System for operating a microfluidic device

Abstract

A system for operating a microfluidic device supports these inputs and outputs. A system includes a first surface configured to interface and operably couple with a microfluidic device and a lid configured to hold the microfluidic device on the first surface, wherein the lid includes a first portion having a first fluid port configured to operably couple with a first fluid inlet / outlet of the microfluidic device to allow a fluid medium to flow into and / or out of the first fluid inlet / outlet of the microfluidic device, and a second portion having a second fluid port configured to operably couple with a second fluid inlet / outlet of the microfluidic device to allow a fluid medium to flow into and / or out of the second fluid inlet / outlet of the microfluidic device.

Description

[Technical field]

[0001]

[0001] Field This application relates generally to systems for use with microfluidic devices. In particular, this application describes a system for operating a microfluidic device. [Background technology]

[0002]

[0002] Background As the field of microfluidics advances, microfluidic devices have become a convenient platform for the processing and manipulation of minute objects, such as biological cells. Microfluidic devices offer several desirable capabilities, including the ability to select and manipulate individual minute objects. Such microfluidic devices require a variety of inputs and outputs (e.g., fluids, pressure, vacuum, heat, cooling, light, etc.) to function. Systems for operating microfluidic devices support these inputs and outputs. Summary of the Invention [Means for solving the problem]

[0003] overview

[0003] This application describes a system for operating a microfluidic device. In an exemplary embodiment, a system for operating a microfluidic device is provided, the system including: a first surface configured to interface and operably couple with the microfluidic device; and a lid configured to hold the microfluidic device on the first surface, the lid including: a first lid portion having a first fluid port operably coupled with a first fluid inlet / outlet of the microfluidic device to allow fluid medium to flow into and / or out of the first fluid inlet / outlet of the microfluidic device; and a second lid portion having a second fluid port operably coupled with a second fluid inlet / outlet of the microfluidic device to allow fluid medium to flow into and / or out of the second fluid inlet / outlet of the microfluidic device, the second lid portion being separable from the first lid portion and movable between a closed position in which the second fluid port of the second lid portion is operably coupled with the second fluid inlet / outlet of the microfluidic device and an open position in which a portion of the microfluidic device including the second fluid inlet / outlet is exposed.

[0004]

[0004] In another exemplary embodiment, a system for operating a microfluidic device is provided, the system including: a support configured to hold and operably couple the microfluidic device to the microfluidic device; a first fluid line having a distal end configured to be fluidly coupled to an inlet port of the microfluidic device when the microfluidic device is held by and operably coupled to the support; a second fluid line having a proximal end configured to be fluidly coupled to an outlet port of the microfluidic device, respectively; at least one flow controller operably coupled to one or both of the first and second fluid lines, the at least one flow controller including a first thermally controlled flow controller operably coupled to flow segments of one or both of the first and second fluid lines to selectively flow fluid to the at least one flow controller; and a light modulation subsystem configured to emit structured light onto the microfluidic device when the microfluidic device is held by and operably coupled to the support.

[0005]

[0005] In yet another exemplary embodiment, a method for analyzing a fluid sample includes connecting a microfluidic device to a system for operating the microfluidic device, the system including a first surface configured to interface and operably couple with the microfluidic device, a first lid portion configured to hold the microfluidic device on the first surface, the first lid portion having a first fluid port configured to operably couple with a first fluid inlet / outlet of the microfluidic device to allow a fluid medium to flow into and / or out of the first fluid inlet / outlet of the microfluidic device, and a second fluid inlet / outlet configured to operably couple with a second fluid inlet / outlet of the microfluidic device to allow a fluid medium to flow into and / or out of the second fluid inlet / outlet of the microfluidic device. and a second lid portion having a second fluid port configured to allow the fluid to flow out of the port, the second lid portion being separable from the first lid portion and a lid movable between a closed position in which the second fluid port of the second portion of the cover is operably coupled to a second fluid inlet / outlet of the microfluidic device and an open position in which a portion of the microfluidic device including the second fluid inlet / outlet is exposed; moving the second lid portion from the closed position to the open position, thereby exposing the second fluid inlet / outlet of the microfluidic device; fluidly connecting the fluid sample to the second fluid inlet / outlet of the microfluidic device; applying suction to the first fluid line, thereby drawing at least a portion of the fluid sample into the microfluidic device; and processing at least a portion of the fluid sample drawn into the microfluidic device.

[0006]

[0006] A partial list of embodiments is as follows:

[0007]

[0007] Embodiment 1. A system for operating a microfluidic device, the system comprising: a first surface configured to interface and operably couple with the microfluidic device; a lid configured to hold the microfluidic device on the first surface, the lid comprising: a first lid portion having a first fluid port operably coupled with a first fluid inlet / outlet of the microfluidic device to allow a fluid medium to flow into and / or out of the first fluid inlet / outlet of the microfluidic device; and a second lid portion having a second fluid port operably coupled with a second fluid inlet / outlet of the microfluidic device to allow a fluid medium to flow into and / or out of the second fluid inlet / outlet of the microfluidic device, the second lid portion being separable from the first lid portion and movable between a closed position in which the second fluid port of the second lid portion is operably coupled with the second fluid inlet / outlet of the microfluidic device and an open position in which a portion of the microfluidic device including the second fluid inlet / outlet is exposed.

[0008]

[0008] Embodiment 2. The system of embodiment 1, wherein the first lid portion holds the microfluidic device on the first surface when the second lid portion is in the open position.

[0009]

[0009] Embodiment 3. The system of embodiment 1 or 2, wherein when the second lid portion is in the open position, the first fluid port of the first lid portion remains operatively coupled to the first fluid inlet / outlet of the microfluidic device.

[0010]

[0010] Embodiment 4. The system of any one of embodiments 1 to 3, wherein the first fluid port of the first lid portion is connected to a pump configured to remove fluid from the microfluidic device.

[0011]

[0011] Embodiment 5. The system of any one of embodiments 1 to 4, wherein the first lid portion further comprises a first fluid line connected to the first fluid port.

[0012]

[0012] Embodiment 6. The system of any one of embodiments 1 to 5, wherein the second lid portion further comprises a second fluid line connected to the second fluid port.

[0013]

[0013] Embodiment 7. The system of any one of embodiments 1 to 6, wherein the lid further comprises a hinge configured to move the second portion of the cover between the open position and the closed position.

[0014]

[0014] Embodiment 8. The system of any one of embodiments 1 to 7, wherein the lid further comprises a latch configured to releasably hold the second lid portion in a closed position.

[0015]

[0015] Embodiment 9. The system of any one of embodiments 1 to 8, further comprising an insert configured to operably couple with a second fluid inlet / outlet of the microfluidic device when the second lid portion is in the open position and to allow fluid medium to flow into the second fluid inlet / outlet of the microfluidic device.

[0016]

[0016] Embodiment 10. The system of embodiment 9, wherein the insert is configured to contact the first lid portion.

[0017]

[0017] Embodiment 11. The system of embodiment 9 or 10, wherein the insert comprises a fluid well configured to be in fluid communication with a second fluid inlet / outlet of the microfluidic device.

[0018]

[0018] Embodiment 12. The system of embodiment 11, wherein the fluid well is configured to hold a fluid sample of about 50 microliters or less, about 45 microliters or less, about 40 microliters or less, about 35 microliters or less, about 30 microliters or less, about 25 microliters or less, about 20 microliters or less, about 15 microliters or less, about 10 microliters or less, about 5 microliters or less, or any range formed by two of these endpoints.

[0019]

[0019] Embodiment 13. The system of embodiment 11, wherein the fluid well is configured to hold a fluid sample in a range of about 5 microliters to about 25 microliters, about 5 microliters to about 20 microliters, about 5 microliters to about 15 microliters, or about 5 microliters to about 10 microliters.

[0020]

[0020] Embodiment 14. The system of any one of embodiments 1 to 13, wherein the first surface is contained in a support (or "nest").

[0021]

[0021] Embodiment 15. The system of embodiment 14, wherein the support comprises a socket configured to receive and interface with the microfluidic device.

[0022]

[0022] Embodiment 16. The system of any one of embodiments 1 to 15, further comprising an electrical signal generation subsystem configured to apply a bias voltage across a pair of electrodes of the microfluidic device when the microfluidic device is operably coupled to the first surface or support.

[0023]

[0023] Embodiment 17. The system of embodiment 16, wherein the electrical signal generation subsystem includes a waveform generator configured to generate a bias voltage waveform that is applied across the electrode pair when the microfluidic device is operably coupled to the first surface or support.

[0024]

[0024] Embodiment 18. The system of embodiment 17, wherein the electrical signal generation subsystem further includes a waveform amplifier circuit configured to amplify the bias voltage waveform generated by the waveform generator.

[0025]

[0025] Embodiment 19. The system of embodiment 17 or 18, wherein the electrical signal generating subsystem further includes an oscilloscope configured to measure the bias voltage waveform, and optionally, data from the measurement is provided as feedback to the waveform generator.

[0026]

[0026] Embodiment 20. The system of any of embodiments 1 to 19, further comprising a thermal control subsystem configured to regulate a temperature of the microfluidic device when the microfluidic device is operably coupled to the first surface or support.

[0027]

[0027] Embodiment 21. The system of embodiment 20, wherein the thermal control subsystem includes a thermoelectric generation module, a Peltier thermoelectric device, and a cooling unit, the thermoelectric generation module configured to regulate a temperature of the Peltier thermoelectric device, and optionally, the Peltier thermoelectric device disposed between the first surface and a surface of the cooling unit.

[0028]

[0028] Embodiment 22. The system of embodiment 21, wherein the cooling unit includes a liquid cooling device, a cooling block, and a liquid pathway configured to circulate chilled liquid between the liquid cooling device and the cooling block, the cooling block including a surface of the cooling unit.

[0029]

[0029] Embodiment 23. The system of embodiment 21 or 22, wherein the Peltier thermoelectric device and the thermoelectric generation module are mounted on and / or integral with a support.

[0030]

[0030] Embodiment 24. The system of any of embodiments 14 to 23, wherein the support further comprises a microprocessor that controls one or both of the electrical signal generation subsystem and the thermoelectric generation module.

[0031]

[0031] Embodiment 25. The system of embodiment 24, wherein the support includes a printed circuit board (PCB), and at least one of the electrical signal generating subsystem, the thermoelectric generation module, and the microprocessor is mounted on and / or integrated with the PCB.

[0032]

[0032] Embodiment 26. The system of embodiment 24 or 25, further comprising an external computing device operatively coupled to the microprocessor, optionally the external computing device including a graphical user interface configured to receive operator input, process and transmit the operator input to the microprocessor, and control one or both of the electrical signal generation subsystem and the thermal control subsystem.

[0033]

[0033] Embodiment 27. The system of embodiment 26, wherein the microprocessor is configured to transmit the detected or received or calculated data and / or information based on the data or information detected or received from one or both of the electrical signal generating subsystem and the thermal control subsystem to an external computing device.

[0034]

[0034] Embodiment 28. The system of embodiment 16 or 27, wherein the microprocessor and / or external computing device is configured to measure and / or monitor the impedance of an electrical circuit across the electrodes of the microfluidic device when the microfluidic device is operably coupled to the support.

[0035]

[0035] Embodiment 29. The system of embodiment 28, wherein the microprocessor and / or external computing device is configured to determine a flow rate of the fluid pathway based on a detected change in the measured and / or monitored impedance of the electrical circuit, the fluid pathway including at least a portion of a microfluidic circuit within the microfluidic device.

[0036]

[0036] Embodiment 30. The system of embodiment 28, wherein the microprocessor and / or external computing device is configured to determine a height of an internal chamber of the microfluidic device based on a detected change in the measured and / or monitored impedance of the electrical circuit.

[0037]

[0037] Embodiment 31. The system of embodiment 28, wherein the microprocessor and / or external computing device is configured to determine one or more properties of the chemical and / or biological material contained within the microfluidic circuit of the microfluidic device based on the detected change in the measured and / or monitored impedance of the electrical circuit.

[0038]

[0038] Embodiment 32. The system of any one of embodiments 1 to 31, further comprising a light modulation subsystem configured to emit structured light onto the microfluidic device when the microfluidic device is operably coupled to the first surface (or support).

[0039]

[0039] Embodiment 33. The system of any one of embodiments 1 to 32, wherein the first surface, the support, and / or the light modulation subsystem are configured to be attached to an optical microscope.

[0040]

[0040] Embodiment 34. The system of any of embodiments 1 to 32, wherein the first surface, the support, and / or the light modulation subsystem are integral components of an optical microscope.

[0041]

[0041] Embodiment 35. The system of any one of embodiments 6 to 34, further comprising at least one flow controller (e.g., two or more, one of which may be a pump) operably coupled to one or both of the first fluid line and the second fluid line.

[0042]

[0042] Embodiment 36. The system of embodiment 35, wherein the at least one flow controller comprises a first thermally controlled flow controller operatively coupled to the first fluid line and / or the second fluid line to selectively flow fluid to the at least one flow controller.

[0043]

[0043] Embodiment 37. The system of embodiment 36, wherein the first thermally controlled flow controller includes a Peltier thermoelectric device configured to controllably reduce or increase a temperature of fluid contained in a flow segment of the first fluid line sufficient to freeze or thaw, respectively, the fluid contained in the flow segment of the first fluid line, thereby selectively preventing or allowing fluid to pass through the first fluid line and into or out of the first fluid inlet / outlet of the microfluidic device.

[0044]

[0044] Embodiment 38. The system of embodiment 37, wherein the first thermally controlled flow controller further comprises a first housing having a first passageway within which a flow segment of a first fluid line extends, the housing further comprising a Peltier thermoelectric device, and / or an insulating material at least partially surrounding the flow segment of the first fluid line, and optionally a first thermally conductive interface coupled to the flow segment of the first fluid line.

[0045]

[0045] Embodiment 39. The system of any one of embodiments 36 to 38, wherein the at least one flow controller includes a second thermally controlled flow controller operatively coupled to the other of the first fluid line and the second fluid line to selectively flow the fluid.

[0046]

[0046] Embodiment 40. The system of embodiment 39, wherein the second thermally controlled flow controller includes a Peltier thermoelectric device configured to controllably reduce or increase a temperature of the fluid contained in the flow segment of the second fluid line sufficient to freeze or thaw, respectively, the fluid contained in the flow segment of the second fluid line, thereby selectively preventing or allowing fluid to flow out of or into the second fluid inlet / outlet of the microfluidic device.

[0047]

[0047] Embodiment 41. The system of embodiment 40, wherein the second thermally controlled flow controller further comprises a second housing having a second passageway within which a flow segment of a second fluid line extends, the housing further comprising a Peltier thermoelectric device, and / or an insulating material at least partially surrounding the flow segment of the second fluid line, and optionally a first thermally conductive interface coupled to the flow segment of the first fluid line.

[0048]

[0048] Embodiment 42. The system of embodiment 35, wherein the at least one flow controller comprises a thermally controlled flow controller operatively coupled to the first fluid line and the second fluid line, the thermally controlled flow controller comprising at least one flow-controlled Peltier thermoelectric device configured to controllably reduce or increase a temperature of a flow segment of the first fluid line and the second fluid line sufficient to freeze or thaw fluid contained in the flow segments of the first fluid line and the second fluid line, respectively, thereby selectively preventing or enabling fluid to pass through the first fluid line and into the first fluid inlet / outlet of the microfluidic device and pass out of the second fluid inlet / outlet of the microfluidic device and through the second fluid line, or vice versa.

[0049]

[0049] Embodiment 43. The system of embodiment 42, wherein the at least one flow control Peltier thermoelectric device includes at least a first flow control Peltier thermoelectric device thermally coupled to a flow segment of the first fluid line and a second flow control Peltier thermoelectric device thermally coupled to a flow segment of the second fluid line.

[0050]

[0050] Embodiment 44. The system of embodiment 42 or 43, wherein the thermally controlled flow controller further includes a housing having a first passage through which a flow segment of the first fluid line extends and a second passage through which a flow segment of the outlet fluid line extends, and wherein the at least one flow-controlling Peltier thermoelectric device is mounted within the housing.

[0051]

[0051] Embodiment 45. The system of embodiment 44, wherein the housing defines an insulating chamber.

[0052]

[0052] Embodiment 46. The system of any of embodiments 32 to 45, wherein the light modulation subsystem includes a digital mirror device (DMD) or a microshutter array system (MSA).

[0053]

[0053] Embodiment 47. The system of any of embodiments 32-45, wherein the light modulation subsystem comprises a liquid crystal display (LCD), a liquid crystal on silicon device (LCOS), a ferroelectric liquid crystal on silicon device (FLCOS), or a scanning laser device.

[0054]

[0054] Embodiment 48. The system of any of embodiments 32 to 47, wherein the light modulation subsystem includes a multi-input light pipe, the light pipe including:

[0055]

[0055] The housing has a plurality of input apertures, each configured to receive light emitted from a respective light source, and the housing further comprises: an output aperture configured to emit light received through the input apertures; a first light propagation path extending within the housing from the first input aperture to the output aperture; a first dichroic filter disposed within the housing at an oblique angle across the first light propagation path, the first dichroic filter being constructed and arranged such that light received through the first light aperture passes through the first dichroic filter as it propagates along the first light propagation path to the output aperture; and a second input aperture extending within the housing from the first dichroic filter. a second light propagation path extending to the dichroic filter, the second propagation path and the first dichroic filter configured and dimensioned such that light received through the second input aperture propagates along the second light propagation path and is reflected by the first dichroic filter onto the first light propagation path towards the output aperture, and each input aperture, first and second light propagation paths, first dichroic filter, and output aperture are sized, dimensioned and configured such that light emitted by at least one light source and received through at least one of the first and second input apertures is emitted from the output aperture with a substantially uniform intensity.

[0056]

[0056] Embodiment 49. The system of embodiment 48, wherein the light pipe includes a second dichroic filter disposed within the housing at an oblique angle across the first light propagation path between the first dichroic filter and the output aperture, the second dichroic filter configured and dimensioned such that light received through the first and second light apertures passes through the second dichroic filter as the received light propagates along the first light propagation path to the output aperture, and a third light propagation path extending within the housing from the third input aperture to the second dichroic filter, the third propagation path and the second dichroic filter configured and dimensioned such that light received through the third input aperture propagates along the third light propagation path and is reflected by the second dichroic filter onto the first light propagation path toward the output aperture.

[0057]

[0057] Embodiment 50. The system of embodiment 48, wherein the light modulation subsystem further includes a first light source having an output optically coupled to the first input aperture of the light pipe.

[0058]

[0058] Embodiment 51. The system of embodiment 50, wherein the first light source includes a plurality of first light source emitting elements.

[0059]

[0059] Embodiment 52. The system of embodiment 51, wherein one or more of the plurality of first light source emitting elements emit light at a first narrowband wavelength.

[0060]

[0060] Embodiment 53. The system of any one of embodiments 50 to 52, wherein the light modulation subsystem further includes a second light source having an output optically coupled to a second input aperture of the light pipe.

[0061]

[0061] Embodiment 54. The system of embodiment 53, wherein the second light source includes a plurality of second light source emitting elements.

[0062]

[0062] Embodiment 55. The system of embodiment 54, wherein one or more of the plurality of second light source emitting elements emit light at the first narrowband wavelength or at a second narrowband wavelength different from the first narrowband wavelength.

[0063]

[0063] Embodiment 56. The system of embodiment 54, wherein the plurality of first light-emitting elements and the plurality of second light-emitting elements collectively comprise a first subset of one or more light-emitting elements that emit light at a first narrowband wavelength and a second subset of one or more light-emitting elements that emit light at a second narrowband wavelength different from the first narrowband wavelength, such that light comprising one or both of the first narrowband wavelength and the second narrowband wavelength can be controllably emitted from the light pipe output aperture by selectively activating one or both of the plurality of first light-emitting elements and the plurality of second light-emitting elements.

[0064]

[0064] Embodiment 57. The system of embodiment 56, wherein the light emitted by the first subset of light emitting elements and received through the first and / or second input aperture is emitted from the output aperture of the light pipe with a first substantially uniform intensity, and the light emitted by the second subset of light emitting elements and received through the first and / or second input aperture is emitted from the output aperture with a second substantially uniform intensity.

[0065]

[0065] Embodiment 58. The system of embodiment 57, wherein the first substantially uniform intensity is different from the second substantially uniform intensity.

[0066]

[0066] Embodiment 59. The system of any of embodiments 56 to 58, wherein the first narrowband wavelength and the second narrowband wavelength are each selected from the group consisting of about 380 nm, about 480 nm, and about 560 nm.

[0067]

[0067] Embodiment 60. The system of any of embodiments 43 to 46, wherein the plurality of light-emitting elements of the first light source includes or consists of all of the first subset of light-emitting elements, and the plurality of light-emitting elements of the second light source includes or consists of all of the second subset of light-emitting elements.

[0068]

[0068] Embodiment 61. The system of any of embodiments 40 to 47, wherein the light modulation subsystem further includes a third light source having an output optically coupled to a third input aperture of the light pipe.

[0069]

[0069] Embodiment 62. The system of embodiment 61, wherein the third light source includes a plurality of third light source emitting elements.

[0070]

[0070] Embodiment 63. The system of embodiment 62, wherein one or more of the plurality of third light source emitting elements emit light at the first narrowband wavelength, at the second narrowband wavelength, or at a third narrowband wavelength different from each of the first narrowband wavelength and the second narrowband wavelength.

[0071]

[0071] Embodiment 64. The system of embodiment 62, wherein the plurality of first light-source emitting elements, the plurality of second light-source emitting elements, and the plurality of third light-source emitting elements collectively include a first subset of one or more light-emitting elements that emit light at a first narrowband wavelength, a second subset of one or more light-emitting elements that emit light at a second narrowband wavelength different from the first narrowband wavelength, and a third subset of one or more light-emitting elements that emit light at a third narrowband wavelength different from each of the first and second narrowband wavelengths, such that light comprising one or more of the first narrowband wavelength, the second narrowband wavelength, and the third narrowband wavelength can be controllably emitted from the light pipe output aperture by selectively activating one or more of the first, second, and third subsets of light-emitting elements.

[0072]

[0072] Embodiment 65. The system of embodiment 64, wherein the light emitted by the first subset of light-emitting elements and received through any of the first, second and third input apertures is emitted from the output aperture with a first substantially uniform intensity, the light emitted by the second subset of light-emitting elements and received through any of the first, second and third input apertures is emitted from the output aperture with a second substantially uniform intensity, and the light emitted by the third subset of light-emitting elements and received through any of the first, second and third input apertures is emitted from the output aperture with a third substantially uniform intensity.

[0073]

[0073] Embodiment 66. The system of embodiment 65, wherein the first substantially uniform intensity is different from one or both of the second substantially uniform intensity and the third substantially uniform intensity.

[0074]

[0074] Embodiment 67. The system of any of embodiments 64 to 66, wherein the first narrowband wavelength is about 380 nm, the second narrowband wavelength is about 480 nm, and the third narrowband wavelength is about 560 nm.

[0075]

[0075] Embodiment 68. The system according to any of embodiments 64 to 67, wherein the plurality of light-emitting elements of the first light source includes or consists of all of the first subset of light-emitting elements, the plurality of light-emitting elements of the second light source includes or consists of all of the second subset of light-emitting elements, and the plurality of light-emitting elements of the third light source includes or consists of all of the third subset of light-emitting elements.

[0076]

[0076] Embodiment 69. A microscope configured to operate a microfluidic device, comprising: a support (e.g., the support of any one of embodiments 14 to 31 or 35 to 45) configured to hold and operably couple to the microfluidic device; a light modulation subsystem configured to emit structured light; and an optical system, wherein when the microfluidic device is held by and operably coupled to the support, the optical system is configured to: (1) focus the structured light emitted by the light modulation subsystem onto at least a first region of the microfluidic device; (2) focus the unstructured light emitted by the unstructured light source onto at least a second region of the microfluidic device; and (3) capture reflected and / or emitted light from the microfluidic device and direct the captured light to a detector.

[0077]

[0077] Embodiment 70. The microscope of embodiment 69, further comprising a detector.

[0078]

[0078] Embodiment 71. The microscope of embodiment 69 or 70, wherein the detector comprises an eyepiece and / or an imaging device.

[0079]

[0079] Embodiment 72. The microscope of any one of embodiments 69 to 71, wherein the light modulation subsystem includes a digital mirror device (DMD) or a microshutter array system (MSA).

[0080]

[0080] Embodiment 73. The microscope of any of embodiments 69-71, wherein the light modulation subsystem includes a liquid crystal display (LCD), a liquid crystal on silicon device (LCOS), a ferroelectric liquid crystal on silicon device (FLCOS), or a scanning laser device.

[0081]

[0081] Embodiment 74. The microscope of any of embodiments 69 to 73, further comprising a controller for controlling the light modulation subsystem.

[0082]

[0082] Embodiment 75. The microscope of any of embodiments 69 to 74, wherein the optical system includes an objective lens configured to focus the structured light onto the first region of the microfluidic device and / or the unstructured light onto the second region of the microfluidic device, the objective lens being selected from the group including a 10x objective lens, a 5x objective lens, a 4x objective lens, and a 2x objective lens.

[0083]

[0083] Embodiment 76. The microscope of any of embodiments 69 to 75, wherein the optical system includes a dichroic filter configured to substantially prevent structured light emitted by the light modulation subsystem (and reflected by the microfluidic device) from reaching a detector.

[0084]

[0084] Embodiment 77. The microscope of any of embodiments 69 to 75, wherein the optical system includes a dichroic filter configured to balance an amount of visible structured light emitted by a light modulation subsystem (and reflected by the microfluidic device) and an amount of visible unstructured light emitted by an unstructured light source (and reflected by the microfluidic device) that reaches a detector.

[0085]

[0085] Embodiment 78. The microscope of any of embodiments 69 to 75, wherein the light modulation subsystem emits structured white light.

[0086]

[0086] Embodiment 79. The microscope of any of embodiments 69 to 75, wherein the light modulation subsystem includes a mercury or xenon arc lamp.

[0087]

[0087] Embodiment 80. The microscope of any of embodiments 69 to 75, wherein the light modulation subsystem includes one or more LEDs.

[0088]

[0088] Embodiment 81. The microscope of any of embodiments 69 to 75, wherein the unstructured light source comprises one or more LEDs.

[0089]

[0089] Embodiment 82. The microscope of embodiment 81, wherein the unstructured light source emits light having a wavelength of about 495 nm or less.

[0090]

[0090] Embodiment 83. The microscope of embodiment 81, wherein the unstructured light source emits blue light.

[0091]

[0091] Embodiment 84. The microscope of embodiment 82 or 83, wherein the optical system includes a dichroic filter configured to at least partially filter visible light having wavelengths longer than 495 nm.

[0092]

[0092] Embodiment 85. The microscope of embodiment 81, wherein the unstructured light source emits light having a wavelength of about 650 nm or greater.

[0093]

[0093] Embodiment 86. The microscope of embodiment 81, wherein the unstructured light source emits red light.

[0094]

[0094] Embodiment 87. The microscope of embodiment 85 or 86, wherein the optical system includes a dichroic filter configured to at least partially filter visible light having a wavelength shorter than 650 nm.

[0095]

[0095] Embodiment 88. The microscope of any of embodiments 69 to 87, wherein the support includes an integrated electrical signal generating subsystem configured to apply a bias voltage across a pair of electrodes in the microfluidic device when the apparatus is carried by and operably coupled to the support.

[0096]

[0096] Embodiment 89. The microscope of any of embodiments 69 to 88, wherein the support includes a thermal control subsystem configured to regulate a temperature of the microfluidic device when the apparatus is held by and operably coupled to the support.

[0097]

[0097] Embodiment 90. A method of analyzing a fluid sample, comprising connecting a microfluidic device to a system for operating the microfluidic device, the system comprising a first surface configured to interface and operably couple with the microfluidic device, a lid configured to hold the microfluidic device on the first surface, a first lid portion having a first fluid port configured to operably couple with a first fluid inlet / outlet of the microfluidic device to allow a fluid medium to flow into and / or out of the first fluid inlet / outlet of the microfluidic device, and a second fluid inlet / outlet configured to operably couple with a second fluid inlet / outlet of the microfluidic device to allow a fluid medium to flow into and / or out of the second fluid inlet / outlet of the microfluidic device. and a second lid portion having a second fluid port configured to allow a fluid sample to exit the microfluidic device, the second lid portion being separable from the first lid portion and movable between a closed position in which the second fluid port of the second portion of the cover is operably coupled to a second fluid inlet / outlet of the microfluidic device and an open position in which a portion of the microfluidic device including the second fluid inlet / outlet is exposed; moving the second lid portion from the closed position to the open position, thereby exposing the second fluid inlet / outlet of the microfluidic device; fluidly connecting the fluid sample to the second fluid inlet / outlet of the microfluidic device; applying suction to the first fluid line, thereby drawing at least a portion of the fluid sample into the microfluidic device; and processing at least a portion of the fluid sample drawn into the microfluidic device.

[0098] Embodiment 91. The method of embodiment 90, further comprising placing an insert in a position previously occupied by the second lid portion in the closed position, the insert including a fluid well configured to be in fluid communication with the second fluid inlet / outlet of the microfluidic device, and providing the fluid sample comprises introducing the fluid sample into the fluid well of the insert.

[0099]

[0099] Embodiment 92. The system is any one of embodiments 1 to 68. The method of embodiment 90 or 91.

[0100]

[0100] Embodiment 93. The method of embodiment 90 or 91, wherein the system is a microscope of any one of embodiments 69 to 89.

[0101]

[0101] Embodiment 94. The method of any one of embodiments 90 to 93, wherein suction is applied sufficient to draw a preselected volume (e.g., about 2 microliters to about 10 microliters or about 3 microliters to about 7 microliters) of the fluid sample into the microfluidic chip, at which point suction is stopped.

[0102]

[0102] Embodiment 95. The method of any one of embodiments 90 to 94, wherein the fluid sample comprises micro-objects, optionally biological micro-objects (eg, cells).

[0103]

[0103] Embodiment 96. The method of any one of embodiments 90 to 95, wherein the microfluidic device includes: (i) a flow region having a plurality of microfluidic channels; and (ii) a plurality of chambers, such as a sequestration pen (e.g., as described in PCT Publications WO 2014 / 070873 and WO 2015 / 061497, the entire contents of each of which are incorporated herein by reference), wherein each chamber of the plurality of chambers is fluidly connected to one of the plurality of microfluidic channels.

[0104]

[0104] Embodiment 97. The method of any one of embodiments 90 to 96, wherein processing at least a portion of the fluid sample includes imaging the sample contained within a microfluidic chip.

[0105]

[0105] Embodiment 98. The method of embodiment 97, wherein imaging includes imaging a minute object contained within at least a portion of the fluid sample.

[0106]

[0106] Embodiment 99. The method of embodiment 96, wherein processing at least a portion of the fluid sample includes performing an assay of microscopic objects contained within at least a portion of the fluid sample.

[0107]

[0107] Embodiment 100. The method of embodiment 99, wherein the assay provides for detection of cell secretions and / or nucleic acids released by the cells (e.g., any of the assays described in PCT publications WO 2014 / 070783, WO 2015 / 061497, WO 2015 / 061506, WO 2015 / 095623, WO 2017 / 181135, WO 2018 / 064640, WO 2018 / 076024, WO 2019 / 075476, and WO 2019 / 133874, or PCT application numbers, International Application No. PCT / US2019 / 041692 and International Application No. PCT / US2019 / 024623, the entire contents of which are incorporated herein by reference).

[0108]

[0108] Embodiment 101. A system for operating a microfluidic device, the system including: a support configured to hold and operably couple to a microfluidic device; a first fluid line having a distal end configured to be fluidly coupled to an inlet port of the microfluidic device when the microfluidic device is held by and operably coupled to the support; a second fluid line having a proximal end configured to be fluidly coupled to an outlet port of the microfluidic device; at least one flow controller (e.g., two or more, one of which can be a pump) operably coupled to one or both of the first and second fluid lines, the at least one flow controller including a first thermally controlled flow controller operably coupled to a flow segment of one or both of the first and second fluid lines to selectively flow fluid to the at least one flow controller; and a light modulation subsystem configured to emit structured light onto the microfluidic device when the microfluidic device is held by and operably coupled to the support.

[0109]

[0109] Embodiment 102. The system of embodiment 101, further comprising an electrical signal generating subsystem configured to apply a bias voltage across the pair of electrodes of the microfluidic device when the microfluidic device is held by and operably coupled to the support.

[0110]

[0110] Embodiment 103. The system of embodiment 101 or 102, comprising any of the elements of the system of any one of embodiments 1 to 68 and 116 to 122 (e.g., alone or in combination) or the microscope of any one of embodiments 69 to 89.

[0111]

[0111] Embodiment 104. The system of embodiments 37 or 101-103, wherein the first thermally controlled flow controller further comprises a thermally conductive interface coupled to the flow segments of the first fluid line and the second fluid line, and a Peltier thermoelectric device in contact with the thermally conductive interface and configured to controllably reduce or increase a temperature of fluid contained in the flow segments of the first fluid line and / or the second fluid line.

[0112]

[0112] Embodiment 105. The system of embodiment 104, wherein the temperature is reduced or increased sufficiently to freeze or thaw, respectively, fluid contained in a flow segment of the first fluid line and / or the second fluid line, thereby selectively preventing or allowing fluid to flow out of or into the first fluid inlet / outlet and / or the second fluid inlet / outlet of the microfluidic device.

[0113]

[0113] Embodiment 106. The system of embodiment 104 or 105, wherein the thermally conductive interface comprises a thermistor.

[0114]

[0114] Embodiment 107. The system of embodiment 106, wherein the thermistor is disposed in a region located between the flow segment of the first fluid line and the flow segment of the second fluid line.

[0115]

[0115] Embodiment 108. The system of any one of embodiments 104 to 107, wherein the thermally conductive interface is located between at least two Peltier thermoelectric devices.

[0116]

[0116] Embodiment 109. The system of embodiment 108, wherein the first thermally controlled flow controller further comprises a conduit for removing heat from one of the at least two Peltier thermoelectric devices.

[0117]

[0117] Embodiment 110. The system of any one of embodiments 104 to 109, wherein the first thermally controlled flow controller further comprises a heat sink.

[0118]

[0118] Embodiment 111. The system of any one of embodiments 104 to 110, wherein the thermally conductive interface is configured to directly contact (e.g., rest on) a top surface of the Peltier thermoelectric device.

[0119]

[0119] Embodiment 112. The system of any one of embodiments 104 to 111, wherein the first thermally controlled flow controller includes a cover including guides for inserting the flow segments of the first fluid line and the second fluid line into the thermally conductive interface.

[0120]

[0120] Embodiment 113. The system of any one of embodiments 104 to 112, further comprising a barrier material located within the thermally controlled flow controller, the barrier material (e.g., an insulating polymer or spray foam) being sufficient to prevent ice formation.

[0121]

[0121] Embodiment 114. The system of embodiment 113, wherein the barrier material substantially fills an empty space that would otherwise be present within the cover of the first thermally controlled flow controller.

[0122]

[0122] Embodiment 115. The system of any one of embodiments 104 to 114, wherein the first thermally controlled flow controller is configured to control fluid flowing into and out of a microfluidic device (e.g., a single microfluidic device).

[0123]

[0123] Embodiment 116. The system of any one of embodiments 1 to 68, wherein the support includes a sensor configured to determine when the second lid portion is in the closed position.

[0124]

[0124] Embodiment 117. The system of embodiment 116, wherein the sensor is further configured to determine when the insert interfaces with the microfluidic device.

[0125]

[0125] Embodiment 118. The system of embodiment 116 or 117, wherein the sensor includes a first optical switch configured to be blocked to indicate when the second lid portion is in the closed position.

[0126]

[0126] Embodiment 119. The system of any one of embodiments 116 to 118, wherein the sensor includes a second optical switch configured to be blocked to indicate when the insert interfaces with the microfluidic device.

[0127]

[0127] Embodiment 120. The system of any one of embodiments 116 to 119, wherein the sensor includes a first extender, the first extender configured to extend to the first optical switch by a first actuator included in the second lid portion to interrupt the first optical switch.

[0128]

[0128] Embodiment 121. The system of any one of embodiments 116 to 120, wherein the sensor includes a second extender, the second extender configured to extend to the second optical switch by a second actuator included in the insert to interrupt the second optical switch.

[0129]

[0129] Embodiment 122. The system of any one of embodiments 116 to 121, wherein the sensor detects when the second lid portion is in an open position, and the insert does not interface with the microfluidic device when the optical paths of the first optical switch and the second optical switch are not blocked.

[0130]

[0130] Embodiment 123. The method of embodiment 90 or 91, wherein the microfluidic device comprises: (i) a flow region having a plurality of microfluidic channels; and (ii) a plurality of chambers, each chamber of the plurality of chambers being fluidly connected to one of the plurality of microfluidic channels.

[0131]

[0131] Embodiment 124. The method of embodiment 123, wherein an import cell density of at least 4x10^6 is provided.

[0132]

[0132] Other aspects and advantages of the disclosed systems, microscopes, and methods will become apparent in the following detailed description and the appended claims.

[0133] BRIEF DESCRIPTION OF THE DRAWINGS The drawings illustrate the design and utility of embodiments of the disclosed system, with similar elements being referred to by common reference numerals. The drawings are not necessarily drawn to scale. To better understand how the above and other advantages and objects are obtained, a more detailed description of the embodiments illustrated in the accompanying drawings is provided. These drawings illustrate only typical embodiments of the disclosed system, and therefore should not be considered as limiting its scope. [Brief description of the drawings]

[0134] [Figure 1A]

[0134] FIG. 1 is a perspective view of a support configured to hold a microfluidic device according to some embodiments. [Figure 1B]

[0135] FIG. 1B is a schematic diagram of the support shown in FIG. 1A with the cover removed for clarity. [Diagram 2]

[0136] FIG. 2 is a schematic diagram of elements of an electrical signal generation subsystem according to some embodiments of the present system. [Diagram 3]

[0137] FIG. 2 is a schematic diagram of a thermal control subsystem according to some embodiments of the present system. [Figure 4]

[0138] FIG. 2 is a circuit diagram illustrating an analog circuit used for thermal control feedback in a thermal control subsystem according to some embodiments of the present system. [Diagram 5]

[0139] 1 is an exemplary screenshot illustrating a graphical user interface (GUI) used to control both the electrical signal generation subsystem and the thermal control subsystem according to some embodiments of the present system. [Figure 6]

[0140] 6 is a schematic diagram of a system for operating a microfluidic device according to some embodiments of the present system. The system shown in Figure 6 includes an optical system having various beam splitters and / or dichroic filters, a first light source, a second light source, a light modulation subsystem, an objective lens, and a detector. [Figure 7A]

[0141] 1 is a schematic diagram of a structured light path in an optical system according to some embodiments of the present system. [Figure 7B]

[0141] FIG. 1 is a schematic diagram of an imaging path in an optical system according to some embodiments of the present system. [Figure 8A]

[0142] One illustration of how structured light can be used to compensate for light vignetting is FIG. 8A, which shows how the light intensity measured at the sample plane can vary across the field of view. [Figure 8B]

[0142] As one illustration showing how structured light can be used to compensate for light vignetting, FIG. 8B shows an inverted function that can be used to control the light intensity output from a light modulation subsystem. [Figure 8C]

[0142] As one illustration showing how structured light can be used to compensate for light vignetting, Figure 8C shows the light intensity measured at the sample plane when an inverted function as shown in Figure 8B is used to control the light intensity output from a light source that would produce the light intensity pattern shown in Figure 8A if this function was not used. [Figure 9]

[0143] FIG. 2 is a schematic diagram of an impedance measurement circuit according to some embodiments of the present system. [Figure 10]

[0144] FIG. 2 is a side view of a freeze valve according to some embodiments of the present system. [Figure 11]

[0144] A perspective view of a freeze valve according to some embodiments of the present system. [Figure 12]

[0145] 1 is a perspective view of a pair of freeze valves according to some embodiments of the present system.As shown, the freeze valves flank a socket that holds a microfluidic device. [Figure 13]

[0146] FIG. 13 is a perspective view of various components of the freeze valve shown in FIG. [Figure 14]

[0147] 1 is a perspective view of a freeze valve according to some embodiments of the present system. [Figure 15]

[0148] FIG. 15 is a top perspective view of the cover of the freeze valve shown in FIG. 14. [Figure 16]

[0148] FIG. 15 is a bottom perspective view of the cover of the freeze valve shown in FIG. [Figure 17]

[0149] FIG. 15 is a perspective view of the bottom of the freeze valve shown in FIG. [Figure 18]

[0150] FIG. 18 is a perspective view of the bottom enclosure of the freeze valve shown in FIG. 17. [Figure 19]

[0151] FIG. 15 is a perspective view of the heat sink of the freeze valve shown in FIG. [Figure 20]

[0152] FIG. 15 is a top view of the sleeve of the freeze valve shown in FIG. 14. [Figure 21] FIG. 15 is a side view of the sleeve of the freeze valve shown in FIG. [Figure 22]

[0153] 22 is a schematic diagram of a system for operating a microfluidic device according to some embodiments of the present system. The system shown in Fig. 22 includes an optical system having various beam splitters and / or dichroic filters, a first light source, a second light source, a light modulation subsystem, an objective lens, and a detector. [Diagram 23]

[0154] 1 is a schematic diagram of two LED arrays according to some embodiments of the present system. [Figure 24]

[0155] 1 is a schematic diagram of a light pipe / optical integrator according to some embodiments of the present system. [Diagram 25]

[0156] 1 is a schematic diagram of a light source according to some embodiments of the present system. [Figure 26]

[0157] 1 is a schematic diagram of a multiple-input light pipe / optical integrator according to some embodiments of the present system. [Figure 27]

[0158] 1 shows several embodiments of a split lid for a system used to operate a microfluidic device. [Figure 28]

[0159] 13 shows another embodiment of a split lid for a system used to operate a microfluidic device. [Figure 29]

[0160] 13 shows yet another embodiment of a split lid for a system used to operate a microfluidic device. [Figure 30A]

[0161] 13 shows an embodiment of a split lid removal component of a system used to operate a microfluidic device. [Figure 30B]

[0161] An embodiment of a split lid removal component of a system used to operate a microfluidic device is shown. [Figure 30C]

[0161] An embodiment of a split lid removal component of a system used to operate a microfluidic device is shown. [Figure 31A]

[0162] 1 illustrates an embodiment of a method for adding a fluid sample to a microfluidic device. [Figure 31B]

[0162] An embodiment of a method for adding a fluid sample to a microfluidic device is provided. [Figure 31C]

[0162] An embodiment of a method for adding a fluid sample to a microfluidic device is provided. [Diagram 32]

[0163] FIG. 13 shows a front view of a freeze valve of another embodiment of the system. [Diagram 33]

[0163] Fig. 1 shows a perspective view of a freeze valve of another embodiment of the system. [Diagram 34]

[0164] 13 shows a perspective view of a freeze valve of yet another embodiment of the system. [Diagram 35]

[0164] A perspective view of a freeze valve of yet another embodiment of the system is shown. [Diagram 36]

[0165] 1A-1D show perspective views of some embodiments of a split lid including a sensor, with the split lid in a closed position. [Figure 37]

[0166] 1A-1D show perspective views of some embodiments of a split lid including a sensor, with the split lid in an open position. [Figure 38]

[0167] 13A-13C show several embodiments of exploded components of a sensor that may be used with a split lid. [Figure 39]

[0168] 13A-13C show several embodiments of assembled components of a sensor that may be used with a split lid. [Diagram 40]

[0169] 1A-1D show side views of some embodiments of a split lid including a sensor, with the split lid in a closed position. [Diagram 41]

[0170] 1A-1D show perspective views of several embodiments of optical switches that may be used in the sensor. [Diagram 42]

[0171] FIG. 1 illustrates a top view of an embodiment of a sensor in an activated position. [Diagram 43]

[0171] A top view of an embodiment of the sensor in an activated position is shown. [Diagram 44]

[0172] 1 illustrates cell / bead distribution within a microfluidic device in accordance with some exemplary embodiments.

[0135] DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS

[0173] This specification describes exemplary embodiments and uses of the present disclosure. However, the disclosure is not limited to these exemplary embodiments and uses, nor to the manner in which they function or are described herein. Furthermore, the drawings may show simplified or partial views, and the dimensions of the elements in the figures may be exaggerated or out of proportion. In addition, when the terms "on", "attached to", "connected to", "coupled to" or similar words are used herein, an element (e.g., a material, layer, substrate, etc.) can be "on", "attached to", "connected to", or "coupled to" another element, regardless of whether the element is directly on, attached to, connected to, or coupled to the other element, or whether there are one or more intervening elements between the element and the other element. Similarly, unless otherwise specified, directions (e.g., above, below, top, bottom, side, up, down, under, over, upper, lower, horizontal, vertical, "x", "y", "z", etc.), when presented, are relative and are presented for ease of description and discussion only, by way of example and not as a limitation. In addition, when referring to a list of elements (e.g., elements a, b, c), such reference is intended to include any one of the listed elements alone, any combination of less than all of the listed elements, and / or all combinations of the listed elements. The division of paragraphs herein is merely for ease of review and does not limit any combination of the elements described.

[0136]

[0174] As used herein, "substantially" means sufficient to function for the intended purpose. The term "substantially" thus allows for minor, inconsequential variations from an absolute or perfect state, dimension, measurement, result, etc., as would be expected by one of ordinary skill in the art, but which have little effect on overall performance. When used in reference to a numerical value, or a parameter or characteristic that can be expressed as a numerical value, "substantially" means within 10 percent.

[0137]

[0175] 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.

[0138]

[0176] As used herein, μm means micrometer; μm 3 means cubic micrometer, pL means picoliter, nL means nanoliter, and μL (or uL) means microliter.

[0139]

[0177] As used herein, a "microfluidic device" or "microfluidic apparatus" is a device that includes one or more individual microfluidic circuits configured to hold a fluid, each microfluidic circuit comprising fluidically interconnected circuit elements including, but not limited to, regions, flow paths, channels, chambers, and / or pens, and at least one port configured to allow fluid (and optionally, microscopic objects suspended in the fluid) to flow into and / or out of the microfluidic device. Typically, the microfluidic circuits of a microfluidic device include a flow region that may include a microfluidic channel and at least one chamber, and hold a volume of fluid 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 fluidly connected to a first port (e.g., an inlet) of the microfluidic device and a second end fluidly connected to a second port (e.g., an outlet) of the microfluidic device. In some embodiments, a microfluidic device may have more than two ports, e.g., three, four, five, six or more ports, and a typical example may have, for example, two inlets and two outlets for fluidly connecting two microfluidic circuits of the same microfluidic device.

[0140]

[0178] A microfluidic device may be referred to herein as a "microfluidic chip" or a "chip."

[0141]

[0179] As used herein, a "microfluidic channel" or "flow channel" refers to a flow region of a microfluidic device that has a length that is significantly longer than both its horizontal and vertical dimensions. The length of a channel is generally defined by the flow path of the channel. For straight channels, the length is the "longitudinal axis" of the channel. The "horizontal dimension" or "width" of a channel is the horizontal dimension when viewed in a cross-section oriented perpendicular to the longitudinal axis of the channel (or, if the channel is curved, perpendicular to an axis tangential to the flow path of the channel in the plane of the cross-section). The "vertical dimension" or "height" of a channel is the vertical dimension when viewed in a cross-section oriented perpendicular to the longitudinal axis of the channel (or, if the channel is curved, perpendicular to an axis tangential to the flow path of the channel in the plane of the cross-section).

[0142]

[0180] The flow channel can be, for example, at least 5 times the length in 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 more. In some embodiments, the length of the flow channel is from about 100,000 microns to about 500,000 microns, including all values ​​therebetween. In some embodiments, the horizontal dimension is from about 100 microns to about 1000 microns (e.g., from about 150 to about 500 microns) and the vertical dimension is from about 25 microns to about 200 microns (e.g., from about 40 to about 150 microns). It is noted that the flow channel may have a variety of different spatial configurations in a microfluidic device and thus is not limited to being a completely linear element. For example, the flow channel may be or may include one or more cross sections having the following configurations: curved, bent, spiral, sloping upward, sloping downward, forked (e.g., multiple different flow paths), and any combination thereof. Additionally, the flow channel may have different cross-sectional areas along its path, expanding and contracting to provide the desired fluid flow within the flow channel. The flow channel may include valves, which may be of any type known in the art of microfluidics. Examples of microfluidic channels including valves are disclosed in U.S. Patent Nos. 6,408,878 and 9,227,200, each of which is incorporated herein by reference in its entirety.

[0143]

[0181] The direction of fluid flow through a flow region (e.g., a channel) or other circuit element (e.g., a chamber) affects the "upstream" and "downstream" orientation of the flow region or circuit element. Thus, an inlet is at an upstream location and an outlet is generally at a downstream location. Those skilled in the art will understand that the designation of an "inlet" or "outlet" may be altered by reversing the flow within the device or by opening one or more additional apertures.

[0144]

[0182] As used herein, "bright field" illumination and / or imaging refers to white light illumination of a microfluidic field with a broad spectrum light source, where contrast is created by absorption of light by objects in the field.

[0145]

[0183] As used herein, "structured light" is projected light that is tailored to provide one or more lighting effects. The first lighting effect can be projected light, e.g., a projected light pattern, that illuminates a portion of the device's surface without (or at least minimally) illuminating adjacent portions of the surface, used to activate DEP forces in a DEP (dielectrophoresis) substrate, as described in more detail below. When a structured light pattern is used to activate DEP forces, the intensity, e.g., the duty cycle of a structured light modulator, such as a DMD (Digital Mirror Device), can be used to change the light power applied to the light-activated DEP actuator, and thus the DEP force, without changing the nominal voltage or frequency. Another lighting effect that can be produced by structured light includes projected light that can be corrected for surface irregularities and irregularities associated with the light projection itself, e.g., falloff at the edges of the illumination field. Structured light is typically produced by a structured light modulator, e.g., a digital mirror device (DMD), a microshutter array system (MSA), or a liquid crystal display (LCD). Illumination of a small area of ​​a surface, e.g., a selected region of interest, with structured light improves the signal-to-noise ratio (SNR), because illuminating only the selected region of interest reduces stray / scattered light, thereby reducing the dark level of the image. An important aspect of structured light is that it can be changed quickly over time. A light pattern with a structured light modulator, e.g., a DMD, can be used to autofocus on difficult targets, such as clean mirrors or surfaces that are barely in focus. Using a clean mirror, some self-test features, such as measurements of the modulation transfer function and field curvature / tilt, can be replicated without the need for a more expensive Shack-Hartmann sensor. In another application of structured light patterns, the power distribution on a sample surface can be measured using a simple power meter instead of a camera. Structured light patterns can also be used as a reference feature for alignment of optical modules / system components, as well as a manual readout for manual focus.Another lighting effect made possible by the use of structured light patterns is the selective curing, for example the solidification of hydrogels in microfluidic devices.

[0146]

[0184] As used herein, the term "micro object" generally refers to any microscopic object that can be separated and / or manipulated according to the present disclosure. Non-limiting examples of micro objects include inanimate micro objects such as microparticles; microbeads (e.g., polystyrene beads, glass beads, amorphous solid substrates, or Luminex™ beads, etc.); magnetic beads; microrods; microwires; quantum dots, etc.; biological micro objects such as cells; biological organelles; vesicles or complexes; synthetic vesicles; liposomes (e.g., synthetic or derived from membrane preparations); lipid nanorafts, etc.; or combinations of inanimate and biological micro objects (e.g., microbeads attached to cells, microbeads coated with liposomes, or magnetic beads coated with liposomes, etc.). Beads may include covalently or non-covalently attached moieties / molecules such as fluorescent labels, proteins (including receptor molecules), carbohydrates, antigens, small molecule signaling moieties, or other chemical / biological species that can be used in assays. In some variations, the bead / solid substrate containing the moiety / molecule may be a capture bead configured to selectively or non-selectively bind nearby molecules, including, for example, small molecules, peptides, proteins, or nucleic acids. In one non-limiting example, the capture bead may include a nucleic acid sequence configured to bind to a nucleic acid having a specific nucleic acid sequence, or the nucleic acid sequence of the capture bead may be configured to bind to a set of nucleic acids having related nucleic acid sequences. Either type of binding may be understood to be selective. The capture bead containing the moiety / molecule may bind non-selectively when binding of structurally different but physicochemically similar molecules occurs. For example, size exclusion beads or zeolites are configured to capture molecules of a selected size or charge. Lipid nanorafts are described, for example, in Ritchie et al. (2009) “Reconstitution of Membrane Proteins in Phospholipid Bilayer Nanodiscs,” Methods Enzymol., 464:211-231.

[0147]

[0185] 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, reptile cells, bird cells, or fish cells, prokaryotic cells, bacterial cells, fungal cells, or protozoan cells, cells isolated from tissues such as muscle, cartilage, fat, skin, liver, lung, nervous tissue, immune cells such as T cells, B cells, natural killer cells, macrophages, embryos (e.g., zygotes), oocytes, eggs, sperm cells, hybridomas, cultured cells, cells of cell lines, cancer cells, infected cells, transformed and / or converted cells, reporter cells, and the like. Mammalian cells can be, for example, from humans, mice, rats, horses, goats, sheep, cows, or primates, and the like.

[0148]

[0186] A colony of biological cells is a "clone" if all living cells in the colony that can reproduce are daughter cells derived from a single parent cell. In certain embodiments, all daughter cells in a clonal colony are derived from a single parent cell by 10 or fewer divisions. In other embodiments, all daughter cells in a clonal colony are derived from a single parent cell by 14 or fewer divisions. In other embodiments, all daughter cells in a clonal colony are derived from a single parent cell by 17 or fewer divisions. In other embodiments, all daughter cells in a clonal colony are derived from a single parent cell by 20 or fewer divisions. The term "clonal cells" refers to cells of the same clonal colony.

[0149]

[0187] As used herein with respect to a fluid medium, "diffuse" and "diffusion" refer to the thermodynamic movement of a component of a fluid medium down a concentration gradient.

[0150]

[0188] The term "medium flow" refers to bulk movement of a fluid medium primarily by any mechanism other than diffusion, and may include perfusion. For example, medium flow may involve movement of a fluid medium from one point to another due to pressure differences between the points. Such flow may include continuous, pulsed, periodic, random, intermittent, or reciprocating liquid flows, or any combination thereof. Flow of one fluid medium into another may result in turbulence and mixing of the media. Flowing may include drawing a solution into a microfluidic channel (e.g., aspiration) or forcing a fluid through a microfluidic channel (e.g., perfusion).

[0151]

[0189] The phrase "substantially no flow" refers to a flow rate of a fluid medium that, averaged over time, is less than the diffusion rate of a component of a substance (e.g., an analyte of interest) into or within the fluid medium. The diffusion rate of a component of such a substance may depend, for example, on temperature, size of the component, and strength of interaction between the component and the fluid medium.

[0152]

[0190] As used herein with respect to different regions within a microfluidic device, the term "fluidically connected" means that when the different regions are substantially filled with a fluid, such as a fluid medium, the fluid in each region is connected to form a single fluid. This does not mean that the composition of the fluid (or fluid medium) in the different regions is necessarily identical. Rather, the fluids in different fluidly connected regions within a microfluidic device may have different compositions (e.g., different concentrations of solutes, such as proteins, carbohydrates, ions, or other molecules) that are in flux as the solutes move down their respective concentration gradients and / or as the fluid flows through the device.

[0153]

[0191] As used herein, a "flow path" refers to one or more fluidly connected circuit elements (e.g., channels, regions, chambers, etc.) that define and are exposed to the flow trajectory of a medium. Thus, a flow path is an example of a sweep region of a microfluidic device. Other circuit elements (e.g., non-sweep regions) may be fluidly connected to a circuit element that includes a flow path without being exposed to the flow of the medium in the flow path.

[0154]

[0192] As used herein, "isolation of a micro-object" refers to confining a micro-object to a defined area within a microfluidic device. The defined area may be, for example, a chamber. As used herein, a "chamber" is a region (e.g., a circuit element) within a microfluidic device that allows one or more micro-objects to be isolated from other micro-objects located within the microfluidic device. Examples of chambers include microwells, which may be regions etched from a substrate (e.g., a planar substrate) as described in U.S. Patent Application Publication No. 2013 / 0130232 (Weibel et al.) and U.S. Patent Application Publication No. 2013 / 0204076 (Han et al.), or regions formed within a multilayer device, such as the microfluidic devices described in WO 2010 / 040851 (Dimov et al.) or U.S. Patent Application Publication No. 2012 / 0009671 (Hansen et al.). Other examples of chambers include valved chambers such as those described in WO 2004 / 089810 (McBride et al.) and US 2012 / 0015347 (Singhal et al.). Further examples of chambers include those described in Somaweera et al. (2013), "Generation of a Chemical Gradient Across an Array of 256 Cell Cultures in a Single Chip", Analyst., Vol. 138(19), pp 5566-5571; US ​​2011 / 0053151 (Hansen et al.); and US 2006 / 0154361 (Wikswo et al.). Further examples of chambers include the isolation pens described herein. In certain embodiments, the chamber may be 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, the chamber may be configured to hold a volume of fluid between 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.

[0155]

[0193] As used herein, "pen" or "penning" specifically refers to placing a micro-object in an isolation pen in a microfluidic device. The force used to pen the micro-object may be any suitable force described herein, such as dielectrophoresis (DEP), e.g., optically driven dielectrophoretic force (OEP); gravity; magnetic force; locally driven fluid flow; or tilt. In some embodiments, penning a plurality of micro-objects may reposition substantially all of the micro-objects. In some other embodiments, a selected number of the plurality of micro-objects may be penned, and the remainder of the plurality of micro-objects may not be penned. In some embodiments, when a selected micro-object is penned, a DEP force, e.g., an optically driven DEP force or a magnetic force, may be used to reposition the selected micro-object. Typically, the micro-object may be introduced into a flow region of a microfluidic device, e.g., a microfluidic channel, and then introduced into a chamber by penning.

[0156]

[0194] As used herein, "unpen" or "unpenning" refers to repositioning a micro-object from within an isolation pen to a new location within a flow region of a microfluidic device, e.g., a microfluidic channel. The force used to unpen a micro-object may be any suitable force described herein, such as dielectrophoresis, e.g., optically driven dielectrophoretic force; gravity; magnetic force; locally driven fluid flow; or gradient. In some embodiments, unpenning a plurality of micro-objects may reposition substantially all of the micro-objects. In some other embodiments, a selected number of the plurality of micro-objects may be unpenned, and the remainder of the plurality of micro-objects may not be unpenned. In some embodiments, when a selected micro-object is unpenned, a DEP force, e.g., an optically driven DEP force or a magnetic force, may be used to reposition the selected micro-object.

[0157]

[0195] As used herein, "export" or "exporting" may include relocating a micro-object from a location within a microfluidic device, e.g., a flow field, microfluidic channel, chamber, etc., to a location outside the microfluidic device, e.g., a well plate, tube, or other receptacle; may consist of relocating a micro-object from a location within a microfluidic device, e.g., a flow field, microfluidic channel, chamber, etc., to a location outside the microfluidic device, e.g., a well plate, tube, or other receptacle; or may consist essentially of relocating a micro-object from a location within a microfluidic device, e.g., a flow field, microfluidic channel, chamber, etc., to a location outside the microfluidic device, e.g., a well plate, tube, or other receptacle. In some embodiments, exporting a micro-object comprises withdrawing (e.g., micropipetting) a volume of medium containing the micro-object from within the microfluidic device and placing the volume of medium in or on a location outside the microfluidic device. In some related embodiments, the microfluidic device is disassembled (e.g., an upper layer, such as a cover or lid of the microfluidic device, is removed from an underlying layer, such as a base portion or substrate of the microfluidic device) prior to withdrawal of the amount of medium to facilitate (e.g., micropipette) access to the interior region of the microfluidic device. In other embodiments, exporting a micro-object involves flowing a volume of fluid containing the micro-object into a flow region of the microfluidic device (e.g., including a microfluidic channel) and out an outlet of the microfluidic device, and depositing the volume of medium in or on a location outside the microfluidic device. In such embodiments, the micro-object within the microfluidic channel may be exported without requiring disassembly (e.g., removal of a cover of the device) or insertion of a tool into the interior region of the microfluidic device to remove the micro-object for further processing. "Export" or "exporting" may further include relocating the micro-object from within a chamber, which may include an isolation pen, to a new location within a flow region, such as a microfluidic channel, as described above with respect to "unpenning."As described herein with respect to the isolation pen, the flat orientation of the chamber relative to the microfluidic channel, such that the chamber opens laterally from the microfluidic channel, allows for easy export of micro-objects that have been positioned or repositioned (e.g., unpenned from the chamber) to be placed within the microfluidic channel.

[0158]

[0196] A microfluidic device may include "sweep" and "non-sweep" regions. As used herein, a "sweep" region comprises one or more fluidically interconnected circuit elements of a microfluidic circuit, each of which receives a flow of a medium as the fluid flows through the microfluidic circuit. The circuit elements of a sweep region may include, for example, all or part of a region, a channel, and a chamber. As used herein, a "non-sweep" region comprises one or more fluidically interconnected circuit elements of a microfluidic circuit, each of which does not substantially receive a flow of a medium as the fluid flows through the microfluidic circuit. A non-sweep region may be fluidly connected to a sweep region, provided that the fluid communication is configured to allow diffusion between the sweep region and the non-sweep region but substantially no flow of a medium. Thus, a microfluidic device may be configured to substantially isolate the non-sweep region from the flow of a medium in the sweep region, while allowing substantially only diffusion fluid communication between the sweep region and the non-sweep region. For example, a flow channel of a microfluidic device is an example of a sweep region, and a separation region of a microfluidic device (described in more detail below) is an example of a non-sweep region.

[0159]

[0197] As used herein, a "non-sweeping" flow rate of a fluid medium means a flow rate sufficient to allow components of a second fluid medium in a separation region of the isolation pen to diffuse into a first fluid medium in the flow region and / or components of the first fluid medium to diffuse into a second fluid medium in the separation region, and further, the first medium does not substantially flow into the separation region.

[0160]

[0198] In some embodiments, the system may include a support (also known as a "nest") configured to hold a microfluidic device. The support may include, for example, a socket configured to interface with and / or hold an optically actuated microfluidic device, a printed circuit board assembly (PCBA), an electrical signal generation subsystem, a thermal control subsystem, or any combination thereof.

[0161]

[0199] In certain embodiments, the support includes a socket that can interface with a microfluidic device, such as an optically actuated microfluidic device. An exemplary socket 106 is included in the support 100 of FIGS. 1A and 1B. However, the shape and function of the socket 106 need not be exactly the same as that shown in FIGS. 1A and 1B. For example, the socket can include a lid). Furthermore, the socket 106 can be tailored as needed to match the size and type of the microfluidic device 110 with which the socket 106 will interface. Various microfluidic devices 110 are known in the art, including devices 110 with optically actuated configurations, such as optoelectronic tweezer (OET) and / or opto-electrowetting (OEW) configurations. Examples of suitable OET configurations are shown in the following U.S. patent documents, each of which is incorporated herein by reference in its entirety as if fully set forth: U.S. Patent RE44,711 (Wu et al.) (originally issued as U.S. Patent 7,612,355); and U.S. Patent 7,956,339 (Ohta et al.). Examples of OEW configurations are shown in U.S. Patent 6,958,132 (Chiou et al.) and U.S. Patent Application Publication No. 2012 / 0024708 (Chiou et al.), both of which are incorporated herein by reference in their entirety as if fully set forth. Yet another example of an optically actuated microfluidic device includes an integrated 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 Publication Nos. WO2015 / 164846 and WO2015 / 164847, all of which are incorporated by reference in their entireties as if fully set forth herein.

[0162]

[0200] The support 100 shown in Figures 1A and 1B also includes a base portion 102 and a cover 104 (omitted in Figure 1B). The support 100 also includes a number of connectors, namely, a first fluid input / output 112; a communication connection 114; a power connection 116; and a second fluid input / output 118. The first and second fluid input / output 112, 118 are configured to deliver cooling fluid to and from a cooling block (shown in Figure 3) used to cool the microfluidic device 110. Whether the first and second fluid input / output 112, 118 are inputs or outputs depends on the direction of fluid flow through the support 100. The first and second fluid input / output 112, 118 are fluidly coupled to the cooling block by first and second fluid connectors 142, 144 disposed within the support 100. The communication connections 114 are configured to connect the support 110 with other components of a system for operating the microfluidic device, as described below. The power connections 116 are configured to provide power (e.g., electricity) to the support 110.

[0163]

[0201] In certain embodiments, the support 100 can include an integrated power generation subsystem 138. The power generation subsystem 138 can be configured to apply a bias voltage between a pair of electrodes in the microfluidic device 110 held by the support 100. The ability to apply such a bias voltage does not mean that the bias voltage is applied at all times when the microfluidic device 110 is held by the support 100. Rather, in most cases, the bias voltage is applied intermittently and only when needed to facilitate the generation of microfluidic forces, such as dielectrophoresis or electrowetting, or to facilitate the measurement of complex impedance in the microfluidic device 110.

[0164]

[0202] Typically, the electrical signal generation subsystem 138 includes a waveform generator 202, as shown in FIG. 2. The power generation subsystem 138 may further include a detection module 208 (e.g., an oscilloscope) and / or a waveform amplifier circuit 204 configured to amplify the waveform received from the waveform generator 202. The detection module 208, if present, may be configured to measure the waveform applied to the microfluidic device 110 held by the support 100. In certain embodiments, the detection module 208 measures the waveform at a location closer to the microfluidic device 110 (and further from the waveform generator 202), thus ensuring a more accurate measurement of the waveform actually applied to the microfluidic device 110. Data obtained from the measurement of the detection module 208 may be provided, for example, as feedback to the waveform generator 202, which may be configured to adjust its output based on such feedback. An example of a suitable combined waveform generator 202 and detection module 208 is the RED PITAYA™.

[0165]

[0203] In certain embodiments, the support 100 can include a thermal control subsystem 140. The thermal control subsystem 140 can be configured to regulate the temperature of the microfluidic device 110 held by the support 100. As shown in FIG. 3, the thermal control subsystem 140 can include proximal components of a Peltier thermoelectric device 304 and a cooling unit 312. The Peltier thermoelectric device 304 can have a first surface 306 configured to contact at least one surface of the microfluidic device 110. The cooling unit can include, for example, a cooling block 322. A second surface 308 (e.g., a surface 308 opposite the first surface 306) of the Peltier thermoelectric device 304 can be configured to mate with a surface of such a cooling block 322. All or a portion of the cooling block 322 (e.g., the portion that interfaces with the Peltier thermoelectric device 304) can be made of a material having a high thermal conductivity. For example, the material can be a metal, such as aluminum. The cooling block 322 can be connected to a fluid pathway 324 configured to circulate a cooled fluid between the fluid cooling device 326 and the cooling block 322. The fluid pathway 324 can include the fluid input / outputs 112, 118 and the fluid connectors 142, 144 described in connection with FIG. 1. The Peltier thermoelectric device 304 and the cooling block 322 can be mounted on the support 100.

[0166]

[0204] The thermal control subsystem 140 may further include a thermoelectric generation module 302, as shown in Figure 3. The thermoelectric generation module 302 may adjust the temperature of the Peltier thermoelectric device 304 to achieve a target temperature of the microfluidic device 110. Feedback to the thermoelectric generation module 302 may include a temperature value provided by an analog circuit 400 as shown in Figure 4. Alternatively, the feedback may be provided by a digital circuit (not shown). The Peltier thermoelectric device 304, the cooling block 322, and the thermoelectric generation module 302 may all be mounted on the support 100.

[0167]

[0205] In certain embodiments, the support 100 may also include or interface with an environmental temperature monitor / regulator in addition to the thermal control subsystem 140 .

[0168]

[0206] The analog circuit 400 shown in FIG. 4 includes a resistor 402, a thermistor 406, and an analog input 404. The analog input is operatively coupled to the electrical signal generation subsystem 138 (e.g., its detection module 208) to provide it with a signal that can be used to calculate the temperature of the microfluidic device 110. The thermistor 406 is configured such that its resistance decreases in a known manner when the temperature of the thermistor 406 decreases and increases in a known manner when the temperature of the thermistor 406 increases. The analog circuit 400 is connected to a power source (not shown) configured to provide a bias voltage to the electrodes 408. In one particular embodiment, the resistor 402 can have a resistance of about 10,000 ohms, the thermistor 406 can have a resistance of about 10,000 ohms at 25° C., and the power source (e.g., a DC power source) can apply a bias voltage of about 5 V. The analog circuit 400 is exemplary and other systems can be used to provide a temperature value as feedback to the thermoelectric generation module 302.

[0169]

[0207] In certain embodiments, the support 100 further comprises a controller 136 (e.g., a microprocessor). The controller 136 can be used to sense and / or control the electrical signal generation subsystem 138. Additionally, to the extent the support 100 includes a thermal control subsystem 140, the controller 136 can be used to sense and / or control the thermal control subsystem 140. Examples of suitable controllers 136 include ARDUINO NANO™ microprocessors, such as the ARDUINO NANO™. The controller 136 can be configured to interface with an external controller (not shown), such as a computer or other computing device, via the plug / connector 134. In certain embodiments, the external controller can include a graphical user interface (GUI) configured to sense and / or control the electrical signal generation subsystem 138, the thermal control subsystem 140, or both. An exemplary GUI 500 configured to control both the electrical signal generation subsystem 138 and the thermal control subsystem 140 is shown in FIG. 5.

[0170]

[0208] In certain embodiments, the support 100 may include a printed circuit board (PCB) 132. The electrical signal generation subsystem 138 may be mounted on and electrically integrated with the PCB 132. Similarly, to the extent the support 100 includes the controller 136 or the thermal control subsystem 140, the controller 136 and / or the thermoelectric generation module 302 may be mounted on and electrically integrated with the PCB 132.

[0171]

[0209] 1A and 1B, the exemplary support 100 can include a socket 106, an interface 134, a controller 136, an electrical signal generation subsystem 138, and a thermal control subsystem 140, all of which are mounted on and electrically integrated with a PCB 132, thereby forming a printed circuit board assembly (PCBA) 130. As discussed above, the socket 106 can be designed to hold microfluidic devices 110 (or "consumables"), including optically operated microfluidic devices.

[0172]

[0210] In certain specific embodiments, the power generation subsystem 138 may include a RED PITAYA™ waveform generator 202 / detection module 208 and a waveform amplifier circuit 204 that amplifies the waveform generated by the RED PITAYA™ waveform generator 202 and sends the amplified waveform (voltage) 206 to the microfluidic device 110. Both the RED PITAYA™ units 202, 208 and the waveform amplifier circuit 204 may be electrically integrated into the PCB 132 as the electrical signal generation subsystem 138, as shown in FIG. 1B. Additionally, the RED PITAYA™ units 202, 208 may be configured to measure the amplified voltage at the microfluidic device 110 and then adjust their own output voltage as necessary so that the voltage measured at the microfluidic device 110 is at a desired value. The amplifier circuit 204 may have a power supply of +6.5V to −6.5V formed, for example, by a pair of DC-DC converters mounted on the PCB 132, resulting in a signal at the microfluidic device 110 of up to 13 Vpp.

[0173]

[0211] In a particular specific embodiment, the support 100 includes a thermal control subsystem 140 (shown in FIG. 3) having a Peltier thermoelectric device 304 disposed between a liquid-cooled aluminum block 322 and the backside of the microfluidic device 110, a POLOLU™ thermoelectric generator (not shown), and an ARDUINO NANO™ controller 136. Feedback for the thermal control subsystem 140 can be an analog voltage divider circuit 400 (shown in FIG. 4) including a resistor 402 (e.g., resistance 10k ohms + / - 0.1%, temperature coefficient + / - 0.02 ppm / °C) and a negative temperature coefficient thermistor 406 (nominal resistance 10k ohms + / - 0.01%). The controller 136 can measure the voltage from the feedback circuit 400 and then use the calculated temperature value as an input (e.g., to an on-board PID control loop algorithm) to drive both directional and pulse width modulated signal pins in the thermoelectric generation module 302, thereby actuating the thermoelectric subsystem 140. The liquid cooling unit 326 can be configured to pump fluid through a cooling path 324 located partially within the support 100 (e.g., the fluid inputs / outputs 112, 118 and fluid connectors 142, 144) and partially around the support 100.

[0174]

[0212] In certain specific embodiments, the support 100 includes a serial port 114 and a Plink tool that together enable the RED PITAYA™ unit to communicate with an external computer. The serial port 114 can also enable the controller 136 to communicate with the external computer. Alternatively, a separate serial port (not shown) can be used to enable the controller 136 to communicate with the external computer. In other embodiments, the support 100 can include a wireless communication device configured to facilitate wireless communication between components of the support 100 (e.g., the controller 136 and / or the power generation subsystem 138) and an external computer, which can include a portable computing device such as a mobile phone, PDA, or other handheld device. A GUI (such as that shown in FIG. 5) on the external computer can be configured for a variety of functions, including but not limited to plotting temperature and waveform data, performing scaling calculations for output voltage adjustment, and updating the controller 136 and the RED PITAYA™ devices 202, 208.

[0175]

[0213] In certain embodiments, the support 100 may also include or be interfaced with an inductance / capacitance / resistance (LCR) meter configured to measure properties of the contents (e.g., fluid contents) of the microfluidic device 110.

[0176]

[0214] For example, the LCR meter can be configured to measure the complex impedance of the system, particularly the complex impedance of the fluid as it flows into, is disposed within, and / or leaves the microfluidic device 110. In some embodiments, the LCR meter can be connected to and / or integrated with the fluid lines that carry the fluid into and out of the microfluidic device 110. In other embodiments, the LCR meter can be connected to or be an integral part of the power generation subsystem 138. Thus, in certain specific embodiments, the RED PITAYA™ waveform generator 202 and the detection module 208 in the support 100 can be configured to function as an LCR meter. In certain embodiments, the electrodes of the microfluidic device 110 configured for use with the power generation subsystem 138 can also be configured for use with the LCR meter. By measuring the impedance of the system, various system characteristics and changes therein can be determined, such as the height of the fluid circuits within the microfluidic device 110, changes in the salt content of the fluids within the microfluidic device 110 (which can be correlated to the state of biological micro-objects therein), and the movement of specific volumes of fluids (having different impedances) through the microfluidic device 110.

[0177]

[0215] In certain embodiments, measuring the impedance of the system can be used to accurately (i.e., close to true) and precisely (i.e., repeatably) detect changes from a first fluid in the system (i.e., microfluidic device 110) to a second fluid in the system. For example, the first fluid can be deionized water (DI) and the second fluid can be saline (e.g., phosphate buffered saline or "PBS"), or vice versa. Alternatively, the first fluid can be saline (e.g., PBS) and the second fluid can be a cell culture medium having a detectably different impedance from saline, or vice versa. In yet another alternative, the first fluid can be a first cell culture medium and the second fluid can be a second cell culture medium having a detectably different impedance from the first cell culture medium. FIG. 9 illustrates an impedance measurement circuit 900 for detecting the impedance of a system. The circuit 900 includes an output 902 from the waveform generator 202 of the power generation subsystem 138 and two inputs 904, 906 to the detection module 208 of the power generation subsystem 138. The circuit 900 includes the microfluidic device 110 (connected via the socket 106 of the support 100) and a shunt resistor 908. The shunt resistor 908 can be selected to make the LCR sufficiently accurate to measure impedances in the range of 0 to about 5,000 ohms (e.g., in the range of 0 to about 4,000, 0 to about 3,000, 0 to about 2,500, 0 to about 2,000, 0 to about 1,500, or 0 to about 1,000 ohms). The microfluidic device 110 functions in the circuit 900 as a measurement cell, with the base portion (e.g., a semiconductor device) and cover (e.g., having an indium tin oxide (ITO) layer) of the microfluidic device 110 functioning as electrodes. In certain specific embodiments, the output 902 of the circuit 900 can be from the waveform generator 202 of the RED PITAYA™ device, and the inputs 904, 906 can originate from the microfluidic device 110 and be received by the detection module 208 of the RED PITAYA™ device. In certain specific embodiments, the shunt resistor 908 can be a 50 ohm resistor.In these embodiments, the power generation subsystem 138 can be switched between an “optical operation mode” and an “LCR mode.” Additionally, when in LCR mode, the power generation subsystem 138 can be connected to a computer running a MATLAB script.

[0178]

[0216] Therefore, the system of the present invention can control the flow rate (V flow) is determined. For example, the microfluidic device 110 is initially filled with a first fluid associated with a first impedance (e.g., DI associated with an impedance of about 450 ohms). A second fluid associated with a second impedance detectably different from the first impedance (e.g., PBS associated with an impedance of about 160 ohms) is then flowed into and through the microfluidic device 110. The second fluid can be flowed into the microfluidic device 110 through a port that can function, for example, as either a fluid inlet port or a fluid outlet port. The system continuously measures the complex impedance of the microfluidic device 110 as the second fluid is flowed into and through the microfluidic device 110. As discussed above, to measure the complex impedance of the microfluidic device 110 at a particular time, the system applies a voltage potential to the microfluidic device 110 and receives a concomitant signal from the microfluidic device 110 that is used to calculate the complex impedance. The voltage potential applied to the microfluidic device can have a frequency of about 10 kHz to about 1 MHz (e.g., about 50 kHz to about 800 kHz, about 100 kHz to about 700 kHz, about 200 kHz to about 600 kHz, about 300 kHz to about 500 kHz, about 350 kHz to about 400 kHz, or about 380 kHz). The particular frequency can be selected based on the properties of the microfluidic device 110 and the first and second fluids to optimize the accuracy of the impedance measurement, minimize the measurement time, and reduce inductive effects. The second fluid is flowed into and through the microfluidic device 110 until the measured complex impedance changes from a first impedance associated with the first fluid to a second impedance associated with the second fluid. The minimum amount of the second fluid required to completely switch the complex impedance of the microfluidic device 110 from the first impedance to the second impedance is determined by the flow rate (V) of the microfluidic device. flowThe amount of the second fluid required to switch the complex impedance of the microfluidic device 110 from the first impedance to the second impedance is a measure of the flow rate (V flow ), (2) the volume of the fluid outlet port of the microfluidic device, and (3) the flow rate of the tube that carries the second fluid from the pump to the microfluidic device 110. The flow rate of the tube and inlet port can be easily distinguished from the flow rate of the microfluidic device 110 because the flow of the second fluid through the tube and fluid outlet port does not change the complex impedance of the microfluidic device 110.

[0179]

[0217] Using the calculated flow rate of the microfluidic device 110, the system further provides a method for reliably exporting one or more micro-objects in discrete volumes of fluid from the microfluidic device 110. flow ), the minimum export volume (V) required to export a microobject (e.g., a biological cell) placed in the channel is determined. ex ) can be estimated by calculating the portion of the flow channel that separates the micro-object from the fluid exit port of the microfluidic device 110. For example, the total length of the flow channel (L tot ) can be determined by tracing the flow path of the microfluidic device 110 from the fluid inlet port to the fluid outlet port. ex ) can be determined by tracing the flow path of the microfluidic device 110 from the position of the micro-object within the flow path to the fluid exit port. The minimum amount of fluid required to export the micro-object from the electrokinetic device 110 (V ex ) is therefore V ex =(L ex / L tot )*V flow Alternatively, the total volume of the flow channel (V) can be calculated from the predicted geometry of the flow channel (e.g., using a CAD drawing). flow-tot) can be estimated; and the total volume of the outflow pathway (V ex-tot ) can similarly be calculated from the predicted geometry of the flow channel. In such an embodiment, the minimum volume of fluid (V ex ) is V ex =(V ex-tot / V flow-tot )*V flow It can be calculated as: V ex Regardless of the method for calculating V, the micro-object is forced to move at least V through the fluid outlet port of the kinetic device 110. ex To ensure reliable export, the micro-objects can be exported from the electrokinetic device 110 by flushing a volume of fluid equal to C*Vex (V ex-rel ), where C is a multiple that is equal to or greater than about 1.1 (e.g., about 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, or more). In some methods, V ex (or V ex-rel ) is the residual volume (V ex (or V ex-rel ) equal to V res minus the leading portion) is discarded before being exported from the microfluidic device 110. For example, V ex (or V ex-rel ) may be equal to 1.0 μL, and the initial volume of 0.5 μL may be discarded, so that the micro-objects are delivered to a final volume V of 0.5 μL. res In this way, small objects can be exported in small but discrete fluid volumes. ex , V ex-rel , or V res can be about 2.0 μL, 1.5 μL, 1.2 μL, 1.0 μL, 0.9 μL, 0.8 μL, 0.7 μL, 0.6 μL, 0.5 μL, 0.4 μL, 0.3 μL, 0.25 μL, or less. Typically, the volume of fluid that contains the micro object (i.e., Vex , V ex-rel , or V res ) is exported through the export tubing, which has a finite internal volume before reaching the collection vessel. Therefore, the calculations used in the method can be adjusted to account for the known or estimated volume of the export tubing. For example, the export tubing may have an internal volume of 5.0 μL. In this case, a V of 1.0 μL ex (or V ex-rel ) can be adjusted to 6.0 μL, and the discarded head volume of 0.5 μL can be adjusted to 5.5 μL, so a V res remains unchanged.

[0180]

[0218] In certain embodiments, the support 100 includes one or more valves coupled to the support 100, the one or more valves configured to limit (e.g., stop) movement of fluids in the microfluidic device 110 coupled to the support 100. Although the flow of fluids into and out of the microfluidic device 110 can be controlled, for example, by a pump, movement of the fluid lines connecting the pump to the microfluidic device 110 can cause undesired movement (e.g., drift and / or vibration) of fluids in the microfluidic device 110, even when the pump is off. This movement can further interfere with processes occurring in the microfluidic device 110, such as detection and / or selection of micro-objects (e.g., for counting, characterization, and / or movement between channels and chambers) or assays performed in the microfluidic device 110. One or more valves located on the support 100 can reduce or prevent such undesired movement of fluids in the microfluidic device 110. A suitable valve can be substantially free of internal dead space (i.e., space within the valve that is accessible to fluid, but is largely not exposed to the fluid flow as the fluid flows through the valve). In certain embodiments, at least one of the one or more valves is a thermally controlled flow controller, such as a freeze valve. Figures 10 and 11 show a thermally controlled flow controller 1000 for use with the support 100 according to one embodiment of the system. The flow controller 1000 includes a temperature adjustment device 1004, a thermally conductive interface 1006, and a flow segment of a fluid line 1008 (hidden). The temperature adjustment device 1004 can include one or more Peltier thermoelectric devices (e.g., a stack of two, three, four, five, or more Peltier devices). The thermally conductive interface 1006 may be formed from a material with a high thermal conductivity that is resistant to thermal damage, such as a metal (e.g., copper). The thermally conductive interface 1006 can be wrapped around the flow segment of the fluid line 1008.The thermally conductive interface 1006 may be, for example, a sleeve or other object that completely surrounds the flow segment of the fluid line 1008, or may have a grooved surface that receives the flow segment of the fluid line 1008 within the groove. The fluid in the fluid line 1008 may be a liquid that freezes a solid at a temperature achievable by the flow controller 1000. The thermally conductive interface 1006 is positioned adjacent to, and preferably in contact with, a thermally conductive surface of the temperature adjustment device 1004 to increase the efficiency of the flow controller 1000.

[0181]

[0219] In certain embodiments, the thermally controlled flow controller 1000 can include a heat sink 1002, which can be made of one or more materials with high thermal conductivity (and low heat capacity), such as aluminum. Alternatively, the flow controller 1000 can be configured to rest on and / or be secured to the heat sink 1002. Additionally, the flow controller 1000 can include an insulating material 1010, which can be configured to prevent moisture from interfering with the function of the flow controller 1000, which can occur when moisture condenses on the thermally conductive interface 1006 and / or the temperature adjustment device 1004. The flow controller 1000 can also include a cover 1012. The cover 1012 or other device (e.g., a clamp) can be configured to press the thermally conductive interface 1006 against the temperature adjustment device 1004, for example, thereby increasing the efficiency of the flow controller 1000.

[0182]

[0220] FIG. 12 illustrates a socket 106 and a pair of valves, each of which is a thermally controlled flow controller 1000, according to another embodiment. The flow controllers 1000 are disposed immediately upstream and downstream of the socket 106. As shown in FIG. 12, each flow controller 1000 includes a heat sink 1002 and an enclosure 1014. Each enclosure 1014 includes a temperature regulator 1004, a thermally conductive interface 1006, and a flow segment of a fluid line 1008. The fluid line 1008 can be seen exiting the flow controller 1000 and entering the socket 106. The enclosure 1014 may be made of a material having low thermal conductivity and / or low gas permeability. The material can be, for example, a polymer, such as PVC. Each of the enclosures 1014 may include a volume at least twice (e.g., 2-10 times, 2-7 times, 2-5 times, 2-4 times, or 2-3 times) the volume of each temperature adjustment device 1004 contained therein. The enclosures may be configured to prevent moisture from interfering with the function of the flow controller 1000, which may occur when moisture condenses on each temperature adjustment device 1004 and / or the thermally conductive interface 1006. FIG. 12 also shows a second heat sink 1020 onto which the flow controller 1000 may be mounted. The second heat sink 1020 is configured to absorb heat from the heat sink 1002 of the flow controller 1000.

[0183]

[0221] Figure 13 shows a heat sink 1002 and enclosure 1014 of a thermally controlled flow controller 1000 such as that shown in Figure 12. The underside of the enclosure 1014 is seen in Figure 13 and shows a groove 1016 configured to accommodate at least a portion of the fluid line 1008 (not shown) and / or the thermally conductive interface 1006. The groove 1016 can be further configured to hold the thermally conductive interface 1006 (not shown) against a temperature adjustment device 1004 (e.g., one or more (e.g., stacked) Peltier thermoelectric devices (not shown)).

[0184]

[0222] FIG. 14 illustrates an exterior view of a thermally controlled flow controller 1000 according to yet another embodiment. As illustrated, the flow controller 1000 includes a cover 1030, a bottom 1040, and a heat sink 1002. The cover 1030 defines a plurality of indicator openings 1034, 1036, respectively, configured to allow an indicator (e.g., an LED) to be observed from a position outside the cover 1030. The indicators can be configured to indicate whether the flow controller 1000 is on or off and / or whether a flow segment of the fluid line 1008 is in an open (i.e., unfrozen) or closed (i.e., frozen) configuration. Additionally, the cover 1030 can define fastener openings 1032 configured to receive fasteners (e.g., screws) for assembling the flow controller 1000. The bottom 1040 defines a plurality of fluid line openings 1042 configured to allow a fluid line (not shown) to enter the interior of the bottom 1040.

[0185]

[0223] 15 and 16 show the top and bottom surfaces, respectively, of the cover 1030 shown in FIG. 14, shown without the bottom portion 1040. Indicator openings 1034, 1036 and fastener openings 1032 are also shown in FIGS. 15 and 16. FIG. 16 also shows a cavity formed in the bottom surface of the cover 1030 configured to hold a PCB (not shown) of the thermally controlled flow controller 1000. The PCB can include circuitry configured to control one or more temperature regulators 1004 (not shown) and / or one or more indicators (not shown). The cover 1030 can be made from a low thermal conductivity material, such as PVC (e.g., PVC).

[0186]

[0224] FIG. 17 shows the bottom 1040 and heat sink 1002 of the thermally controlled flow controller 1000 shown in FIG. 14, shown without the cover 1030. The bottom 1040 includes a sleeve 1050 and an enclosure 1044 configured to hold the sleeve 1050. The bottom 1040 also defines fastener openings 1048 configured to receive fasteners (e.g., screws) for attaching the cover 1030 and bottom 1040 onto the heat sink 1002. In addition to holding the sleeve 1050, the enclosure 1044 also defines a plurality of fluid line openings 1042 (shown in FIG. 18) that correspond to a plurality of fluid line openings 1052 (shown in FIG. 21) in the sleeve 1050. The fluid line openings 1042 pass completely through the enclosure 1044 within the horizontal plane of the enclosure 1044. FIG. 18 is a perspective view from below of the enclosure 1044. The perspective view angle shows two corresponding sets of fluid line openings 1042 and two cavities 1046 formed in the underside of the enclosure 1044. The cavities 1046 of the enclosure 1044 are each configured to hold a temperature adjustment device 1004 (e.g., each having one or more (e.g., two or more) Peltier thermoelectric devices in a stack; not shown) and associated wiring (not shown).

[0187]

[0225] 19 illustrates a heat sink 1002 optionally defining two cavities 1060 each configured to hold a temperature adjustment device 1004 (e.g., having one or more (e.g., two or more) Peltier thermoelectric devices in a stack). The heat sink 1002 is optionally configured to be coupled to a second heat sink 1020 or a support 100 that can function as a second heat sink.

[0188]

[0226] 20 and 21 show a sleeve 1050 configured to hold two fluid lines 1008 (e.g., an inlet and an outlet; not shown). The sleeve 1050 can be configured to completely surround the flow segments of the fluid lines 1008. Alternatively, the sleeve 1050 can have grooves configured to accommodate the flow segments of the fluid lines 1008. The sleeve 1050 is thus an embodiment of a thermally conductive interface 1006. The sleeve 1050 thus facilitates maintaining the flow segments of the fluid lines 1008 in close proximity to the temperature adjustment device 1004 (not shown). The sleeve 1050 can be made from a material with high thermal conductivity (and low heat capacity), such as a metal (e.g., copper). The side view of FIG. 21 shows the openings 1052 of the fluid lines 1008 defined by the sleeve 1050. As shown, the fluid line openings 1052 pass completely through the sleeve 1050 in the horizontal plane of the sleeve 1050. The fluid line openings 1052 are substantially aligned with corresponding fluid line openings 1042 of the enclosure 1044 (as shown in FIG. 18 ) such that when the sleeve 1050 is placed within the enclosure 1044 (as shown in FIG. 17 ), the fluid line 1008 can pass through both the enclosure 1044 and the sleeve 1050. Furthermore, when the sleeve 1050 is placed within the enclosure 1044 (as shown in FIG. 17 ), the sleeve 1050 is placed in contact with the tops of both temperature adjustment devices 1004 (e.g., one or more (e.g., two or more in a stack) Peltier thermoelectric devices, respectively; not shown).

[0189]

[0227] In certain embodiments, the thermally controlled flow controller 1000 also includes a thermistor (not shown) configured to monitor the temperature of the sleeve and / or temperature regulating device 1004 (or a surface thereof). The monitored temperature can provide feedback indicative of an open or closed state of the flow controller 1000.

[0190]

[0228] In certain embodiments, the thermally controlled flow controller 1000 also includes or is operably coupled to a printed circuit board (PCB; not shown), as discussed above. The PCB can be configured to interface with a thermistor. The PCB may also be configured to regulate the current (e.g., DC) supplied to the temperature adjustment device 1004. Additionally, the PCB may be configured to step down the current supplied to the temperature adjustment device 1004.

[0191]

[0229] The thermally controlled flow controller 1000 described above is robust and substantially eliminates (compared to other fluid valves) dead spaces where bacteria or other debris can accumulate and / or grow. Additionally, the flow controller 1000 reduces microbial contamination associated with other types of valves. Additionally, the flow controller 1000 limits fluid movement within a microfluidic device connected to it (e.g., microfluidic device 110) that would otherwise result from kinking of fluid lines connected to the inlets and outlets of the microfluidic device. To optimize the system to minimize fluid movement within the microfluidic device, the flow controller 1000 should be located as close as possible to the inlets and outlets of the microfluidic device.

[0192]

[0230] Unfortunately, the thermally controlled flow controller 1000 has some limitations. In some configurations, the thermally controlled flow controller 1000 may take a long time to cool to the desired temperature and freeze the fluid lines, thereby preventing precise control of the flow of fluid to and from the microfluidic device. In some cases, it may take about 45 to about 90 seconds to cool to the desired temperature. Also, over an extended period of time, the thermally controlled flow controller 1000 may accumulate moisture and form ice, thereby increasing the time required to thaw the fluid in the fluid lines and reopen the valves. In some cases, it may be difficult to precisely control the temperature because the thermistor is not in close proximity to the fluid lines. Also, the thermally controlled flow controller 1000 includes many components required to connect the fluid lines to the Peltier device.

[0193]

[0231] In other embodiments, the thermally controlled flow controller may include one or more freeze valves that are not subject to these constraints. Examples of these thermally controlled flow controllers are shown in Figures 32-33. As shown in Figure 32 (vertical cross section), the thermally controlled flow controller 2000 includes a base portion 2016 (or substrate) that may be attached to a heat sink (not shown), which may be the second heat sink 1020 or the support 100. In some configurations, the base portion 2016 may be configured as the heat sink itself and need not be attached to a separate heat sink.

[0194]

[0232] The base portion 2016 may be connected to the conduit 2012. The conduit 2012 surrounds some of the other components and captures and conducts heat to the base portion 2016. The base portion 2016 may dissipate the heat if configured as a heat sink, or may conduct the heat to a separate heat sink (e.g., the second heat sink 1020 or the support 100). The base portion 2016 may be connected to the conduit 2012 using any connector, including the screw 2010 shown in FIG. 32. Other connectors may also be used, such as pins, or clamps.

[0195]

[0233] The top of the base portion 2016 and the bottom of the conduits 2012 are each configured to abut or be adjacent to the Peltier device 2004. The Peltier device 2004 may include a two layer Peltier stack as shown. Alternatively, the Peltier device 2004 may include a three, four, or more layer stack. Although two Peltier devices are shown in FIG. 32, additional Peltier devices may be used. The hot side of each Peltier device 2004 is positioned to abut or be adjacent to the conduction 2012 and base portion 2016, respectively, so that heat can be removed from the Peltier device 2004. In some configurations, the base portion 2016 and the conduits 2012 may be configured with one or more indentations 2009 that help stabilize the Peltier device 2004 after the thermally controlled flow controller 2000 is assembled.

[0196]

[0234] The Peltier devices 2004 can be configured to be adjacent and / or abut a thermally conductive interface 2014. In some configurations, the thermally conductive interface 2014 is referred to as a cold head because it interfaces with one or more fluid lines (e.g., inlet and / or outlet fluid lines) and thereby defines a flow segment of the fluid line 1008 to be cooled / frozen. The thermally conductive interface 2014 can be configured to maximize contact with adjacent Peltier devices 2004. The thermally conductive interface 2014 can include one or more (e.g., 2, 3, 4, 5, 6, 7, 8 or more) openings 2011 that can be used to contain fluid lines (not shown) used to input and remove fluids from one or more microfluidic devices, as described herein. A thermally controlled flow controller 2000 having a thermally conductive interface 2014 with two or more (e.g., three, four, six, eight or more) openings 2011 can be used to control flow to one or more (e.g., two, three, four or more) microfluidic devices 110. For example, a thermally controlled flow controller 2000 having a thermally conductive interface 2014 with two openings 2011 can controllably freeze / thaw flow segments of a pair of inlet and outlet fluid lines leading to a single microfluidic device 110 or two each of two inlet (or outlet) fluid lines leading to two separate microfluidic devices 110. Similarly, a thermally controlled flow controller 2000 having a thermally conductive interface 2014 with four openings 2011 can controllably freeze / thaw flow segments of two pairs of inlet and outlet fluid lines leading to each of two microfluidic devices 110, or can controllably freeze / thaw each of four inlet (or outlet) fluid lines leading to four separate microfluidic devices 110.

[0197]

[0235] In certain embodiments, the thermally conductive interface 2014 also includes a central portion that can be coupled to a thermal sensor (e.g., a thermistor). The central portion can include a hole, such as central hole 2013, and the thermal sensor can be located within the hole. The thermal sensor is used to measure the temperature of the fluid line located within the opening 2011.

[0198]

[0236] The various components of the thermally controlled flow controller 2000 may be enclosed within a cover 2022, as shown in FIG. 33 (another vertical cross section with the controller 2000 rotated 90° about the z-axis relative to FIG. 32). The cover 2022 may be made of any material with low thermal conductivity, such as plastic. The thermally controlled flow controller 2000 may also include a barrier material located within any desired cavity of the controller 2000 (e.g., within the cover 2022). In the configuration shown in FIG. 33, the barrier material 2024 may be inserted to surround the thermally conductive interface 2014 and any gaps between the base portion 2016 and the conduit 2012, as well as any other gaps between components of the controller 2000 and / or between the components and the cover 2022. The barrier material 2024 prevents or reduces the ability of moisture to collect and form ice on any internal parts of the controller 2000. In some embodiments, the barrier material 2024 may include a polymer, such as, for example, polyurethane. In some embodiments, the barrier material may include spray foam or foam slices made from expanding foam (eg, urethane foam).

[0199]

[0237] 33, the thermally controlled flow controller 2000 may also include a guide 2020. The guides may be located on either side of the thermally conductive interface 2014 and may be used in feeding fluid lines (not shown) into the thermally conductive interface 2014. The guides 2020 may be made of any material with low thermal conductivity, such as plastic. The guides 2020 may be part of a cover 2022 included in the thermally controlled flow controller 2000.

[0200]

[0238] The conduit 2012 of the thermally controlled flow controller 2000 includes a bridge-like design. The conduit 2012 can conduct heat from the hot side of the two sandwiched Peltier devices to the same heat sink. Thus, its ability to transfer heat from the Peltier devices is improved compared to the thermally controlled flow controller 1000.

[0201]

[0239] Other embodiments of the thermally controlled flow controller are shown in Figures 34-35. In these embodiments, the thermally controlled flow controller 3000 includes a base portion 3016 (or substrate) that can be attached to a heat sink (not shown), such as the second heat sink 1020 or the support 100. In some configurations, the base portion 3016 can be configured as the heat sink itself and does not need to be attached to a separate heat sink.

[0202]

[0240] The base portion 3016 includes a top surface having portions configured to mate with other components of the controller 3000. This top mating portion 3012 of the base portion 3016 may be configured to mate and abut with the bottom of the Peltier device 3004 and / or to mate with the bottom of the cover 3022. The Peltier device 3004 shown in FIG. 34 has three Peltier stacks, but fewer (e.g., two) or more (e.g., four or more) Peltiers may be included. In some cases, as few as one Peltier may be used in the Peltier device 3004. Each layer of the Peltier stack increases the absolute temperature difference between the hot and cold sides of the Peltier device, thereby reducing the total heat flux that the Peltier device stack must sustain. When the thermally controlled flow controller 3000 is activated, the liquid in the fluid lines is cooled (sometimes supercooled) to a temperature sufficient to nucleate ice formation. This result can be achieved using a single layer Peltier device, but may be more easily achieved using a multi-layer structure.

[0203]

[0241] The Peltier device 3004 is configured to have a thermally conductive interface 3014 (or cold head) that rests on the layered structure (e.g., on the top surface of the layered structure). In some configurations, the thermally conductive interface 3014 may be configured to be similar in size to the top Peltier of the multi-layered structure. The thermally conductive interface 3014 may include one or more (e.g., two, three, four, six, eight) openings 3011 that may be used to contain one or more fluid lines (not shown) each used to input fluid to and / or remove fluid from one or more microfluidic devices as described herein. In a preferred embodiment, the thermally conductive interface 3014 includes two openings 3011 configured to contain a pair of inlet and outlet fluid lines leading to a single microfluidic device 110. In other embodiments, the thermally conductive interface 3014 includes two openings 3011 configured to contain two inlet (or outlet) fluid lines each leading to two separate microfluidic devices 110. Similarly, a thermally controlled flow controller 3000 having a thermally conductive interface 3014 with four openings 3011 can controllably freeze / thaw flow segments of two pairs of inlet and outlet fluid lines leading to two microfluidic devices 110 each, or can controllably freeze / thaw four inlet (or outlet) fluid lines leading to four separate microfluidic devices 110, etc.

[0204]

[0242] In certain embodiments, the thermally conductive interface 3014 also includes a central portion that may be coupled with a thermal sensor 3015 (e.g., a thermistor). The central portion may include a hole, such as central hole 3013, and the thermal sensor 3015 may be located within the hole. The thermal sensor 3015 is used to measure the temperature of the fluid line located within the opening 3011.

[0205]

[0243] As shown in FIG. 35 (perspective view of vertical cross section), various components of the thermally controlled flow controller 3000 may be surrounded by a cover 3022. The cover 3022 may be made of a material with low thermal conductivity, such as plastic. The thermally controlled flow controller 3000 may also include a barrier material located within any desired cavity of the controller 3000 (e.g., within the cover 3022). In the configuration shown in FIG. 35, a barrier material (not shown) may be inserted to surround the thermally conductive interface 3014 and any gaps between the Peltier stack 3004 and the cover 3022 and any other gaps between the components of the controller 3000. The barrier material prevents or reduces the ability of moisture to collect and form ice in any internal portion of the controller 3000. In some embodiments, the barrier material may include a polymer, such as polyurethane. In some embodiments, the barrier material may include spray foam or foam slices made of any expanding foam (e.g., urethane foam).

[0206]

[0244] 35, the thermally controlled flow controller 3000 may include a guide 3020. The guide 3020 may be located on either side of the thermally conductive interface 3014 to assist a user in feeding a fluid line (not shown) into the thermally conductive interface 3014. The guide 3020 may be made of a material with low thermal conductivity, such as plastic. The guide 3020 may be part of a cover 3022 included in the thermally controlled flow controller 3000.

[0207]

[0245] The thermally controlled flow controller 2000 and the thermally controlled flow controller 3000 are approximately 9 mm 2 From about 25 mm 2 , or about 10 mm 2 From about 20 mm 2 , or about 13 mm 2 From about 18 mm 2 In a particular embodiment, the thermally conductive interface may have a contact surface (e.g., a surface that contacts the Peltier device) of about 20 mm. 3 From about 60 mm3 , or about 25 mm 3 From about 50 mm 3 , or about 30 mm 3 From about 40 mm 3 The thermally controlled flow controller 2000 may have a volume of about 100 sq. m. This small thermal mass, coupled with the relatively large surface area in contact with the Peltier device, may reduce the time required to cool (or supercool) the fluid line and the fluid flowing therein. In certain embodiments, the thermally controlled flow controller 2000 and the thermally controlled flow controller 3000 may achieve freezing of the fluid in the flow segment of the fluid line in about 35 seconds or less (e.g., about 30 seconds or less, about 27 seconds or less, about 25 seconds or less, about 23 seconds or less, or about 20 seconds, or in a range of about 20 seconds to about 35 seconds, about 20 seconds to about 30 seconds, or about 23 seconds to about 27 seconds). In certain related embodiments, the thermally controlled flow controller 2000 and the thermally controlled flow controller 3000 can achieve thawing of frozen fluid within a flow segment of a fluid line in about 40 seconds or less (e.g., about 35 seconds or less, about 32 seconds or less, about 30 seconds or less, about 28 seconds or less, or about 25 seconds or less, or in a range of about 25 seconds to about 40 seconds, about 25 seconds to about 35 seconds, or about 28 seconds to about 32 seconds).

[0208]

[0246] Thermally controlled flow controller 2000 and thermally controlled flow controller 3000 also contain fluid line openings to the microfluidic devices. Associated guides in these devices allow blind guiding of the fluid line tubes to the coldhead, simplifying their assembly.

[0209]

[0247] Thermally controlled flow controller 2000 and thermally controlled flow controller 3000 also include a barrier material that acts as a moisture barrier, allowing the Peltier device (and thus controller 2000 and controller 3000) to operate over long periods of time without the accumulation of ice that would reduce performance.

[0210]

[0248] In certain embodiments, the support 100 can also include or interface with O2 and CO2 sources configured to maintain culture conditions. In certain embodiments, the support 100 can also include or interface with a humidity monitor / regulator.

[0211]

[0249] The support 100 can have dimensions of about 6-10 inches (or about 150-250 mm) by about 2.5-5 inches (or about 60-120 mm) by about 1-2.5 inches (or about 25-60 mm). Depending on the function incorporated into the support 100, it may be desirable to maintain the dimensions of the support 100 substantially within these exemplary dimensions, although the dimensions may be smaller or larger than the exemplary dimensions. Although the exemplary support 100 has been described as including certain components configured for specific functions, supports according to other embodiments can include different components performing various combinations and subcombinations of the described functions.

[0212]

[0250] In certain embodiments, the light modulation subsystem 634 includes one or more of a digital mirror device (DMD), a liquid crystal display or device (LCD), a liquid crystal on silicon device (LCOS), and a ferroelectric liquid crystal on silicon device (FLCOS). The light modulation subsystem 634 may be, for example, a projector (e.g., a video projector or a digital projector). One example of a suitable light modulation subsystem is the MOSAIC™ system from ANDOR TECHNOLOGIES™. In other embodiments, the light modulation subsystem 634 may include a micro-shutter array system (MSA) that may provide an improved contrast ratio. In yet other embodiments, the light modulation subsystem 634 may include a scanning laser device. In certain embodiments, the light modulation subsystem 634 may be capable of emitting both structured and unstructured light.

[0213]

[0251] In certain embodiments, support 100 and light modulation subsystem 634 are each individually configured to mount to a microscope, such as a standard research-grade optical or fluorescent microscope. For example, support 100 can be configured to mount to a stage of the microscope. Light modulation subsystem 634 can be configured to mount to a port of the microscope.

[0214]

[0252] Thus, in certain embodiments, the system can employ a method for converting an optical microscope into a microscope configured to operate a microfluidic device 110. The method can include mounting a system including a support 100 (e.g., as described herein) and a light modulation subsystem 634 (e.g., as described herein) to a suitable microscope. The support 100 can be mounted to a stage of the optical microscope, and the light modulation subsystem 634 can be mounted to a port of the optical microscope. In certain embodiments, the converted optical microscope can be configured to operate an optically actuated microfluidic device 110 (e.g., a microfluidic device having an OET and / or OEW configuration).

[0215]

[0253] In other embodiments, the support 100 and light modulation subsystem 634 described herein can be integral components of an optical microscope. For example, a microscope having an integrated support 100 and an integrated light modulation subsystem 634 can be configured to operate an optically actuated microfluidic device 110 (e.g., a microfluidic device having an OET and / or OEW configuration).

[0216]

[0254] In certain related embodiments, the system provides a microscope configured to operate the microfluidic device 110. The microscope can include a support 100 configured to hold the microfluidic device 110, a light modulation subsystem 634 configured to receive light from a first light source and emit structured light, and an optical system. The optical system can be configured to (1) receive structured light from the light modulation subsystem 634 and focus the structured light onto at least a first region of the microfluidic device 110 when the device 110 is held by the support 100, and (2) receive reflected and / or emitted light from the microfluidic device 110 and focus at least a portion of such reflected and / or emitted light onto the detector 602. The optical system can also be configured to receive unstructured light from a second light source 622 and focus the unstructured light onto at least a second region of the microfluidic device 110 when the device 110 is held by the support 100. In certain embodiments, the first and second regions of the microfluidic device 110 can be overlapping regions. For example, the first region can be a subset of the second region.

[0217]

[0255] In certain embodiments, the microscope of the system may further include one or more detectors 602. The detectors 602 may include, but are not limited to, a charge-coupled device (CCD), a complementary metal oxide semiconductor (CMOS), a scientific complementary metal oxide semiconductor (SCMOS), a camera (e.g., a digital camera or a film camera), or any combination thereof. When at least two detectors 602 are present, one detector 602 may be, for example, a high frame rate camera, and the other detector 602 may be a high sensitivity camera. The microscope may also include an eyepiece configured for visualization by a user. Additionally, the optical system may be configured to receive reflected and / or emitted light from the microfluidic device 110 and focus at least a portion of the reflected and / or emitted light on the additional detector 602. The optical system of the microscope may also include different tube lenses for the different detectors 602 such that the final magnification of each detector 602 may be different.

[0218]

[0256] In certain embodiments, the light modulation subsystem 634 of the microscope of the present system may include one or more of a digital mirror device (DMD), a liquid crystal display / device (LCD), a liquid crystal on silicon device (LCOS), a ferroelectric liquid crystal on silicon device (FLCOS), and a scanning laser device. Additionally, the DMD, LCD, LCOS, FLCOS, and / or a scanning laser device may be part of a projector (e.g., a video projector or a digital projector). In other embodiments, the light modulation subsystem 634 may include a micro-shutter array system (MSA) that may provide an improved contrast ratio. In certain embodiments, the microscope of the present system may include an embedded or external controller (not shown) for controlling the light modulation subsystem 634. Such a controller may be, for example, an external computer or other computing device.

[0219]

[0257] In certain embodiments, the system 600 / microscope of the present system is configured to use at least two light sources 622, 632. For example, the first light source 632 can be used to generate structured light 650, which is then modulated by a light modulation subsystem 634 to form modulated structured light 652 for optically actuated electrokinesis and / or fluorescence excitation. The second light source 622 can be used to provide background illumination (e.g., using unstructured light 654) for bright-field or dark-field imaging. An example of such a configuration is shown in FIG. 6. The first light source 632 is shown to provide structured light 650 to the light modulation subsystem 634, which provides modulated structured light 652 to the optics of the microscope. The second light source 622 is shown to provide unstructured light 654 to the optics via a beam splitter 624. The modulated structured light 652 from the light modulation subsystem 634 and the unstructured light 654 from the second light source 622 travel together through the optical system and reach the beam splitter 606, where the light 652, 654 is reflected through the objective lens 608 (which may be a lens) to the sample plane 610. The reflected and / or emitted light 662, 664 from the sample plane 610 then travels through the objective lens 608, through the beam splitter 606, to the dichroic filter 604. The light 662, 664 can be the modulated structured light 652 and the unstructured light 654, respectively, reflected from the sample plane 610. Alternatively, the light 662, 664 can originate at or below the sample plane 610. Only a small portion of the light 662, 664 that reaches the dichroic filter 604 passes through the filter 604 and reaches the detector 602. Depending on how the system is being used, beam splitter 606 may be replaced with a dichroic filter (eg, for detecting fluorescent emissions occurring at or below sample plane 610).

[0220]

[0258] As shown in FIG. 6, the second light source 622 emits blue light. The blue light reflected from the sample surface 610 can pass through the dichroic filter 604 and reach the detector 602. In contrast, the structured light coming from the light modulation subsystem 634 is reflected from the sample surface 610 but does not pass through the dichroic filter 604. In this example, the dichroic filter 604 filters out visible light having wavelengths longer than 495 nm. Such filtering of the light from the light modulation subsystem 634 is complete (as shown) only if the light emitted from the light modulation subsystem 634 does not contain wavelengths shorter than 495 nm. In fact, if the light coming from the light modulation subsystem 634 contains wavelengths shorter than 495 nm (e.g., blue wavelengths), some of the light from the light modulation subsystem 634 will pass through the filter 604 and reach the detector 602. In such a scenario, the filter 604 acts to change the balance of the amount of light reaching the detector 602 from the first light source 632 and the second light source 622. This can be beneficial when the first light source 632 is significantly stronger than the second light source 622 .

[0221]

[0259] One alternative to the configuration shown in FIG. 6 that achieves the same goal of varying the balance of the amount of light reaching the detector 602 from the first light source 632 and the second light source 622 is to have the second light source 622 emit red light and have the filter 604 filter out visible light having wavelengths shorter than 650 nm.

[0222]

[0260] In certain embodiments, the microscope (or system) of the system further comprises a first light source 632 and / or a second light source 622 .

[0223]

[0261] In certain embodiments, the first light source 632 can emit a broad spectrum of wavelengths (e.g., "white" light). The first light source 632 can emit at least one wavelength suitable for, for example, excitation of phosphors. The first light source 632 can be sufficiently powerful such that the structured light emitted by the light modulation subsystem 634 can activate light-actuated electrophoresis of the optically actuated microfluidic device 110. In certain embodiments, the first light source 632 can include high intensity discharge arc lamps, such as those containing metal halide, ceramic discharge, sodium, mercury, and / or xenon. In other embodiments, the first light source 632 can include one or more LEDs (e.g., an array of LEDs, such as a 2×2 array of four LEDs or a 3×3 array of nine LEDs). The LEDs can include broad spectrum "white" light LEDs (e.g., UHP-T-LED-White by PRIZMATIX), or various narrow band wavelength LEDs (e.g., emitting wavelengths of about 380 nm, 480 nm, or 560 nm). In yet other embodiments, the first light source 632 may incorporate a laser configured to emit light at a selectable wavelength (eg, for OET and / or fluorescence).

[0224]

[0262] In certain embodiments, the second light source 622 is suitable for bright field illumination. Thus, the second light source 622 can include one or more LEDs (e.g., an array of LEDs, such as a 2×2 array of four LEDs or a 3×3 array of nine LEDs). The LEDs can be configured to emit white (i.e., broad spectrum) light, blue light, red light, etc. In some embodiments, the second light source 622 can emit light having a wavelength of 495 nm or less. For example, the second light source 622 can emit light having a wavelength of substantially 480 nm, substantially 450 nm, or substantially 380 nm. In other embodiments, the second light source 622 can emit light having a wavelength of 650 nm or more. For example, the second light source 622 can emit light having a wavelength of substantially 750 nm. In yet other embodiments, the second light source 622 can emit light having a wavelength of substantially 560 nm.

[0225]

[0263] In certain embodiments, the optics of the microscope of the system includes a dichroic filter 604 that at least partially filters visible light having a wavelength longer than 495 nm. In other embodiments, the optics of the microscope of the system includes a dichroic filter 604 that at least partially filters visible light having a wavelength shorter than 650 nm (or shorter than 620 nm). More generally, the optics can also include a dichroic filter 604 configured to reduce or substantially prevent structured light from the first light source 632 from reaching the detector 602. Such a filter 604 can be located (along the optics) near the detector 602. Alternatively, the optics can include one or more dichroic filters 604 configured to balance the amount of structured light (e.g., visible structured light) from the light modulation subsystem 634 and the amount of unstructured light (e.g., visible unstructured light) from the second light source 622 that reaches the detector 602. Such balancing can be used to ensure that the structured light does not overwhelm the unstructured light at detector 602 (or in the image obtained by detector 602).

[0226]

[0264] In certain embodiments, the microscope optics of the system can include an objective lens 608 configured to focus structured and unstructured light onto the microfluidic device 110, the objective lens being selected from a 100x, 60x, 50x, 20x, 10x, 5x, 4x, or 2x objective lens. These magnifications are listed for purposes of illustration and are not intended to be limiting. The object can have any magnification.

[0227]

[0265] The microscope of the system can include any of the supports 100 described herein. Thus, for example, the support 100 can include an integrated electrical signal generation subsystem 138 configured to at least intermittently set a bias voltage between a pair of electrodes in the microfluidic device 110 when the device 110 is held by the support 100. Alternatively or in addition, the support 100 can include a thermal control subsystem 140 configured to regulate a temperature of the microfluidic device 110 when the device 110 is held by the support 100.

[0228]

[0266] Any of the systems or microscopes described herein may further include a microfluidic device 110. The microfluidic device 110 may be a microfluidic device 110, such as a microfluidic device 110 configured to support dielectrophoresis or a microfluidic device 110 configured to support electrowetting. The microfluidic device 110 may be an optically actuated microfluidic device (e.g., a microfluidic device having an OET and / or OEW configuration).

[0229]

[0267] FIG. 7A illustrates a structured light path 700 of an optical system according to some embodiments of the present system. The structured light path 700 illustrated in FIG. 7A begins with a DMD 702 that includes a glass cover 704 (e.g., a 20 mm glass plate). The DMD 702 may be part of a light modulation subsystem, such as the light modulation subsystem 634 illustrated in FIG. 6. The DMD 702 modifies light from a light source (not shown) to form structured light 708. The structured light 708 is then focused by a tubular lens 706 toward an objective lens 710 (which may be a lens). The objective lens 710 then focuses the structured light 708 onto a cover 712 (e.g., a cover glass). The cover 712 may be a cover for a microfluidic device 110, such as an optically actuated microfluidic device. In the latter embodiment, the structured light may actuate and / or operate the optically actuated microfluidic device 110, as described below.

[0230]

[0268] FIG. 7B illustrates an imaging optical path 750 of an optical system according to some embodiments of the present system. The imaging optical path 750 illustrated in FIG. 7B begins at a sample plane 752, which may coincide with the cover 712 of the microfluidic device 110. The sample plane 752 may be similar to the sample plane 610 illustrated in FIG. 6. Thus, the light 758 in the imaging optical path 750 may be reflected from the sample plane 752. Alternatively, the light 758 may pass through the sample plane 752. From the sample plane 752, the light 758 is focused by an objective lens 754 and an achromatic tube lens 756 toward a camera plane 760. The camera plane 760 may coincide with a detector (not shown), similar to the detector 602 illustrated in FIG. 6. In this manner, the imaging optical path 750 may be used to visualize a sample or a portion thereof disposed at the sample plane 752 (e.g., contained within the microfluidic device 110).

[0231]

[0269] FIG. 22 illustrates a system 600 having an optical system similar to that illustrated in FIG. 6. In the system 600 illustrated in FIG. 22, the second light source 622 and the beam splitter 624 are positioned in the main optical path between the sample plane 610 and the detector 602, rather than off to the side of the main optical path as in FIG. 6. In such an embodiment, the second light source is sized, shaped, and configured so as not to interfere with the reflected and / or emitted light 662, 664 from the sample plane 610. Additionally, the beam splitter 624 may function only as a filter to modify the unstructured light 654 from the second light source 622 without redirecting the unstructured light 654. In other embodiments, the system 600 may not include the beam splitter 624.

[0232]

[0270] In certain embodiments, the second light source 622 comprises a light pipe and / or one or more LEDs (eg, an LED array, such as a 2×2 of a 3×3 array of LEDs).

[0233]

[0271] FIG. 23 shows two LED arrays that may be used as light sources in the system 600 described herein. The first LED array 1102 includes a 2×2 array of four LEDs. The second LED array 1104 includes a 3×3 array of nine LEDs. The square array increases the light intensity per unit area compared to a non-square array. The LEDs in the array may have the same color / wavelength (e.g., ultraviolet, 380 nm, 480 nm, or 560 nm). Alternatively, various subsets of the LEDs in the array may have different colors / wavelengths. Additionally, the LEDs may originally emit a narrowband wavelength (e.g., 450 nm wavelength) but may be coated with a phosphorescent material to emit white light upon excitation at the narrowband wavelength.

[0234]

[0272] FIG. 24 shows a light pipe (or optical integrator) 1112 that may be configured to receive and propagate light from a light source, such as one of the LED arrays 1102, 1104 shown in FIG. 23. The light pipe 1112 (also known as a "non-imaging collecting optic") is configured to propagate light from one end (i.e., input aperture) to the other end (i.e., output aperture) such that the light emitted from the output aperture has a substantially uniform intensity (i.e., the luminous flux passing through a first area of ​​a defined size in the plane of the output aperture is substantially the same as the luminous flux passing through any other area in the plane of the output aperture having the same defined size). The body wall of the light pipe 1112 may be constructed of transparent glass or transparent plastic. Light pipes 1112 are available, for example, from EDMOND OPTICS.

[0235]

[0273] FIG. 25 illustrates a light source 1122 including a plurality of 3×3 LED arrays 1104 coupled to a surface 1124. The surface 1124 may be an LED board. The light source 1122 may be disposed within a system such that it is movable relative to an aperture configured to receive light emitted from the light source 1122. For example, the system may include a light pipe / optical integrator 1112, where an input aperture of the light pipe 1112 may be configured to receive light emitted from one of the plurality of LED arrays 1104 coupled to the surface 1124. Thus, depending on the relative position of the surface 1124 of the light source 1122 and the light pipe / optical integrator 1112, different LED arrays 1104 may be available as light sources (e.g., via the light pipe / optical integrator 1112).

[0236]

[0274] FIG. 26 illustrates a multiple-input light pipe / optical integrator 1132. The multiple-input light pipe 1132 has a plurality of (e.g., three) input apertures, each associated with a light propagation path and a respective light source 1134, 1136, 1138, and one less (e.g., two) dichroic filters 1140, 1142. Each dichroic filter 1140, 1142 is configured to reflect light from a corresponding light source 1136, 1138. The multiple-input light pipe 1132 illustrated in FIG. 26 has first, second, and third light sources 1134, 1136, 1138, any of which may be an array of LEDs (e.g., a 2×2 or 3×3 array of LEDs). The first light source 1134 may be an array of LEDs emitting light at about 380 nm. The second light source 1136 may be an array of LEDs emitting light at about 480 nm. The third light source 1138 may be an array of LEDs emitting light at about 560 nm. Thus, by selectively activating the first, second, and third light sources 1134, 1136, 1138, the wavelength of light emitting from the multiple-input light pipe 1132 can be controlled. The multiple-input light pipe 1132 is configured such that light from any one of the light sources 1134, 1136, 1138, or any combination thereof, incident on a corresponding input aperture, is of substantially uniform intensity when emitted from the output aperture. The body wall of the multiple-input light pipe 1132 may be constructed of clear glass or clear plastic.

[0237]

[0275] In certain embodiments, the microscope of the present system is configured to use a single light source (e.g., a white light LED; not shown) that is received by the light modulation subsystem 634 and transmitted to the optical system. The single light source can be used to provide structured light for light-activated electrokinesis, phosphor excitation, and bright field illumination. In such a configuration, structured illumination can be used to compensate for light vignetting or any other accidental non-uniformity in the illumination. Light vignetting is the gradual falloff of illumination 804 toward the edge of the field of view 802 (e.g., FIG. 8A). The light intensity of the single light source can be measured pixel by pixel, and that information is used to generate an inverted light vignetting function 814 (e.g., FIG. 8B). The inverted light vignetting function 814 can then be used to adjust the light output from the light modulation subsystem, thereby generating a uniformly illuminated field 824 in the field of view 802 (e.g., FIG. 8C).

[0238]

[0276] The system further provides a method of using light to manipulate a micro-object in an optically actuated microfluidic device 110. The method includes placing the optically actuated microfluidic device 110 on the support 100 of any one of the systems or microscopes described herein, placing a micro-object on or in the optically actuated microfluidic device 110, focusing structured light from the light modulation subsystem 634 onto a first region on a surface of the optically actuated microfluidic device 110, and moving the focused structured light to a second region on the surface of the optically actuated microfluidic device 110. If the micro-object is placed in proximity to the first region, moving the focused light can induce a directed movement of the micro-object. The focused structured light can be used, for example, to form an optical cage around the micro-object. Alternatively, the focused structured light can be used to at least partially contact a fluid droplet containing the micro-object.

[0239]

[0277] In another embodiment of the method of using light to manipulate micro-objects in an optically actuated microfluidic device 110, a light pattern is spatially fixed and the optically actuated microfluidic device 110 is moved relative to the light pattern. For example, the optically actuated microfluidic device 110 can be moved using a motorized or mechanical microscope stage that can be automatically controlled by a computer, manually controlled by a user, or semi-automatically controlled by a user with the aid of a computer. In another similar embodiment, the spatially fixed light pattern can form a geometric pattern such as a "cage" or box configured to move a micro-object (e.g., a droplet optionally containing biological cells or a micro-object of interest) on a steerable stage.

[0240]

[0278] In other embodiments, a system for operating a microfluidic device may be configured with access for introducing a fluid sample directly (e.g., manually or robotically) into the microfluidic device. In the above embodiments, the fluid sample is introduced (and removed) through the first fluid input / output line 112 and the second fluid input / output line 118. The internal volume of the microfluidic device may be limited, for example, to less than 50 microliters (e.g., less than 40 microliters, less than 30 microliters, less than 25 microliters, less than 20 microliters, less than 15 microliters, or less than 10 microliters, or about 10 to about 50 microliters, about 10 to about 40 microliters, about 10 to about 30 microliters, about 5 to about 25 microliters, about 5 to about 20 microliters, about 5 to about 15 microliters, about 2 to about 10 microliters, or about 2 to about 5 microliters). In some cases, only about half of the fluid volume (e.g., about 25 microliters or less, about 20 microliters or less, about 15 microliters or less, about 10 microliters or less, or about 2 to about 10 microliters, or about 1 to about 5 microliters) typically flows through the microfluidic device because the other half of the fluid remains relatively fixed by the microfluidic device for analysis. Because only a limited amount of fluid can be inserted into the microfluidic device at any given time, the fluid sample flowing into (and out of) the microfluidic device through the first or second fluid input / output lines ideally forms relatively separate fluid packets. However, the length of the fluid lines between the pumps and the microfluidic device of the first and second fluid input / output lines can be long (e.g., about 50 cm, about 75 cm, about 100 cm, about 125 cm, about 150 cm or more) and can have an internal volume significantly larger than about 5 microliters. As a result, the initially small volume of fluid sample may become diluted or dispersed before traveling through the fluid lines and being introduced into the microfluidic device.Furthermore, when a sample is dispersed, micro-objects (eg, cells or beads) within the sample may become unevenly distributed within the fluid sample, leading to uneven loading of the micro-objects between channels in a microfluidic device.

[0241]

[0279] The exemplary embodiments shown in Figures 27-31 allow the sample to be introduced directly into the microfluidic device, thus reducing or preventing dispersion of the fluid sample and associated non-uniform distribution of micro-objects in the fluid sample. In these embodiments, the microfluidic device is held by a socket that is part of the support. The socket includes a lid that can be separated into two (or more) parts. One of these parts can be separated from the other so that it no longer covers (or is in contact with) the microfluidic device, allowing the fluid sample to be introduced directly into the microfluidic device, without the need for the sample to flow through fluid lines. At the same time, the other part of the lid remains in place, holding the microfluidic device in the socket.

[0242]

[0280] 27, a system for operating a microfluidic device of these embodiments includes a support 1200 substantially similar to support 100, a socket 1206 substantially similar to socket 106, a first fluid input / output line 1212 substantially similar to first fluid input / output line 112, and a second fluid input / output line 1218 substantially similar to second fluid input / output line 118. In certain embodiments, socket 1206 includes a surface 1203 configured to support a microfluidic device 1210 and a lid 1204 configured to secure the microfluidic device 1210 within socket 1206. Surface 1203 of socket 1206 may include a substantially flat region that interfaces with a corresponding substantially flat bottom surface of microfluidic device 1210. The resulting interface may operably couple microfluidic device 1210 with socket 1206, for example, thereby establishing a functional interconnection, such as an electrical connection. Alternatively or additionally, the socket 1206 may include features (e.g., pins, recesses) extending from or to the surface 1203. These features may interface with the microfluidic device 1210 and control the position of the microfluidic device 1210 within the socket 1206 and / or operably couple the microfluidic device 1210 to the socket 1206, for example, thereby establishing a functional interconnection, such as an electrical connection. The lid 1204 may interface with a top surface of the microfluidic device 1210. The resulting interface may operably couple the microfluidic device 12 to one or both of the first and second fluid input / output lines 1212, 1218. In certain embodiments, the lid 1204 may be connected to the surface 1203, for example, by a hinge. In certain related embodiments, the lid 1204 may include a latch (or other fastening mechanism, such as, for example, a screw, pin, or clamp) configured to hold the lid 1204 in a closed position. Thus, the latch can facilitate the formation of an interface between the lid 1204 and the top surface of the microfluidic device 1210.

[0243]

[0281] One or both of the first and second input / output lines 1212, 1218 may be connected to a pump at one end and to a fluid port (not shown) included in the lid 1204 at the other end. The fluid port may interface with both an end of the fluid line and an inlet / outlet of the microfluidic device 1210, thereby forming fluid communication between the fluid line and the inlet / outlet. Alternatively or additionally, one of the first or second fluid input / output lines 1212, 1218 may be connected to a fluid port at one end and to a container, such as a waste container or a container for holding a sample (e.g., a sample to be imported into the microfluidic device 1210 or a sample exported from the microfluidic device 1210) at the other end. The fluid port optionally includes a seal, compression fitting, or the like to ensure a leak-tight connection between its respective fluid line and the microfluidic device.

[0244]

[0282] In some embodiments, the lid 1204 includes two parts, a first part 1204A and a second part 1204B. As shown in FIG. 27, the second part 1204B of the lid can be separated from the first part 1204A. This allows the second part 1204B to move from the position shown in FIG. 27 (closed position) to an open position that allows access to the inlets / outlets of the microfluidic device (e.g., inlets / outlets located on the top surface of the microfluidic device 1210). An example of an open position of the second part 1204B of the lid is shown in FIG. 28 and FIG. 29. The second part 1204B can be moved to any number of open positions away from the microfluidic device 1210, not just the positions shown in FIG. 28 and FIG. 29. When the second part 1204B is in the open position, the first part 1204A of the lid remains in place and holds the microfluidic device 1210 on the surface of the support 1200 as the second part 1204B moves. While the second part 1204B of the lid is in the open position, the first fluid input / output line 1212 may be connected to a fluid port of the first part of the lid 1204A and may therefore remain in place (e.g., may maintain fluid communication between the first fluid input / output line 1212 and a corresponding inlet / outlet of the microfluidic device 1210). The second fluid input / output line 1218, which may be connected to a fluid port of the second part 1204B of the lid, moves with the second part 1204B, thereby isolating fluid communication between the second fluid input / output line 1218 and the corresponding inlet / outlet of the microfluidic device 1210.

[0245]

[0283] In certain embodiments, when the second portion 1204B of the lid is in the open position, an insert may be placed in the location that the second portion 1204B of the lid occupied in its closed position. In some configurations, the insert may be shaped substantially similarly to the second portion 1204B of the lid. In other configurations, it is contemplated that the insert may be shaped differently. The insert may serve multiple functions. The first function is to prevent possible contamination resulting from an uncovered inlet / outlet of the microfluidic device 1210 that would otherwise be covered when the second portion 1204B of the lid is in the closed position. In certain embodiments, the insert includes a fluid inlet (such as a well) by which a fluid sample can be directly introduced into the microfluidic device 1210. This fluid inlet of the insert is positioned to interface with an inlet / outlet of the microfluidic device 1210 that interfaces with a fluid port 1222 of the second fluid input / output line 1218 when the second portion 1204B of the lid is in the closed position. One example of an insert is insert 1207 shown in FIG. 28, which includes a custom fluid well for inserting a fluid sample. Another example of an insert is insert 1209 shown in FIG. 29, which includes a non-custom fluid well. As can be seen, the fluid well of insert 1207 is larger than the fluid well of insert 1209 and includes a funnel-like design. Regardless of its exact shape, the fluid well can be configured to hold a fluid sample of about 50 microliters or less (e.g., about 45 microliters, about 40 microliters, about 35 microliters, about 30 microliters, about 25 microliters, about 20 microliters, about 15 microliters, about 10 microliters, about 5 microliters, or any range formed by two of the foregoing endpoints, e.g., about 5 microliters to about 25 microliters).

[0246]

[0284] In some configurations, the second portion of the lid 1204B can be moved using the process illustrated in FIGS. 30A-30C. In these embodiments, the second portion of the lid 1204B includes a latch 1205 and hinge 1225, which can be configured as shown in FIG. 30A. The latch 1205 is configured to releasably hold the second portion of the lid 1204B in a closed position. The latch 1205 can be pulled up as shown by the arrow in FIG. 30B. This action releases the second portion of the lid 1204B from its closed position over the microfluidic device 1210. Of course, the latch 1205 and its actuation can have any number of other configurations and a securing mechanism other than a latch can replace the latch 1205, such as a clamp, a friction lock, a screw, or a magnet. Once the second portion of the lid 1204B is released from its position covering the microfluidic device 1210, it can be moved to any desired position, including the position shown in FIG. 30C, by rotating the second portion of the lid 1204B about the hinge 1225. The position shown in FIG. 30C is rotated about 180° from the closed position, however, any degree of rotation that exposes a portion of the top surface of the microfluidic device 1210 and allows access to the second fluid inlet / outlet of the microfluidic device 1210 is sufficient. For example, the second portion of the lid 1204B can be rotated at least about 60°, about 75°, about 90°, about 105°, about 120°, about 135°, about 150°, or more to achieve an open position of the section portion 1204B. As shown in FIG. 30C, an insert, such as insert 1209, can be placed where the second portion of the lid 1204B was previously located.

[0247]

[0285] An insert, including insert 1207 or insert 1209, may be configured to operably couple with socket 1206 and / or microfluidic device 1210 such that flow of fluid media to the second fluid inlet / outlet of the microfluidic device can be reliably achieved. The insert may be configured to interface with, for example, the first portion of the lid 1204B. The insert typically includes features useful in (i) securing and / or removing the insert from socket 1206 and / or microfluidic device 1210, and / or (ii) aligning the insert with the microfluidic device 1210. One such feature is a retention feature 1215 (shown in FIG. 31B ) that helps to hold the second portion of the lid 1204B in place. In some configurations, the retention feature includes one or more magnets oriented to form an attractive interaction with one or more corresponding magnets in matching positions on the surface of the first portion of the lid 1204A and / or microfluidic device 1210. A single magnet that interfaces with a corresponding magnet (not shown) on the first portion 1204A of the lid is shown in FIG. 31B. Another possible feature of the insert is an alignment feature that serves to align the insert in the correct position on the microfluidic device so that the second fluid input / output line of the microfluidic device 1210 is operatively connected with the fluid inlet of the insert. This alignment feature may include, for example, one or more pins 1217 that fit into alignment holes 1213 on the underside and, optionally, extend into the insert. However, instead of pins, alignment features may be used for this alignment function. As shown in FIG. 31B (including corresponding retention features 1216 (e.g., magnets) and alignment features 1214 (e.g., holes into which pins 1217 fit)) and elsewhere herein, similar retention and / or alignment features may facilitate proper positioning and / or alignment of the second portion 1204B of the lid with the first portion 1204A of the lid.

[0248]

[0286] With an insert, such as insert 1207 shown in FIG. 31A in place, a small volume of fluid sample can be introduced into the microfluidic device 1210 for analysis. In some configurations, this small volume of fluid sample can be introduced manually, for example, using a pipette / micropipette 1220, as shown in FIG. 31C. In alternative embodiments, the small volume of fluid sample can be introduced robotically, for example, using a pipette / micropipette 1220. The small volume of fluid sample can be introduced into the microfluidic device 1210 using a fluid inlet, such as well 1219 shown in FIG. 31B. In these configurations, the first input / output fluid line 1212 is interfaced with a fluid port in the lid first portion 1204A and remains fluidically connected to the inlet / outlet of the microfluidic device 1210. The first input / output fluid line 1212 also remains connected to the pump, so that the first input / output fluid line 1212 can ensure that the pump can draw (using suction or other force) at least a portion of the fluid sample from the insert's fluid inlet (e.g., well 1219) into and through the microfluidic device 1210. This operation maintains a desired rate of fluid flow within the microfluidic device, allowing all or a portion of the fluid sample to be analyzed by the microfluidic device 1210. The suction (or other force) can be sufficient to draw a preselected amount of sample fluid into the microfluidic device 1210. The preselected amount can be, for example, equal to the flow rate in the microfluidic device + / - about 100%. The flow rate is the amount of the microfluidic device that is subject to flow as the medium flows through the microfluidic device (i.e., the sweep area as described in U.S. Pat. No. 10,010,882). In certain embodiments, the preselected amount can be from about 1 microliter to about 25 microliters (e.g., from about 1.5 microliters to about 20 microliters, from about 2 microliters to about 15 microliters, from about 2.5 microliters to about 10 microliters, from about 3 microliters to about 7 microliters, or any range defined by two of the aforementioned endpoints) of fluid sample after which aspiration is stopped.

[0249]

[0287] In some embodiments, it may be useful to know if the second portion of the split lid is in place, if the insert 1207 is in place, or if neither the second portion of the split lid nor the insert is in place on the microfluidic device. In these embodiments, the system may be modified with a sensor to detect if the second portion of the split lid or the insert is present above the microfluidic device. Some embodiments of this sensor are shown in Figures 36-43.

[0250]

[0288] As shown in FIG. 36, a system for operating the microfluidic device 1210 may include a support 1200, a split lid 1204 having a first portion 1204A and a second portion 1204B, a socket 1206 including a base portion 1201, and a sensor 1300. The second portion 1204B of the split lid may include a latch 1205 as shown in FIG. 36 and may be movable to an open position as shown in FIG. 37. The lid 1204 may further include attachment features 2015 (e.g., magnets) and / or alignment features 2017 (e.g., pins) that facilitate attachment and alignment of the second portion 1204B of the lid with the first portion 1204A of the lid. The microfluidic device 1210 and the base portion 1201 of the socket 1206 may be located on the support 1200, which may include a substrate 1390, such as a printed circuit board (PCB).

[0251]

[0289] An exemplary sensor 1300 is shown in FIG. The sensor 1300 may include a sensor cover 1302, a magnet assembly 1304, an extender 1310, a housing 1312, and a connector 1314. The sensor cover 1302 operates to protect and insulate some of the components of the sensor 1300. The magnet assembly 1304 may include one, two, or more magnets 1308 located within a first housing 1306. The magnets 1308 may be used in the process of detecting the presence of a lid or insert. The first housing 1306 insulates and protects the magnets 1308. In certain embodiments, the first housing 1306 includes a bottom that includes one or more openings for contacting each magnet 1308 with the top surface of the extender 1310. In certain embodiments, the first housing can interface with the second housing 1312 via a fastening mechanism, such as an opening in the first housing 1306 made with a post, bolt, clamp, or glue, or the like, and any combination thereof, in the second housing 1310. A first end of each extender 1310 is configured to be attached to the second housing 1312, such as via an opening that fits over a post (as shown in FIG. 38 ) or bolt, or the like, in the second housing 1312. A second, opposite end of each extender is configured to controllably extend downwardly through an opening in the bottom of the housing 1312. The various components of the sensor 1300 can be attached to one another using connectors 1314 (e.g., screws as shown).

[0252]

[0290] The various components of the sensor are shown assembled in Figure 39. The sensor cover 1302 is transparent so that the remaining components can be seen. Figure 39 also shows how the sensor 1300 can be mounted in the socket 1206, for example in a perimeter or corner location.

[0253]

[0291] A side view of the sensor 1300 mounted on the base portion 1201 of the socket 1206 and in operation can be seen in Fig. 40. This view shows the split cover 1204 mounted on the base portion 1201 of the socket 1206 resting on a substrate 1390 included in the support 1200. As shown in Fig. 40, the sensor 1300 is located at the interface between the edge of the split lid 1204 and the base portion 1201. The second portion 1204B of the split lid 1204 is provided with an actuator 1355 (e.g. a screw or pin, etc.) that contacts one of the extenders 1310 and pushes that extender 1310 downwards when the second portion 1204B is in the closed position.

[0254]

[0292] As shown at the bottom of FIG. 40, the end of the extender 1310 is pressed downwards, blocking the optical switch by blocking light from a first element (e.g., an LED) of the optical switch from reaching a second element (e.g., a phototransistor) of the optical switch. The optical switch 1365 is shown in FIG. 41 without the rest of the system other than the substrate 1390 on which the optical switch 1365 is located. Two optical switches 1365 are shown in FIG. 41 corresponding to the two extenders 1310 shown in FIG. 38. However, depending on the desired functionality of the sensor, the sensor can include a single optical switch 1365 and a single extender 1310 or three or more optical switches 1365 and corresponding extenders 1310. The optical switches can be connected to an electrical circuit that is part of the electrical signal generation subsystem of the support 1200. Each optical switch 1365 is located under a single extender 1310. As each extender 1310 is pressed downward, it blocks the optical signal of optical switch 1365 , signaling the presence of actuator 1355 .

[0255]

[0293] As shown in Figures 42 and 43, the split cover 1204 and the inserts 1207, 1209 can each be configured to include an actuator at a different location. The split cover 1204 can be configured with an actuator at a first location 1361 located above the first extender 1310. The insert 1207 can be configured with an actuator at a second location 1362 located above the second extender 1310. Of course, the actual location of the actuator can be changed, as long as one knows which location is associated with the split cover 1204 and which location is associated with the insert 1207. A similar configuration of optical switches and extenders can also be used to determine whether the insert 1207 or the insert 1209 is located on the microfluidic device.

[0256]

[0294] The presence of the sensor 1300 allows the system to detect the presence of the split cover, the movable part of the insert, or even when neither is present. When the second part 1204B of the split cover 1204 is located on the microfluidic device 1210, the actuator in the movable part 1204B can be located, for example, in a first position 1361, and can push the extender 1310 of the underlying optical sensor 1365 downward, blocking the signal between the associated optical switch. When the inserts 1207, 1209 are located on the microfluidic device 1210, the actuator in the inserts can be located, for example, in a second position 1362, and can push the extender 1310 of the underlying optical sensor 1365 downward, blocking the signal between the associated optical switch. When neither the second part 1204B of the split cover nor the insert is present, neither extender 1310 is pushed downward, and the signal between any optical switch is not blocked. The magnet 1308 holds the extender 1310 in an up position (i.e., a position that does not block the associated light switch. Without the magnet, the extender would be in a down position, blocking the light switch. The magnetic force of the magnet is strong enough to hold the extender in this up position when the split lid is open and no insert is present, but weak enough that the actuators on the lid and insert can be overcome. Thus, in this manner, the extender 1310 can be made from any magnetic material that will work with the magnet 1308.

[0257]

[0295] Other types of detection and interruption mechanisms can be used to indicate the presence of the split cover second portion or insert, including magnetic proximity switches, mechanical switches, or conductive contact switches.

[0258]

[0296] These embodiments allow small volumes of fluid samples to be introduced directly into the microfluidic device 1210 without dilution or dispersion. In these embodiments, samples of typically small volumes (e.g., 200 microliters, 150 microliters, 100 microliters, 50 microliters or less) containing small numbers of precious cells (e.g., 200,000 or less) may be introduced. Such fluid samples typically cannot be analyzed and / or recovered using conventional techniques, such as fluorescence activated cell sorters or microfluidic chips that use flow alone to sort cells, without significant loss of material. EXAMPLES

[0259] Working Example

[0297] Example 1: Sample import into a microfluidic chip using a split lid

[0298] Plasma cells were isolated from mice and loaded into OptoSelect™ chips (Berkeley Lights, Inc.) using a Beacon® system (Berkeley Lights, Inc.). To test the effect of well import on cell density and distribution within the channels of the microfluidic chip, plasma cells were loaded into OptoSelect™ 11k and 14k chips using (i) a small volume import method with a Beacon® system with a standard nested lid, (ii) a small volume import method with a Beacon® system with a split-lid nest, or (iii) a well import method with a Beacon® system with a split-lid nest in an open configuration and an insert with a well fluidically connected to the inlet / outlet of the microfluidic chip. The small volume import method involved drawing individual approximately 5 microliter cell samples into the microfluidic chip. The cell samples were followed by a 7.5 microliter air bubble in the fluid line leading to the inlet of the microfluidic device to limit dilution and dispersion of the cells in the sample. In contrast, the well import method involved manually pipetting approximately 3.5 microliters of cell sample into the well of an insert in a split lit (open configuration) and using negative pressure to draw the cell sample into the microfluidic chip. After loading, the fluid flow was stopped and the cells were counted in each channel of the microfluidic chip to determine the import density and coefficient of variation (CV) under each condition.

[0260]

[0299] As shown in Figure 44, for the well import method, the average import density was higher for both the OptoSelect™ 11k and 14 chips (4.8x10^6 and 4.5x10^6, respectively) compared to the low-volume import method (average import density was 2.8x10^6 and 2.1x10^6, respectively, for the Beacon® system with the split lid nest in the closed position, and 2.7x10^6 and 2.4x10^6, respectively, for the Beacon® system with the standard lid nest). In addition, the average CV for both the OptoSelect™ 11k and 14 chips was significantly lower for the well import (8% and 10%, respectively) compared to the low-volume import method (average CV was 26% and 30%, respectively, for the Beacon® system with the split lid nest in the closed position, and 26% and 27%, respectively, for the Beacon® system with the standard lid nest).

[0261]

[0300] Thus, the well import method with the split-lid nest provided a surprising improvement in cell loading. Results similar to those shown in Figure 44 are expected for any of the system / microscope embodiments disclosed herein with a split-lid nest.

[0262]

[0301] While particular embodiments of the disclosed system have been shown and described herein, it will be understood by those skilled in the art that they are not intended to limit the invention, and that it will be apparent to those skilled in the art that various changes and modifications can be made (e.g., dimensions of various parts) without departing from the scope of the disclosed invention, which is defined solely by the following claims and their equivalents. Accordingly, the specification and drawings are to be regarded in an illustrative rather than a restrictive sense.

Claims

1. A system for operating a microfluidic device having a microfluidic channel configured for flow of a fluid medium containing a biological micro-object, comprising: a first surface configured to operably couple to a microfluidic device; a lid configured to hold the microfluidic device on the first surface, a first lid portion having a first fluid port operatively coupled to a first fluid inlet / outlet of the microfluidic device and configured to allow the fluid medium to flow into and / or out of the first fluid inlet / outlet of the microfluidic device; a second lid portion having a second fluid port operatively coupled to a second fluid inlet / outlet of the microfluidic device and configured to allow the fluid medium to flow into and / or out of the second fluid inlet / outlet of the microfluidic device; Equipped with the second lid portion is separable from the first lid portion and movable between a closed position in which the second fluid port of the second lid portion is operably coupled to the second fluid inlet / outlet of the microfluidic device and an open position in which a portion of the microfluidic device comprising the second fluid inlet / outlet is exposed. The lid and an insert configured to operatively couple to the second fluid inlet / outlet of the microfluidic device when the second lid portion is in the open position and to allow the fluid medium to flow into the second fluid inlet / outlet of the microfluidic device; A system comprising:

2. The system of claim 1 , wherein the first lid portion holds the microfluidic device on the first surface when the second lid portion is in the open position.

3. 2. The system of claim 1, wherein when the second lid portion is in the open position, the first fluid port of the first lid portion remains operatively coupled to the first fluid inlet / outlet of the microfluidic device.

4. The system of claim 3 , wherein the first fluid port of the first lid portion is connected to a pump configured to remove fluid from the microfluidic device.

5. the first lid portion further comprising a first fluid line connected to the first fluid port; the second lid portion further comprising a second fluid line connected to the second fluid port. The system of claim 3.

6. The system of claim 1 , wherein the lid further comprises a hinge configured to move the second lid portion between the open and closed positions.

7. The system of claim 1 , wherein the lid further comprises a latch configured to releasably retain the second lid portion in the closed position.

8. The system of claim 1 , wherein the insert is configured to contact the first lid portion.

9. The system of claim 1 , wherein the insert comprises a fluid well configured to be in fluid communication with the second fluid inlet / outlet of the microfluidic device.

10. The system of claim 9 , wherein the fluid well is configured to hold a fluid sample of about 25 microliters or less.

11. the first surface comprises a support; the support comprises a socket configured to receive and interface with the microfluidic device; The system of claim 1 .

12. 12. The system of claim 11, further comprising an electrical signal generating subsystem configured to apply a bias voltage across a pair of electrodes of the microfluidic device when the microfluidic device is operably coupled to the support.

13. The system of claim 11 , further comprising a thermal control subsystem configured to regulate a temperature of the microfluidic device when the microfluidic device is operably coupled to the support.

14. The system of claim 11 , further comprising a light modulation subsystem configured to emit structured light onto the microfluidic device when the microfluidic device is operably coupled to the support.

15. The system of claim 14 , wherein the first surface, the support, and / or the light modulation subsystem are configured to be attached to an optical microscope.

16. The system of claim 14 , wherein the first surface, the support, and / or the light modulation subsystem are integral components of an optical microscope.

17. The system of claim 5 , further comprising at least one flow controller operatively coupled to one or both of the first fluid line and the second fluid line.

18. 20. The system of claim 17, wherein the at least one flow controller comprises a first thermally controlled flow controller operably coupled to the first fluid line and / or the second fluid line to selectively flow fluid to the at least one flow controller.

19. 1. A microscope configured to operate a microfluidic device, comprising: A support according to claim 11; a light modulation subsystem configured to emit structured light; Optical system and Equipped with When the microfluidic device is held by and operably coupled to the support, the optical system (1) focusing structured light emitted by the light modulation subsystem onto at least a first region of the microfluidic device; (2) focusing unstructured light emitted by an unstructured light source onto at least a second region of the microfluidic device; (3) capturing reflected and / or emitted light from the microfluidic device and directing the captured light to a detector; A microscope constructed as follows.

20. A method for analyzing a fluid sample containing biological micro-objects, comprising: connecting a microfluidic device having a microfluidic channel configured for flow of the fluid sample to a system for operating the microfluidic device, The system further comprises: a first surface configured to operably couple to a microfluidic device; a lid configured to hold the microfluidic device on the first surface, a first lid portion having a first fluid port operatively coupled to a first fluid inlet / outlet of the microfluidic device and configured to allow the fluid sample to flow into and / or out of the first fluid inlet / outlet of the microfluidic device; a second lid portion having a second fluid port operatively coupled to a second fluid inlet / outlet of the microfluidic device and configured to allow the fluid sample to flow into and / or out of the second fluid inlet / outlet of the microfluidic device; Equipped with the second lid portion is separable from the first lid portion and movable between a closed position in which the second fluid port of the second lid portion is operatively coupled to the second fluid inlet / outlet of the microfluidic device and an open position in which a portion of the microfluidic device comprising the second fluid inlet / outlet is exposed. The lid and Equipped with To connect and moving the second lid portion from the closed position to the open position, thereby exposing the second fluid inlet / outlet of the microfluidic device; placing an insert in a position previously occupied by the second lid portion in the closed position, the insert comprising a fluid well configured to be in fluid communication with the second fluid inlet / outlet of the microfluidic device; introducing the fluid sample into the fluid well of the insert to fluidically connect the fluid sample to the second fluid inlet / outlet of the microfluidic device; applying suction to a first fluid line connected to the first fluid port, thereby drawing at least a portion of the fluid sample into the microfluidic device; processing the biological micro-objects contained in at least a portion of the fluid sample drawn into the microfluidic device; A method comprising:

21. The method of claim 20, wherein the system comprises a microscope according to claim 19.

22. 21. The method of claim 20, wherein suction is applied sufficient to draw a volume of the fluid sample into the microfluidic device of about 2 microliters to about 10 microliters.

23. the microfluidic device comprises a flow region having the microfluidic channel; 21. The method of claim 20, wherein the microfluidic channel is provided with a density of the introduced biological micro-objects of at least about 4x10^6.

24. 24. The method of claim 23, wherein the coefficient of variation (CV) of the density of the introduced biological micro-object in the microfluidic channel is less than about 20%.

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