved to an end position. In this way, the starting position and the end position partially define the trajectory of a micro-object moved using the conveyor light sequence. However, in some instances (e.g. when a light sequence is initially projected on a microfluidic device), some of the plurality of the light bars in a conveyor light sequence are initially projected at a position other than the starting position and moved to the end position. In some embodiments, when a light bar is moved to the end position, a new light bar can be projected at the starting position. The distance between the starting position and the end position (as well as shape, length and width of the light bars) defines the area of the conveyor light sequence and the corresponding portion of the microfluidic device the conveyor light sequence is projected on. 
     In various embodiments, the conveyor light sequence will comprise a plurality of light bars that are spaced at intervals and move in substantially the same direction towards the end position. The light bars can be spaced at even intervals (i.e. spaced substantially equidistant from each other) or spaced at irregular intervals (i.e. spaced at varying distances from each other). In most embodiments, the conveyor light sequence will comprise light bars that are spaced at intervals ranging from about 20 microns to about 200 microns. In some embodiments, the conveyor light sequence will comprise light bars that are spaced at intervals ranging from about 30 microns to about 100 microns. In some specific embodiments, the conveyor light sequence will comprise light bars that are spaced at intervals ranging from about 40 microns to about 50 microns. However, the intervals at which the light bars in the conveyor light sequence are spaced can vary based on the size of the DEP electrode and the size of the micro-objects that are moved. 
     Depending on the embodiment, the velocity at which the light bars in the conveyor light sequence are moved can vary according to the size and type of the micro-objects to be moved and speed required for the desired functionality (e.g. time limitations in moving micro-objects to an area of the microfluidic device). In most embodiments, the velocity at which the light bars in the conveyor light sequence move will range from about 1 microns/second to about 50 microns/second. In some embodiments, the velocity at which the light bars in the conveyor light sequence are moved will range from about 5 microns/seconds to about 30 microns/second. In some specific embodiments, the velocity at which the light bars in the conveyor light sequence are moved will range from about 10 microns/second to about 20 microns/second. 
       FIGS. 4A-4D  provide a schematic illustration of some embodiments of a conveyor light sequence  430  used to export micro-object(s) from a sequestration pen into a channel  422  adjacent to the sequestration pen at consecutive time points.  FIG. 4A  depicts the conveyor light sequence  430  at a first time point. As shown in  FIG. 4A , the conveyor light sequence  430  comprises nine light bars  410   a - i  projected on the surface of the microfluidic device to provide a DEP force. The light bars  410   a - i  are positioned substantially parallel to each other and move from their initial positions to an end position  454  in a channel  422  proximal to the sequestration pen. As the light bars are moved from their initial positions to the end position  454 , the light bars  410   a - i  provide DEP force sufficient to move the some of the micro-objects out of the sequestration pen. 
       FIG. 4B  depicts the same conveyor light sequence  430  at a second consecutive time point, where the light bars  410   b - i  have advanced from their initial positions towards the end position  454  such that each light bar  410   b - i  occupies a new position that is closer to the end position  454  than in  FIG. 4A . Light bar  410   a  is no longer projected on the microfluidic device after it reaches the end position  454  and a new light bar  410   j  is projected at the starting position  452 . As illustrated in  FIG. 4B , the micro-objects may be moved by different light bars  410   b - j  as the conveyor light sequences advances and new light bars are projected at the starting position  452 . However, as can be appreciated,  FIGS. 4A-4D  are stylized diagrams and a differing number of micro-objects may be moved by the conveyor light sequence  530  in actual implementation. 
       FIG. 4C  depicts the same conveyor light sequence  430  at a third consecutive time point, where the light bars  410   c - j  have again advanced from their positions illustrated in  FIG. 4B  towards the end position  454 . Light bar  410   b  is no longer projected on the microfluidic device after it reaches the end position  454  and a new light bar  410   k  is projected at the starting position  452 . As illustrated in  FIG. 4C , a number of the micro-objects are moved by the conveyor light sequence  430  to the end position  454  where they are located in the channel  422 . 
       FIG. 4D  depicts the same conveyor light sequence  430  at a third consecutive time point, where the light bars  410   d - k  have advanced from their positions illustrated in  FIG. 4C  towards the end position  454 . Light bar  410   c  is no longer projected on the microfluidic device after it reaches the end position  454  and a new light bar  410   l  is projected at the starting position  452 . As illustrated in  FIG. 4D , all of the micro-objects have been moved from the sequestration pen  424  to the channel  422 . Moving micro-objects from a sequestration pen  424  to a channel  422  or other area proximal to a sequestration pen  424  is referred to herein as “exporting” the micro-objects from the sequestration pen  424 . As understood by the skilled artisan, the process illustrated in  FIGS. 4A-4D  can be continued until all of the desired micro-objects are removed from the sequestration pen  424 . 
       FIGS. 5A and 5B  illustrate some embodiments of a conveyor light sequence  530  used to move micro-objects from a channel  522  to one or more sequestration pens  524 ,  526  at consecutive time points. The conveyor light sequence illustrated in  FIGS. 5A and 5B  has a starting position  552  in the channel  522  and an end position  554  in the sequestration pens  524 ,  526 .  FIG. 5A  illustrates the conveyor light sequence  530  at a first time point. As depicted in  FIG. 5A , the conveyor light sequence  530  comprises a five light bars  510   e - i  within the channel  522  and four light bars  510   a - d  within the sequestration pens  524 ,  526 . In the configurations shown in  FIG. 5A , the light bars  510   e - i  within the channel  522  are contiguous (i.e. are joined to substantially form a single structure) and span a plurality of sequestration pens  524 ,  526 . The light bars  510   e - i  within the channel further are curved or angled to create a “V-shape”  545  where the point or indentation of the V-shape corresponds to the vertical axis defined by the walls forming the sequestration pens. The indentations function to direct micro-objects away from the walls  560  (or similarly areas) between the sequestration pens  524 ,  526  and into the sequestration pens  524 ,  526 . As discussed below with respect to  FIGS. 14A-14C , in some embodiments, static light bars may be projected on the vertical axis defined by the walls forming sequestration pens (or other area between sequestration pen) in order to direct micro-objects away from the walls (or area between sequestration pens). 
     As illustrated in  FIGS. 5A and 5B , the light bars  510   a - d  within the sequestration pens  524 ,  526  are not contiguous (i.e., are disjointed). However, as discussed below, in alternate embodiments, the light bars  510   a - d  within the sequestration pens  524 , 526  may be contiguous to form a single line that is projected on several of the sequestration pens  524 ,  526  at the same time. 
       FIG. 5B  shows the conveyor light sequence  530  at a second consecutive time point in which the light bars have advanced from the starting position  552  to the end position  554 . At the time point illustrated in  FIG. 5B , a new light bar  510   j  is protected at the starting position  552 . As illustrated in  FIG. 5B , light bars may change morphology (i.e. height, width and shape) as they are moved (i.e. advanced) in the conveyor light sequence. Specifically, light bar  510   e  changes morphology from the curved, contiguous line shown at the time point illustrated in  FIG. 5A  to the disjointed straight lines in the sequestration pens  524 ,  526  shown at the time point illustrated in  FIG. 5B . As shown in  FIG. 5B , the conveyor light sequence  530  moves the micro-objects from the channel into the sequestration pens. As understood by the skilled artisan, the process illustrated in  FIGS. 5A-5B  can be continued until all of the desired micro-objects are moved the sequestration pen  424 . 
     In some embodiments, conveyor light sequences may be combined with other light sequences to move micro-objects from channels to sequestration pens.  FIGS. 6A and 6B  illustrate some embodiments where a conveyor light sequence  630  is combined with a single static (i.e., unmoving) light bar  670  (referred to herein as a “barrier light bar”) used to position micro-objects in a specific portion of the sequestration pen. The conveyor light sequence  630  illustrated in  FIGS. 6A and 6B  has a starting position  652  in a channel  622  and an end position  654  proximal to the distal openings of the sequestration pens  624 ,  626 . As the conveyor light sequence moves micro-objects from the channel  622  into the sequestration pens  624 ,  626 , the micro-objects are subjected to force from the barrier light bar  670  which functions to prevent most—if not substantially all—of the micro-objects from crossing the barrier light bar  670  to enter the proximal portion of the sequestration pens  624 ,  626 . 
     As shown in  FIGS. 6A and 6B , a barrier light bar  670  is projected at an upper portion of the sequestration pens  624 ,  626 . However, in other embodiments, the barrier light bar can be projected at any portion of the sequestration pen. The use of a barrier light bar may be beneficial in instances where it is desirable to retain micro-objects in a certain portion of the sequestration pen. In some embodiments, it may be desirable to retain micro-objects in a swept portion of a sequestration pen so that micro-objects may be provided the same media as the channel. Similarly, it may be desirable to concentrate micro-objects in a swept portion of a channel using a conveyor light sequence in combination with a barrier light bar. For example, it may be desirable to provide cells in an upper, distal portion of a sequestration pen media used to assay the cells. 
     In some embodiments, conveyor light sequences may be combined with other light sequences used to export micro-objects from sequestration pens into channels.  FIG. 7  illustrates some embodiments where a conveyor light sequence  730  is projected near the center of a sequestration pen  724  and has a starting position  752  within the sequestration pen and an end position  754  in the channel  722 . The conveyor light sequence  730  is projected in combination with light bars  780 ,  782  projected near the sides of a sequestration pen and used to direct micro-objects towards the center of the sequestration pen  724  for export into the channel. In the embodiments illustrated in  FIGS. 6A and 6B , the light bars  780 ,  782  are static (i.e., are not moving). However, as discussed below with respect to  FIG. 8 , the light bars  780 ,  782  projected near the sides of the sequestration pen  724  may be moved to direct micro-objects towards the conveyor light sequence. 
       FIG. 8  illustrates embodiments where light bars  880 ,  882 ,  884  projected near a side of the sequestration pen and near terminus  890 ,  892 ,  894  are used to move and direct micro-objects to a conveyor light sequence  830  used to export the micro-objects from sequestration pens  824 ,  826 ,  828  to a channel  822 . In the embodiments illustrated in  FIG. 8 , the light bars  880 ,  882 ,  824  projected near the side of the sequestration pens  824 ,  826 ,  828  move over a trajectory  870  having a starting position  872  near the side of the sequestration pens  824 ,  826 ,  828  to an end position  874  proximal to the conveyor light sequence  830 . 
     The light bars projected near the terminus  890 ,  892 ,  894  of the sequestration pens move over a trajectory  860  having a starting position  862  near or at the terminus to an end position  864  proximal to the trajectory of the light bars  880 ,  882 ,  824  projected near the sides of the sequestration pens  824 ,  826 ,  828 . As discussed above with respect to conveyor light sequences, the light bars  880 ,  882 ,  884  projected near the sides and the light bars  890 ,  892 ,  894  projected near the termini of the sequestration pens may follow the same motion from the starting positions to the end positon and be re-projected at the starting position once the light bars reach the end position. 
     Depending on the embodiments and the shape of the sequestration pen, any combination of light bars may be projected near the sides of the termini of sequestration pens in order to direct micro-objects to a conveyor light sequence used to export micro-objects from the sequestration pen. For example, in embodiments where the sequestration pen has several isolation regions and/or termini, several light bars may be used to move micro-objects from each isolation region and/or terminus. 
     In the embodiments illustrated in  FIG. 8 , the light bars  890 ,  892 ,  894 ,  880 ,  882 ,  824  are projected near the terminus and near the sides of the sequestration pen concurrently with the conveyor light sequence. However, in other embodiments, light bars  890 ,  892 ,  894 ,  880 ,  882 ,  824  are projected near the terminus and near the sides of the sequestration pen are projected and used to move micro-objects prior to the conveyor light sequence being used. 
     In some embodiments, multiple conveyor light sequences may be combined to provide various functionalities.  FIG. 9  illustrates some embodiments in which two conveyor light sequences  930 ,  932  are projected proximal to each other in the same sequestration pen  924 . The conveyor light sequences  930 ,  932  have starting positions  952 ,  956  and end positons  954 ,  958  that are staggered such that the light bars of the conveyor light sequence are also staggered. 
     In other embodiments, multiple conveyor light sequences may be combined in different configurations. In addition, the conveyor light sequences need not necessary follow a linear trajectory but instead may contain light bars that are not substantially parallel but instead are angled to move micro-objects along a non-linear trajectory. For example, as discussed below with respect to  FIGS. 18A-18E , a conveyor light sequence may include light bars that move micro-objects at right angles relative to other light bars. In other configurations, a conveyor light sequence may include light bars that move micro-objects at obtuse or acute angles relative to other light bars. 
     In various embodiments, conveyor light sequences may be combined with other light sequences used to separate micro-objects as they are moved to a desired area (e.g. a channel, chamber or sequestration pen). In specific embodiments, conveyor light sequences may be combined with light sequences as they are exported from a sequestration pen to a channel. 
       FIG. 10  illustrates embodiments where a conveyor light sequence  1030  has a starting position  1052  in a sequestration pen  1024  and an end position  1054  at the junction between the sequestration pen  1024  and a channel  1022 . The conveyor light sequence  1030  is combined with light bars  1040 ,  1042 ,  1044 ,  1046 ,  1048  and used to move and/or separate the micro-objects. Each of the light bars  1040 ,  1042 ,  1044 ,  1046 ,  1048  may have a trajectory that includes a starting position (not shown) and an end position (not shown). In some embodiments, the light bars  1040 ,  1042 ,  1044 ,  1046 ,  1048  may be positioned to move the micro-objects according to a pre-specified linear or non-linear trajectory. In other embodiments, the light bars  1040 ,  1042 ,  1044 ,  1046 ,  1048  may be positioned (or repositioned) at random to separate the micro-objects. 
     While  FIG. 10  illustrates the use of light bars to separate the micro-objects, any light sequence, including a light cage, may be used to separate and/or move micro-objects. In some embodiments, the light sequences may separate the micro-object without first determining the positions of the micro-objects. In other embodiments, the positions of the micro-objects may be determined first and then a light sequence used to move the micro-objects in a determined trajectory (i.e., from a fixed starting point to a fixed end point). For example, the micro-objects may be identified using image processing techniques and light cage(s) may be used to separate micro-objects and re-position the micro-objects into new sequestration pens according to a pre-determined trajectory. Methods of using imaging processing techniques to identify micro-objects, separate the micro-objects and re-position the micro-objects are discussed in detail in U.S. patent application Ser. No. 14/963,230, the entirety of which is incorporated herein by reference. In other embodiments, the positions of the micro-objects may be determined and light cage(s) used to move the micro-objects according to a random trajectory (i.e. from a fixed starting point to an unknown end point). As discussed below with respect to  FIGS. 19A-19E , after the position of a micro-object(s) has been identified, a light bar(s) may be used to move the micro-object(s) from an initial position determined by automatically identifying the micro-object(s) to an unknown end position. 
     In various embodiments, conveyor light sequences may be combined with other types of force aside from light sequences. For example, conveyor light sequences may be combined with a flow in a channel, gravitational, centrifugal, or any other force described herein. In some embodiments, a conveyor light sequence may be combined with an oscillatory (or alternating) flow in a channel used to separate the micro-object(s).  FIG. 11  illustrates the use of a conveyor light sequence  1130  combined with an oscillatory flow to separate micro-objects. The conveyor light sequence  1130  has a starting position  1152  within the sequestration pens  1124 ,  1126 ,  1126 ,  1127  and an end position  1154  within the channel  1122  proximal to the sequestration pens and is used to export micro-objects from the sequestration pens  1124 ,  1126 ,  1126 ,  1127 . Once the micro-objects are exported from the sequestration pens  1124 ,  1126 ,  1126 ,  1127  to the channel  1122 , the micro-objects are subject to an oscillatory flow in the channel  1122 , which can separate the micro-objects. 
     In some embodiments, light sequences will be selectively used to export micro-objects from sequestration pens. For example, only micro-objects having a specified characteristic (e.g., cells having a specific assay result) may be exported from sequestration pens using conveyor light sequences. 
     In some embodiments, a conveyor light sequence may only be used when there is a sufficient number of micro-objects in the sequestration pen to necessitate the use of a conveyor light sequence.  FIG. 12  illustrates processes performed to determine whether to use a conveyor light sequence to export micro-objects from a sequestration pen, according to some embodiments of the disclosure. Those skilled in the art will appreciate that other processes may be performed and the processes illustrated in  FIG. 12  may be performed in a different sequence or order. 
     At box  1202 , a density value that represents the actual or approximate number of micro-objects within a sequestration pen is determined. In some embodiments, the density value is equal to the number of micro-objects (e.g. cells or beads) within a sequestration pen or within a specific area of the sequestration pen. In these embodiments, the number of micro-objects within the sequestration pen can be determined by using image processing techniques to identify micro-objects within the sequestration pen. In other embodiments, the density value may be an approximation of the number of micro-objects within a sequestration pen or area thereof. In some embodiments, the density value may be equal to an area of the sequestration pen that is occupied by micro-objects. For example, the intensity of one or more pixels corresponding to an image of a sequestration pen may be used to determine whether the pixel corresponds to an empty portion of the sequestration pen or a portion of the sequestration pen occupied by a micro-object. In this way, the number of pixels corresponding to a portion of the sequestration pen occupied by micro-objects may be used as a density value that approximates the number of micro-objects within the sequestration pen. In various embodiments, other methods of determining the density value may be used alone, or in combination with this process to provide the best approximation of the number of micro-objects within a sequestration pen. 
     At box  1204 , the density value is compared to a pre-specified threshold value. Depending on the embodiments and the method used to determine the density value, the pre-specified threshold value may vary. For example, in embodiments where the density value is equal to the number of automatically-identified micro-objects in a sequestration pen, the pre-specified threshold value could range from 3-10 micro-objects (i.e., at least 3, 4, 5, 6, 7, 8, 9, 10 micro-objects). In other embodiments, such as embodiments with a significantly larger sequestration pen, the density value could range from 10 to 20 micro-objects. 
     At box  1206 , a conveyor light sequence is used to export the micro-objects from the sequestration pen responsive to determining that the density of micro-objects in the sequestration pen is greater than the pre-specified threshold value. 
     At box  1208 , if the density of micro-objects in the sequestration pen is less than the pre-specified threshold value, the position of the one or more micro-objects in the sequestration pen is identified. In those embodiments where automated micro-object detection is used to determine a density value for the sequestration pen, the positions of the one or more micro-objects may be identified during micro-object detection. 
     At box  1210 , a trajectory may be identified for each the micro-object to an area where the micro-object is to be moved. Depending on the embodiments, the type of sequestration pen used and the location of the micro-object, the trajectory can vary. As discussed above, the trajectory can be a random trajectory or a deterministic trajectory and can be linear or non-linear. 
     At box  1212 , the micro-object is moved along its identified trajectory to the area where the micro-object is to be moved. Depending on the embodiments, the micro-object may be moved using any type of light sequence such as the light bars and/or light cages described above. 
     In some instances, the density of micro-objects within a sequestration pen may be too great for a conveyor light sequence to be used. Accordingly, in some instances, it may be beneficial to use a “staged conveyor light sequence” where the starting position of the conveyor light sequence can vary according to the density of the micro-objects.  FIGS. 13A-13C  illustrate a staged conveyor light sequence at consecutive time points in a sequestration pen. In  FIG. 13A , a conveyor light sequence  1430  is illustrated at a first time point. The conveyor light sequence  1330  has a starting position  1352  within a sequestration pen  1324  that has a high density of micro-objects and an end position  1354  in a channel  1322  proximal to the sequestration pen  1324 . As illustrated in  FIG. 13A , the starting position  1352  of the conveyor light sequence  1330  is located at a position within the distal portion of the sequestration pen  1324  that does not have a high density of micro-objects relative to the other portions of the sequestration pen  1324 . By initially moving only the micro-objects in the portion of the sequestration pen  1324  above the starting position  1452 , the overall density of the micro-objects in the sequestration pen  1324  is decreased, making it easier to use a conveyor light sequence to move micro-objects in other portions of the sequestration pen. 
       FIG. 13B  illustrates the same conveyor light sequence  1330  at a second consecutive time point. At the second consecutive time point illustrated in  FIG. 14B , the starting position  1352  is located further towards the proximal end of the sequestration pen  1324  than at the time point illustrated in  FIG. 13A . Specifically, the starting position  1352  is located at a position in the middle portion of the sequestration pen  1324  that does not have a high density of micro-objects relative to the lowest portion of the sequestration pen. 
       FIG. 13C  illustrates the same conveyor light sequence  1330  at a third consecutive time point. At the third consecutive time point illustrated in  FIG. 13C , the starting position  1352  is located near the proximal end of the sequestration pen  1324  and thus can be used to move objects in all portions of the sequestration pen  1324 . As discussed above, by changing or “staging” the starting position  1352  of the sequestration pen, the density of micro-objects in the sequestration pen is decreased at the third consecutive time point such that conveyor light sequence may be used in the sequestration pen. 
     In some embodiments, a staged conveyor light sequence will have a number of pre-defined starting positions corresponding to different portions of a sequestration pen or another area of a microfluidic device. In some embodiments, different starting positions used in the staged conveyor light sequence may be determined based on the identified density of micro-objects at different portions of the sequestration pen. As discussed herein, the density in a portion of the sequestration pen may be determined using micro-object identification/counting or other methods. 
     Light patterns for effective isolation of selected micro-objects. In other aspects of the disclosure, the sequences of light patterns project a shape which can surround one or more micro-objects (including cells), thereby selecting that set of one or more cells specifically. As used herein, such a shape surrounding the one or more micro-objects may be referred to as a “light cage”. A light cage may—but does not have to—be a light pattern forming a continuous outlined shape around the one or more micro-objects. In some embodiments, a light cage may be a pattern of light having interruptions or irregularities along its outlined shape. This type of light cage can still function to surround the one or more micro-objects substantially in the same manner as a continuous light cage because the individually activated electrodes or phototransistors create a dielectrophoresis field at each point, and the sum of the collective forces activated can act to surround and capture the one or more selected micro-objects. The dielectrophoresis configuration as may be used herein can repel a micro-object within its field, even if there are visible gaps to the light pattern used to create the light cage to surround the one or more selected micro-objects. The overall effect of a light cage incorporating gaps between illuminated segments of the cage can still provide a force to repel the one or more micro-objects to a desired location within the light cage. The dielectrophoresis forces activated by the light pattern of the light cage can provide sufficient force to transport the one or more selected micro-objects to a selected location. 
     It can be useful to generate specifically formatted shapes for a light cage to assist with transport of the one or more selected cells, while preventing non-selected micro-objects from being “pushed ahead” of a light cage through its trajectory from one location to a second location. If a light cage has a flat leading aspect to its shape, the repelling forces of the dielectrophoresis field can propel the non-selected micro-object to the location where the selected one or more micro-objects are to be disposed. In some configurations, light cages having a shape including an angled leading edge can provide improved selectivity in moving, delivering and disposing only the one or more selected micro-objects, while repelling non-selected micro-objects away from the transiting selected group. 
     In some configurations, the light cage with a leading angled edge may have an overall dimension of about 75 microns by about 75 microns in an x-axial and y-axial plane, yielding a bounding box having sufficient interior area to hold sufficient selected numbers of micro-objects. The light cage with a leading angle edge may be generated within the enclosure of the microfluidic device using structured light as described herein, and each segment of the light cage shape may have a width of about 8 microns, about 10 microns, about 12 microns, about 14 microns, or any value therebetween. The width of the segment may be proportional to the number of electrodes/phototransistors activated and may therefore be proportional to the strength of the field surrounding the one or more micro-objects. 
     This application also describes methods of moving a plurality of micro-objects in a microfluidic device by projecting a plurality of light bars on a portion of the microfluidic device, wherein each light bar has a first position within the portion of the microfluidic device and the plurality of micro-objects are positioned within the portion of the microfluidic device, and moving each of the plurality of light bars of the plurality along a trajectory towards a second position for each light bar, wherein each of the light bars provides sufficient force to move one or more of the plurality of micro-objects. In some embodiments, the plurality of light bars comprises 2-10 light bars. In some embodiments, each of the plurality of light bars is spaced from an adjacent light bar at intervals ranging from about 20 microns to about 200 microns, from about 30 microns to about 100 microns, and even from about 40 microns to about 50 microns. 
     In some embodiments, each of the plurality of light bars are moved from the first position to the second position at a velocity ranging from about 1 micron/second to about 50 microns/second, from about 5 microns/second to about 30 microns/second, and even from about 10 microns/second to about 20 microns/second. In some embodiments, the plurality of light bars is moved at substantially the same speed. 
     In some embodiments, the trajectory comprises a linear trajectory. In some embodiments, some or all of the plurality of light bars are positioned substantially parallel to each other. In some embodiments, the trajectory comprises a non-linear trajectory. In some embodiments, one or more of the plurality of light bars is not positioned substantially parallel to another light bar in the plurality of light bars. 
     In some embodiments, the first position is associated with a starting position that partially defines the trajectory over which some of the light bars of the plurality are moved. In some embodiments, at least one of the plurality of light bars is moved from the starting position to the second position which is associated with an end position. 
     In some embodiments, a first light bar of the plurality of light bars is no longer projected on the portion of the microfluidic device after the first light bar is moved to the end position. In some embodiments, the methods comprise projecting a second light bar of the plurality of light bars at the starting position when the first light bar reaching the end position. 
     In some embodiments, the plurality of light bars is associated with a plurality of starting positions or a plurality of end positions. In some embodiments, the plurality of starting positions or the plurality of end positions are determined based on a density value associated with the starting positions. In some embodiments, the density value comprises the number of micro-objects present within a selected portion in an area of the microfluidic device. In some embodiments, the density value represents 3-10 micro-objects present in the area of the microfluidic device. 
     In some embodiments, a light bar of the plurality of light bars or a portion thereof is substantially rectangular. In some embodiments, a light bar of the plurality of light bars is curved or bent at one or more portions along the length of the light bar. 
     In some embodiments, the microfluidic device comprises a sequestration pen and a channel proximal to the sequestration pen and one of the plurality of light bars is projected on a first portion of sequestration pen and moved towards the channel. In some embodiments, the methods comprise projecting a first light sequence comprising at least two lights bars that separates micro-objects in the channel or in the sequestration pen. In some embodiments, the first light sequence moves the micro-objects from a known starting position to a known end position. In some embodiments, the known starting position is in the sequestration pen and the known end position is in the channel. In some embodiments, the methods comprise applying an oscillatory flow to the channel. 
     In some embodiments, the microfluidic device comprises multiple sequestration pens and the first light sequence is moved substantially in parallel along the sequestration pens. In some embodiments, the methods comprise projecting a second light sequence on a second portion of the sequestration pen proximal to the first portion of the sequestration pen, wherein the second light sequence comprises a light bar that moves towards the plurality of light bars projected on the first portion of the sequestration pen. In some embodiments, the microfluidic device comprises one or more sequestration pens and a channel proximal to the one or more sequestration pens and part of the plurality of light bars is projected on a portion of the channel and moved towards the one or more sequestration pens. In some embodiments, the microfluidic device comprises multiple sequestration pens and the plurality of light bars is moved substantially in parallel from the channel towards the sequestration pens. 
     In some embodiments, the plurality of micro objects comprises a colony of cells. In some embodiments, the plurality of light bars comprise one or more indentations, each indentation corresponding to an axis defined by a wall of a sequestration pen and functioning to direct micro-objects away from the wall of the sequestration pen. In some embodiments, the methods comprise projecting a static light bar on an axis defined by a wall of a sequestration pen, wherein the static light bar functions to direct micro-objects away from the wall of the sequestration pen. In some embodiments, the methods comprise projecting a static light bar on a second portion of the microfluidic device proximal to the first portion of the microfluidic device. In some embodiments, the static light bar maintains the micro-objects in the second portion of the microfluidic device. 
     This application also describes methods of transporting one or more micro-objects in a microfluidic device by identifying one or more micro-objects disposed within an enclosure of the microfluidic device, wherein the enclosure comprises a flow region and a substrate comprising a dielectrophoresis configuration; generating a light cage having a size configured to partially surround the one or more micro-objects and a shape comprising an angled leading edge, and transporting the one or more micro-objects from a first location to a second location within the enclosure of the microfluidic device. In some embodiments, the methods comprise orienting the angled leading edge of the light cage shape towards a direction of transport of the one or more micro-objects. In some embodiments, generating the light cage comprises activating dielectrophoresis forces within the enclosure of the microfluidic device. In some embodiments, transporting the one or more micro-objects from the first location to the second location comprises activating dielectrophoresis forces along a trajectory from the first location to the second location. 
     In some embodiments, the dielectrophoresis forces are sufficient to repel at least one micro-object. In some embodiments, the methods comprise repelling at least one non-selected micro-object away from the trajectory from the first location to the second location. In some embodiments, the methods comprise excluding the at least one non-selected micro-object from transport to the second location. 
     In some embodiments, as shown in  FIG. 20B , the shape of the light cage comprises a substantially polygonal shape with the leading angled edge being a vertex of the polygon. In some embodiments, the vertex of the polygon comprises a convex shape. In some embodiments, as shown in  FIG. 20A , the polygonal light cage comprises a substantially triangular shape with the leading angled edge being a vertex of the triangle. In some embodiments, the triangular light cage is an equilateral triangular light cage. 
     In some embodiments, the polygonal light cage comprises more than three sides. In some embodiments, the polygonal light cage comprises five to eight sides. 
     In some embodiments, the angled leading edge of the light cage comprises a non-linear angled leading edge. In some embodiments, the angled leading edge comprises at least one arc. In some embodiments, the angled leading edge of the light cage comprises a convex vertex. 
     In some embodiments, the shape of the light cage is an irregular polygon and the leading angled edge comprises a convex vertex of the irregular polygon. In some embodiments, the polygonal light cage comprises more than three sides. In some embodiments, the polygonal light cage comprises five to eight sides. 
     In some embodiments, the shape of the light cage comprises a substantial teardrop shape with the leading angled edge being the apical point of the teardrop. In some embodiments, the enclosure comprises at least one sequestration pen, wherein an end of the sequestration pen opens to the flow region. In some embodiments, the flow region comprises a microfluidic channel. 
     In some embodiments, the enclosure comprises a plurality of sequestration pens, wherein an end of each of the sequestration pens opens to the flow region. In some embodiments, the plurality of sequestration pens is disposed in a row adjacent to each other along the length of the flow region. 
     EXPERIMENTAL 
     System and device: An OptoSelect™ device, a nanofluidic device manufactured by Berkeley Lights, Inc. and controlled by an optical instrument which was also manufactured by Berkeley Lights, Inc. were employed. The instrument includes: a mounting stage for the chip coupled to a temperature controller; a pump and fluid medium conditioning component; and an optical train including a camera and a structured light source suitable for activating phototransistors within the chip. The OptoSelect device includes a substrate configured with OptoElectroPositioning (OEP™) technology, which provides a phototransistor-activated OET force. The chip also included a plurality of microfluidic channels, each having a plurality of NanoPen™ chambers (or sequestration pens) fluidically connected thereto. The volume of each sequestration pen is around 1×10 6  cubic microns. 
     Biological cells. OKT3 cells, a murine myeloma hybridoma cell line, were obtained from the ATCC (ATCC® Cat. #CRL-8001™). In culture, the cells behave as a suspension cell line. Cultures were maintained by seeding about 2×10 4  to about 5×10 5  viable cells/mL and incubating at 37° C., in 20 ml Iscove&#39;s Modified Dulbecco&#39;s Medium (IMDM) with 20% Fetal Bovine Serum (FBS) and 1% penicillin-streptomycin, using 5% carbon dioxide gaseous environment. Cells were split every 2-3 days. OKT3 cell number and viability were counted and cell density was adjusted to 5×10 5 /ml for loading the cells onto the OptoSelect device. 
     Device priming. 250 microliters of 100% carbon dioxide is flowed in to the OptoSelect device at a rate of 12 microliters/sec, followed by 250 microliters of PBS containing 0.1% Pluronic® F27 (Life Technologies® Cat #P6866) flowed in at 12 microliters/sec, and finally 250 microliters of PBS flowed in at 12 microliters/sec. Introduction of the culture medium follows. 
     Media perfusion. Medium is perfused through the OptoSelect device according to either of the following two methods:
         1. Perfuse at 0.01 microliters/sec for 2 h; perfuse at 2 microliters/sec for 64 sec; and repeat.   2. Perfuse at 0.02 microliters/sec for 100 sec; stop flow 500 sec; perfuse at 2 microliters/sec for 64 sec; and repeat.       

       FIGS. 14A-14C  depict the use of a conveyor light sequence, inducing dielectrophoretic forces, to move a plurality of micro-objects into NanoPen chambers in parallel.  FIG. 14A  depicts a channel of a OptoSelect device after OKT3 cells were introduced to the device. As shown in  FIG. 14A , some of the cells settled into the NanoPen chambers proximal to the channel but a number of cells remained within the channel.  FIG. 14B  depicts the conveyor light sequence used to move cells into the NanoPen chambers. The conveyor light sequence comprised a number of curved contiguous light bars with indentations or “V-shapes” that were projected on to the surface of the channel and the NanoPen chambers, as well as a single contiguous straight light bar that was projected onto the NanoPen chambers. The light bars in the conveyor light sequence were substantially parallel and were spaced at a 60 micron intervals (measured between the non-curved sections of the light bars) to have an overall distance between the starting position to the end position of 360 microns. The light bars in the conveyor light sequence were moved at a velocity of 3.23 microns/second. In addition, a number of static light bars were projected on the microfluidic material (here PPS walls) between the NanoPen chambers along with the conveyor light sequence. 
       FIG. 14C  depicts the cells after completion of dielectrophoretic transport from the channel into the NanoPen chambers. Although  FIGS. 14A-C  depict a single channel, in the example depicted in  FIGS. 14A-C , the conveyor light sequence illustrated in  FIG. 14B  was used to move 70,000 cells into NanoPen chambers within the same OptoSelect device. 
       FIGS. 15A and 15B  depict a conveyor light sequence inducing dielectrophoretic forces, which was used to export select cells from NanoPen chambers. As illustrated in  FIG. 15A , the conveyor light sequence comprised light bars that are straight disjoined lines. The light bars were selectively projected onto NanoPen chambers that contain micro-objects selected for export based on assay results. The conveyor light sequence had a starting position just below the bottom of the NanoPen chambers and an end position at the proximal opening of the NanoPen chambers. The light bars in the conveyor light sequence were spaced at 60 micron intervals and are moved at a velocity of 1.5 microns/second. 
       FIGS. 16A-16C  depict a conveyor light sequence used to move cells from an upper region of a channel to a lower region of the channel proximal to NanoPen chambers. As shown in  FIGS. 16A-16C , the conveyor light sequence comprised two contiguous curved light bars that were moved from a starting position at the top of a channel to an end position at a mid-way point between the top of the channel and the NanoPen chambers beneath the channel. The conveyor light sequence comprised two contiguous curved light bars with indentations or “V-shapes” corresponding to the vertical plane defined by the walls between the NanoPen chambers. The contiguous curved light bars were moved at a velocity of 10 microns/second and are spaced at 60 micron intervals.  FIG. 16A  depicts the top-most light bar of the conveyor light sequence at its starting position and  FIG. 16B  depicts the bottom-most light bar of the conveyor light sequence at its end position.  FIG. 16C  depicts the cells after completion of moving the majority of cells by the conveyor light sequence to the lower region of the channel. As shown in  FIGS. 16A-16C , the conveyor light sequence was combined with two static barrier light bars which prevented the cells from moving into the NanoPen chambers proximal to the channel. 
       FIGS. 17A-17D  depict the use of a conveyor light sequence in conjunction with light sequences used to separate cells as they were exported by the conveyor light sequence into a channel. As shown in  FIGS. 17A-17D , a conveyor light sequence having a starting position just below the bottom of a NanoPen chamber and an end position at the proximal opening on the NanoPen chamber to the channel was used to export cells. The light bars in the conveyor light sequence were moved at a velocity of 10 microns/second and were spaced at 60 micron intervals. The conveyor light sequence was combined with a light sequence comprising a curved light bar and multiple dots of light that was moved laterally within the channel over the NanoPen chamber (i.e. perpendicular to the light bars in the conveyor belt). 
       FIG. 17A  illustrates the conveyor light sequence at a first time point during cell export. At the time point depicted in  FIG. 17A , the light sequence comprising a curved line and dots was projected above the NanoPen chamber.  FIG. 17B  illustrates the conveyor light sequence at a second time point during cell export. At the time point depicted in  FIG. 17B , the light sequence comprising a curved line and dots was projected above and to the left of the NanoPen chamber relative to the light sequence in  FIG. 17A .  FIG. 17C  illustrates the conveyor light sequence at a third consecutive time point at which the light sequence comprising the curved line and dots was projected above and to the left of the NanoPen chamber.  FIG. 17D  illustrates the conveyor light sequence at a fourth consecutive time point at which the light sequence comprising the curved line and dots was again projected above the NanoPen chamber. As shown in  FIGS. 17A-17D , moving the light sequence comprising the dots and the curved light bar laterally within the channel helped to disperse and separate the cells as they were exported from their sequestration pens. 
       FIGS. 18A-F  depict a conveyor light sequence having a non-linear trajectory. The conveyor light sequence depicted in  FIGS. 18A-F  had a starting position just beneath the bottom of the NanoPen chamber. The light bars within the conveyor light sequence depicted in  FIGS. 18A-F  were moved at a velocity of 10 microns/second and were spaced at 60 micron intervals. As the light bars entered the channel, the light bars changed morphology from a length that spans the NanoPen chambers to a shorter length. At the top-left of the channel, each light bar was rotated ninety degrees to the right so that it is perpendicular to the light bars projected on the NanoPen chamber. The rotated light bar was moved from left to right. At the top-right of the channel, each light bar was again rotated ninety degrees to the right so that it is parallel with the light bars projected on the NanoPen chamber. However, instead of moving upwards from the NanoPen chamber to the channel, each light bar was moved from the top of the channel towards the NanoPen chamber. Each light bar was again rotated ninety degrees to the right so that is it perpendicular to the light bars projected on the sequestration pen. The light bar was then moved from the right to left. 
     As shown in  FIGS. 18A-F , by rotating the light bars within the conveyor light sequence, the cells were moved and repositioned as they were exported from the NanoPen chamber into a channel. However, as discussed above, in other embodiments, the lights bars within the conveyor light sequence may be rotated at various angles to move micro-objects over a trajectory that is non-linear. 
       FIGS. 19A-19E  depict the use of a conveyor light sequence with light sequences that were used to separate cells once they enter the channel.  FIG. 19A  depicts a conveyor light sequence with a starting position just beneath the bottom of a NanoPen chamber and an end position in a channel. As depicted in  FIG. 19A , the conveyor light sequence was used to move cells into the channel. In the sequence shown in  FIGS. 19A and 19B , the light bars in the conveyor light sequence moved at a velocity of 10 microns/second and were spaced at 60 micron intervals.  FIG. 19B  depicts cells within the channel that were moved by the conveyor light sequence of  FIG. 19A . 
       FIG. 19C  depicts the cells being moved from a known starting point along a random trajectory using light bars. In the image shown in  FIG. 19C , the positions of one set of cells within the channel were identified and the position information of the cells was used to generate a set of light bars that that were used to separate the cells into two sets. Specifically, the light bars were initially projected at a midpoint within the set of cells, and then the light bars were moved apart from each other to separate the cells into two sets. The midpoint position was determined based on the midpoint of the automatically-identified positions of the cells within the selected set. As discussed above, because the light bars merely pushed cells into two sets and do not position each cell at a fixed location, the trajectory that the cells follow has a fixed starting point but not a fixed end point and therefore is a random trajectory. As also discussed above, in alternate embodiments the cells can be moved according to a fixed trajectory. 
       FIG. 19D  depicts two sets of cells formed in  FIG. 19C  again being moved by two sets of light bars that were generated based on the identified mid-point positions of the two sets of cells. The two sets of light bars were used to separate the two sets of cells into four sets of cells by moving the cells along a random trajectory.  FIG. 19E  depicts the cells again being moved by a plurality of sets of light bars along a random trajectory. As the cells were separated into smaller sets, the mid-point could be identified by drawing a line between individual cells, as opposed to determining the midpoint of a set of cells. 
     In certain embodiments, the disclosure further provides machine-readable storage devices for storing non-transitory machine readable instructions for carrying out the foregoing methods. The machine-readable instructions can further control the imaging device used to obtain the images. 
     Although specific embodiments and applications of the disclosure have been described in this specification, these embodiments and applications are exemplary only, and many variations are possible.