Systems for operating microfluidic devices

Abstract

A system for operating a microfluidic device is described. The system includes: a first surface configured to engage and operably couple with the microfluidic device; and a cover configured to retain the microfluidic device on the first surface. The cover includes: a first portion having a first fluid port, the first fluid port configured to operably couple with a first fluid inlet / outlet of the microfluidic device and allow fluid media to flow into and / or out of the first fluid inlet / outlet; and a second portion having a second fluid port, the second fluid port configured to operably couple with a second fluid inlet / outlet of the microfluidic device and allow fluid media to flow into and / or out of the second fluid inlet / outlet. The second portion of the cover is separable from the first portion and is movable between a closed position and an open position.

Description

Technical Field

[0001] The present application generally relates to systems for using microfluidic devices. In particular, the present application describes systems for operating microfluidic devices. Background Art

[0002] As the field of microfluidics continues to advance, microfluidic devices have become a convenient platform for handling and manipulating micro-targets such as biological cells. Microfluidic devices provide some desirable functions, including the ability to select and manipulate individual micro-targets. Such microfluidic devices require various inputs and outputs (e.g., fluids, pressures, vacuums, heat, cooling, light, etc.) to function. Systems for operating microfluidic devices facilitate these inputs and outputs. Summary of the invention

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

[0004] In other exemplary embodiments, a system for operating a microfluidic device is provided, the system comprising: a support configured to hold the microfluidic device and be operably coupled to the microfluidic device; a first fluid line and a second fluid line, when the microfluidic device is held by the support and is operably coupled to the support, the first fluid line having a distal end configured to be fluidically coupled to an inlet port of the microfluidic device, and the second fluid line having a proximal end configured to be fluidically coupled to an outlet port of the microfluidic device; at least one flow controller operably coupled to one or both of the first fluid line and the second fluid line, the at least one flow controller comprising a first thermally controlled flow controller operably coupled to a flow segment of one or both of the first fluid line and the second fluid line to selectively allow fluid to flow therethrough; and a light modulation subsystem configured to emit structured light onto the microfluidic device when the microfluidic device is held by the support and is operably coupled to the support.

[0005] In yet another exemplary embodiment, a method for analyzing a fluid sample is provided, the method comprising: connecting a microfluidic device to a system for operating the microfluidic device, wherein the system comprises: a first surface configured to engage with and be operably coupled to the microfluidic device; and a cover configured to hold the microfluidic device on the first surface, the cover comprising: a first cover portion having a first fluid port, the first fluid port being configured to be operably coupled to a first fluid inlet / outlet of the microfluidic device and allowing a fluid medium to flow into and / or out of the first fluid inlet / outlet of the microfluidic device; and a second cover portion having a second fluid port, the second fluid port being configured to be operably coupled to a second fluid inlet / outlet of the microfluidic device and allowing a fluid medium to flow into and / or out of the first fluid inlet / outlet of the microfluidic device. A second fluid inlet / outlet of a microfluidic device is provided, wherein the second cover portion is separable from the first cover portion and is movable between a closed position and an open position, in which the second fluid port of the second cover portion is operably coupled to the second fluid inlet / outlet of the microfluidic device, and in the open position, the portion of the microfluidic device containing the second fluid inlet / outlet is exposed; moving the second cover portion from the closed position to the open position, thereby exposing the second fluid inlet / outlet of the microfluidic device; providing a fluid sample in fluid communication with the second fluid inlet / outlet of the microfluidic device; applying suction to the first fluid line, thereby pulling at least a portion of the fluid sample into the microfluidic device; and processing at least a portion of the fluid sample pulled into the microfluidic device.

[0006] A partial list of examples follows:

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

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

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

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

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

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

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

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

[0015] Embodiment 9. The system according to any one of Embodiments 1 to 8 further includes an insert, which is configured to be operably coupled to a second fluid inlet / outlet of the microfluidic device and allow fluid medium to flow into the second fluid inlet / outlet of the microfluidic device when the second cover portion is in an open position.

[0016] Embodiment 10. The system of Embodiment 9, wherein the insert is configured to engage with the first cover portion.

[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] Example 12. A system according to Example 11, wherein the fluid trap 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 of fluid samples formed by two of these endpoints.

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

[0020] Embodiment 14. The system of any one of Embodiments 1 to 13, wherein the first surface comprises a support (or "restoration member").

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

[0022] Embodiment 16. The system according to any one of Embodiments 1 to 15 further includes an electrical signal generating subsystem, which is configured to apply a bias voltage to an electrode pair in the microfluidic device when the microfluidic device is operably coupled to a first surface or support.

[0023] Example 17. A system according to Example 16, wherein the electrical signal generating subsystem includes a waveform generator, which is configured to generate a bias voltage waveform to be applied to the electrode pair when the microfluidic device is operably coupled to the first surface or support member.

[0024] Embodiment 18. The system according to embodiment 17, wherein the electrical signal generating subsystem further comprises a waveform amplifying circuit configured to amplify the bias voltage waveform generated by the waveform generator.

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

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

[0027] Embodiment 21. A system according to embodiment 20, wherein the thermal control subsystem includes a thermoelectric power module, a Peltier thermoelectric device and a cooling unit, wherein the thermoelectric power module is configured to adjust the temperature of the Peltier thermoelectric device, and optionally, wherein the Peltier thermoelectric device is inserted between the first surface and the surface of the cooling unit.

[0028] Embodiment 22. A system according to Embodiment 21, wherein the cooling unit includes a liquid cooling device, a cooling block and a liquid channel, the liquid channel is configured to circulate cooling liquid between the liquid cooling device and the cooling block, and wherein the cooling block includes a surface of the cooling unit.

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

[0030] Embodiment 24. The system of any one 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 power module.

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

[0032] Example 26. The system of Example 24 or 25 further includes an external computing device operably coupled to the microprocessor, optionally wherein the external computing device includes a graphical user interface configured to receive operator input, process the operator input and transmit the operator input to the microprocessor to control one or both of the electrical signal generating subsystem and the thermal control subsystem.

[0033] Embodiment 27. A system according to Embodiment 26, wherein the microprocessor is configured to send data and / or information sensed or received by one or both of the electrical signal generating subsystem and the thermal control subsystem to an external computing device, or alternatively, to perform calculations based on the sensed or received data or information.

[0034] Example 28. A system according to Example 16 or 27, wherein the microprocessor and / or external computing device is configured to measure and / or monitor the impedance of a circuit across electrodes of the microfluidic device when the microfluidic device is operably coupled to a support.

[0035] Example 29. A system according to Example 28, wherein the microprocessor and / or external computing device is configured to determine the flow volume of a fluid path based on changes detected in measured and / or monitored impedance of the circuit, and the fluid path includes at least a portion of a microfluidic circuit within the microfluidic device.

[0036] Example 30. A system according to Example 28, wherein the microprocessor and / or external computing device is configured to determine the height of the internal chamber of the microfluidic device based on changes detected in the measured and / or monitored impedance of the circuit.

[0037] Example 31. A system according to Example 28, wherein the microprocessor and / or external computing device is configured to determine one or more characteristics of chemical and / or biological materials contained within a microfluidic circuit of the microfluidic device based on changes detected in measured and / or monitored impedance of the circuit.

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

[0039] Embodiment 33. A system according to any one of Embodiments 1 to 32, wherein the first surface, the support member and / or the light modulation subsystem are configured to be mounted on a light microscope.

[0040] Embodiment 34. A system according to any one of Embodiments 1 to 32, wherein the first surface, the support and / or the light modulation subsystem are integral components of the light microscope.

[0041] Embodiment 35. A system according to 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] Embodiment 36. A system according to Embodiment 35, wherein at least one flow controller includes a first thermally controlled flow controller operably coupled to the first fluid line and / or the second fluid line to selectively allow fluid to flow therethrough.

[0043] Example 37. A system according to Example 36, wherein the first thermally controlled flow controller includes a Peltier thermoelectric device configured to controllably lower or raise the temperature of a fluid contained in a flow segment of a first fluid line, wherein the temperature is sufficiently lowered or raised to freeze or thaw the fluid contained in the flow segment of the first fluid line, respectively, thereby selectively preventing or allowing the fluid to flow through the first fluid line and into or out of a first fluid inlet / outlet of the microfluidic device.

[0044] Embodiment 38. A system according to Embodiment 37, wherein the first thermal control flow controller further comprises: a first shell having a first channel, the flow segment of the first fluid pipeline extending through the first channel, the shell also comprising a Peltier thermoelectric device; and / or insulating material at least partially surrounding the flow segment of the first fluid pipeline; and optionally, a first thermally conductive interface coupled to the flow segment of the first fluid pipeline.

[0045] Embodiment 39. A system according to any one of Embodiments 36 to 38, wherein at least one flow controller includes a second thermally controlled flow controller operably coupled to the other of the first fluid line and the second fluid line to selectively allow fluid to flow therethrough.

[0046] Example 40. A system according to Example 39, wherein the second thermally controlled flow controller includes a Peltier thermoelectric device configured to controllably lower or raise the temperature of a fluid contained in a flow segment of the second fluid line, wherein the temperature is lowered or raised sufficient to freeze or thaw the fluid contained in the flow segment of the second fluid line, respectively, thereby selectively preventing or allowing the fluid to flow out of or into a second fluid inlet / outlet of the microfluidic device.

[0047] Embodiment 41. A system according to embodiment 40, wherein the second thermal control flow controller further includes: a second shell having a second channel, the flow segment of the second fluid pipeline extending through the second channel, the shell also containing a Peltier thermoelectric device; and / or insulating material, at least partially surrounding the flow segment of the second fluid pipeline; and optionally, a first thermally conductive interface, coupled to the flow segment of the first fluid pipeline.

[0048] Example 42. A system according to Example 35, wherein at least one flow controller includes a thermal control flow controller operably coupled to the first fluid line and the second fluid line, the thermal control flow controller comprising: at least one flow control Peltier thermoelectric device, configured to controllably lower or raise the temperature of the flow segments of the first fluid line and the second fluid line, wherein the temperature is lowered or raised to a level sufficient to freeze or thaw the fluid contained in the flow segments of the first fluid line and the second fluid line, respectively, thereby selectively preventing or allowing the fluid to flow through the first fluid line into the first fluid inlet / outlet of the microfluidic device and flow out of the second fluid inlet / outlet of the microfluidic device through the second fluid line, and vice versa.

[0049] Embodiment 43. A system according to embodiment 42, wherein 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 a first fluid pipeline; and a second flow control Peltier thermoelectric device thermally coupled to a flow segment of a second fluid pipeline.

[0050] Embodiment 44. A system according to embodiment 42 or 43, wherein the thermal control flow controller also includes a housing having a first channel, through which a flow segment of a first fluid line extends, and a second channel, through which a flow segment of an outflow fluid line extends, wherein at least one flow control Peltier thermoelectric device is installed in the housing.

[0051] Embodiment 45. A system according to Embodiment 44, wherein the shell defines an insulated chamber.

[0052] Embodiment 46. A system according to any one of Embodiments 32 to 45, wherein the light modulation subsystem includes a digital mirror device (DMD) or a micro-shutter array system (MSA).

[0053] Embodiment 47. A system according to any one of Embodiments 32 to 45, 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.

[0054] Embodiment 48. The system of any one of Embodiments 32 to 47, wherein the light modulation subsystem comprises a multi-input light pipe comprising:

[0055] A housing having a plurality of input apertures, each of the input apertures being configured to receive light emitted from a respective light source, and 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 positioned within the housing at an oblique angle across the first light propagation path, the first dichroic filter being configured and positioned such that light received through the first optical aperture passes through the first dichroic filter as it propagates along the first light propagation path to the output aperture; and a second light propagation path extending within the housing from the second input aperture to the first dichroic filter, the second propagation path and the first dichroic filter being 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 to reach the output aperture, wherein the respective input apertures, the first light propagation path and the second light propagation path, the first dichroic filter and the output aperture are dimensioned, designed and configured such that light emitted by at least one light source and received through at least one of the first input aperture and the second input aperture is emitted from the output aperture with a substantially uniform intensity.

[0056] Embodiment 49. According to the system of embodiment 48, the light pipe also includes: a second dichroic filter, which is positioned in the housing at an inclined angle across the first light propagation path between the first dichroic filter and the output hole, and the second dichroic filter is configured and positioned so that when the received light propagates along the first light propagation path to the output hole, the light received through the first light hole and the second light hole passes through the second dichroic filter; and a third light propagation path, which extends from the third input hole to the second dichroic filter in the housing, and the third propagation path and the second dichroic filter are configured and designed so that the light received through the third input hole propagates along the third light propagation path and is reflected by the second dichroic filter onto the first light propagation path to reach the output hole.

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

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

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

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

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

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

[0063] Embodiment 56. According to the system of embodiment 54, the plurality of first light source emitting elements and the plurality of second light source emitting elements together include a first subset of one or more light emitting elements that emit light of a first narrowband wavelength, and a second subset of one or more light emitting elements that emit light having a second narrowband wavelength different from the first narrowband wavelength, so that light including one or both of the first narrowband wavelength and the second narrowband wavelength can be controllably emitted out of the light pipe output hole by selectively activating one or two of the plurality of first light source emitting elements and the plurality of second light source emitting elements.

[0064] Embodiment 57. A system according to Embodiment 56, wherein light emitted by a first subset of light-emitting elements and received via a first input hole and / or a second input hole is emitted out of an output hole of the light pipe with a first substantially uniform intensity, and light emitted by a second subset of light-emitting elements and received via the first input hole and / or the second input hole is emitted out of the output hole with a second substantially uniform intensity.

[0065] Embodiment 58. A system as described in Embodiment 57, wherein the first substantially uniform intensity is different from the second substantially uniform intensity.

[0066] Embodiment 59. The system of any one 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] Embodiment 60. According to the system described in any one of Embodiments 43 to 46, the multiple light-emitting elements of the first light source include all the light-emitting elements of the first subset or consist of all the light-emitting elements of the first subset, and the multiple light-emitting elements of the second light source include all the light-emitting elements of the second subset or consist of all the light-emitting elements of the second subset.

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

[0069] Embodiment 62. According to the system of Embodiment 61, the third light source includes a plurality of third light source emitting elements.

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

[0071] Embodiment 64. A system according to embodiment 62, wherein a plurality of first light source emitting elements, a plurality of second light source emitting elements and a plurality of third light source emitting elements together include a first subset of one or more light-emitting elements that emit light of a first narrowband wavelength, a second subset of one or more light-emitting elements that emit light of a second narrowband wavelength different from the first narrowband wavelength, and a third subset of one or more light-emitting elements that emit light of a third narrowband wavelength different from each of the first narrowband wavelength and the second narrowband wavelength, so that light including one or more of the first narrowband wavelength, the second narrowband wavelength and the third narrowband wavelength can be controllably emitted out of the light pipe output hole by selectively activating one or more of the first subset, the second subset and the third subset of the light-emitting elements.

[0072] Embodiment 65. A system according to embodiment 64, wherein light emitted by the light-emitting elements of the first subset and received through any one of the first input hole, the second input hole and the third input hole is emitted out of the output hole with a first substantially uniform intensity, light emitted by the light-emitting elements of the second subset and received through any one of the first input hole, the second input hole and the third input hole is emitted out of the output hole with a second substantially uniform intensity, and light emitted by the light-emitting elements of the third subset and received through any one of the first input hole, the second input hole and the third input hole is emitted out of the output hole with a third substantially uniform intensity.

[0073] Embodiment 66. A system as described in 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] Embodiment 67. A system according to any one 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] Embodiment 68. According to the system described in any one of Embodiments 64 to 67, the multiple light-emitting elements of the first light source include all the light-emitting elements of the first subset or consist of all the light-emitting elements of the first subset, the multiple light-emitting elements of the second light source include all the light-emitting elements of the second subset or consist of all the light-emitting elements of the second subset, and the multiple light-emitting elements of the third light source include all the light-emitting elements of the third subset or consist of all the light-emitting elements of the third subset.

[0076] Embodiment 69. A microscope configured to operate a microfluidic device, the microscope comprising: a support configured to hold the microfluidic device and be operably coupled to the microfluidic device (for example, a support according to any one of Embodiments 14 to 31 or 35 to 45); a light modulation subsystem configured to emit structured light; and an optical system, wherein when the microfluidic device is held by the support and is operably coupled to the support, the optical system is configured to: (1) focus structured light emitted by the light modulation subsystem onto at least a first region of the microfluidic device, (2) focus unstructured light emitted by an 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] Embodiment 70. According to embodiment 69, the microscope also includes a detector.

[0078] Embodiment 71. A microscope according to embodiment 69 or 70, wherein the detector includes an eyepiece and / or an imaging device.

[0079] Embodiment 72. A microscope according to any one of Embodiments 69 to 71, wherein the light modulation subsystem includes a digital mirror device (DMD) or a micro-shutter array system (MSA).

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

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

[0082] Embodiment 75. A microscope according to any one of Embodiments 69 to 74, wherein the optical system includes an objective lens configured to focus the structured light on the first area of ​​the microfluidic device and / or to focus the unstructured light on the second area of ​​the microfluidic device, and wherein the objective lens is selected from the group consisting of a 10x objective lens, a 5x objective lens, a 4x objective lens, and a 2x objective lens.

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

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

[0085] Embodiment 78. A microscope according to any one of Embodiments 69 to 75, wherein the light modulation subsystem emits structured white light.

[0086] Embodiment 79. A microscope according to any one of Embodiments 69 to 75, wherein the light modulation subsystem comprises a mercury lamp or a xenon arc lamp.

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

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

[0089] Embodiment 82. A microscope according to Embodiment 81, wherein the unstructured light source emits light having a wavelength of approximately 495 nm or shorter.

[0090] Embodiment 83. A microscope according to Embodiment 81, wherein the unstructured light source emits blue light.

[0091] Embodiment 84. A microscope according to embodiment 82 or 83, wherein the optical system includes a dichroic filter, and the dichroic filter is configured to at least partially filter out visible light with a wavelength longer than 495nm.

[0092] Embodiment 85. A microscope according to Embodiment 81, wherein the unstructured light source emits light having a wavelength of about 650 nm or longer.

[0093] Embodiment 86. A microscope according to Embodiment 81, wherein the unstructured light source emits red light.

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

[0095] Embodiment 88. A microscope according to any one of Embodiments 69 to 87, wherein the support member includes an integrated electrical signal generating subsystem, and the integrated electrical signal generating subsystem is configured to apply a bias voltage to a pair of electrodes in the microfluidic device when the device is held by the support member and operably coupled to the support member.

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

[0097] Embodiment 90. A method for analyzing a fluid sample, the method comprising: connecting a microfluidic device to a system for operating the microfluidic device, wherein the system comprises: a first surface configured to engage with and be operably coupled to the microfluidic device; and a cover configured to hold the microfluidic device on the first surface, the cover comprising: a first cover portion having a first fluid port, the first fluid port being configured to be operably coupled to a first fluid inlet / outlet of the microfluidic device and allowing a fluid medium to flow into and / or out of the first fluid inlet / outlet of the microfluidic device; and a second cover portion having a second fluid port, the second fluid port being configured to be operably coupled to a second fluid inlet / outlet of the microfluidic device and allowing a fluid medium to flow into and / or out of the microfluidic device The invention relates to a microfluidic device comprising a first cover portion and a second fluid inlet / outlet disposed in the microfluidic device, wherein the second cover portion is separable from the first cover portion and is movable between a closed position and an open position, in which the second fluid port of the second cover portion is operably coupled to the second fluid inlet / outlet of the microfluidic device, and in the open position, the portion of the microfluidic device containing the second fluid inlet / outlet is exposed; moving the second cover portion from the closed position to the open position, thereby exposing the second fluid inlet / outlet of the microfluidic device; providing a fluid sample in fluid communication with the second fluid inlet / outlet of the microfluidic device; applying suction to the first fluid line, thereby pulling at least a portion of the fluid sample into the microfluidic device; and processing at least a portion of the fluid sample pulled into the microfluidic device.

[0098] Example 91. The method according to Example 90 also includes: placing an insert in a position previously occupied by a second cover portion in a closed position, the insert comprising a fluid well, the fluid well being configured to be fluidically connected to a second fluid inlet / outlet of the microfluidic device; wherein providing a fluid sample includes introducing the fluid sample into the fluid well of the insert.

[0099] Embodiment 92. A method according to embodiment 90 or 91, wherein the system is a system according to any one of embodiments 1 to 68.

[0100] Embodiment 93. A method according to Embodiment 90 or 91, wherein the system is a microscope according to any one of Embodiments 69 to 89.

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

[0102] Embodiment 95. A method according to any one of Embodiments 90 to 94, wherein the fluid sample includes micro-objects, optionally biological micro-objects (e.g., cells).

[0103] Example 96. A method according to any one of Examples 90 to 95, wherein the microfluidic device comprises (i) a flow region having a plurality of microfluidic channels, and (ii) a plurality of chambers, e.g., an isolation fence (e.g., an isolation fence as described in PCT Publications WO2014 / 070873 and WO2015 / 061497, the entire contents of each of which are incorporated herein by reference), wherein each of the plurality of chambers is fluidically connected to one of the plurality of microfluidic channels.

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

[0105] Embodiment 98. A method according to Embodiment 97, wherein imaging includes imaging micro-objects contained within at least a portion of the fluid sample.

[0106] Embodiment 99. A method according to Embodiment 96, wherein processing at least a portion of the fluid sample includes performing an assay on micro-objects contained within at least a portion of the fluid sample.

[0107] Example 100. A method according to Example 99, wherein the assay provides detection of nucleic acids secreted by cells and / or released by cells (e.g., any assay described in PCT publications WO2014 / 070783, WO2015 / 061497, WO2015 / 061497, WO2015 / 061506, WO2015 / 095623, WO2017 / 181135, WO2018 / 064640, WO2018 / 076024, WO2019 / 075476 and WO2019 / 133874 or PCT / US2019 / 041692 and PCT / US2019 / 024623, the entire contents of each of which are incorporated herein by reference).

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

[0109] Embodiment 102. The system according to Embodiment 101 further includes an electrical signal generating subsystem, which is configured to apply a bias voltage to an electrode pair in the microfluidic device when the microfluidic device is held by and operably coupled to the support.

[0110] Embodiment 103. A system according to Embodiment 101 or 102, wherein the system includes any element (e.g., alone or in combination) of the system according to any one of Embodiments 1 to 68 and 116 to 122 or the microscope according to any one of Embodiments 69 to 89.

[0111] Embodiment 104. A system according to Embodiments 37 or 101 to 103, wherein the first thermal control flow controller further comprises: a thermally conductive interface coupled to the flow segments of the first fluid pipeline and the second fluid pipeline; and a Peltier thermoelectric device configured to contact the thermally conductive interface and controllably lower or raise the temperature of the fluid contained in the flow segments of the first fluid pipeline and / or the second fluid pipeline.

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

[0113] Embodiment 106. A system according to embodiment 104 or 105, wherein the thermally conductive interface includes a thermistor.

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

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

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

[0118] Embodiment 111 The system of any one of Embodiments 104 to 110, wherein the thermally conductive interface is configured to directly contact (eg, rest on) an upper surface of the Peltier thermoelectric device.

[0119] Embodiment 112. A system according to any one of Embodiments 104 to 111, wherein the first thermal control flow controller includes a cover, the cover including a guide, and the guide is used to insert the flow segments of the first fluid pipeline and the second fluid pipeline into the thermal interface.

[0120] Embodiment 113. A system according to any one of Embodiments 104 to 112, further comprising a barrier material located inside the thermal control flow controller, wherein the barrier material (e.g., an insulating polymer or spray foam) is sufficient to prevent the formation of ice.

[0121] Embodiment 114. The system of Embodiment 113, wherein the barrier material substantially fills any empty space that would exist within the cover of the first thermal control flow controller in the absence of the barrier material.

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

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

[0124] Embodiment 117. A system according to Embodiment 116, wherein the sensor is further configured to determine when the insert is engaged with the microfluidic device.

[0125] Embodiment 118. A system according to any one of Embodiments 116 to 117, wherein the sensor includes a first optical switch, and the first optical switch is configured to be interrupted when the second cover portion is in the closed position and to indicate when the second cover portion is in the closed position.

[0126] Embodiment 119. A system according to any one of Embodiments 116 to 118, wherein the sensor includes a second optical switch, and the second optical switch is configured to be interrupted when the insert is engaged with the microfluidic device and to indicate when the insert is engaged with the microfluidic device.

[0127] Embodiment 120. A system according to any one of Embodiments 116 to 119, wherein the sensor includes a first extension member, and the first extension member is configured to extend into the first optical switch through a first actuator included in the second cover portion, thereby interrupting the first optical switch.

[0128] Embodiment 121. A system according to any of Embodiments 116 to 120, wherein the sensor includes a second extension member, and the second extension member is configured to extend into the second optical switch through a second actuator included in the insert, thereby interrupting the second optical switch.

[0129] Embodiment 122. A system according to any one of Embodiments 116 to 121, wherein the sensor detects when the second cover portion is in the open position and when the insert is not engaged with the microfluidic device when the optical path of the first optical switch and the second optical switch is not interrupted.

[0130] Embodiment 123. A method according to 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, wherein each of the plurality of chambers is fluidically connected to one of the plurality of microfluidic channels.

[0131] Example 124. A method according to Example 123, wherein the method produces an introduced cell density of at least 4x10^6.

[0132] Other aspects and advantages of the disclosed systems, microscopes, and methods will be apparent from the following detailed description and the appended claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0133] The design and utility of embodiments of the disclosed system are illustrated in the accompanying drawings, in which similar elements are represented by the same reference numerals. The drawings are not necessarily drawn to scale. In order to better understand how the above and other advantages and objects are obtained, a more particular description of the embodiments will be presented, which will be illustrated in the accompanying drawings. These drawings depict only typical embodiments of the disclosed system and are therefore not to be considered as limiting the scope thereof.

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

[0135] Figure 1B yes Figure 1A Schematic diagram of a support member shown in , with the cover removed for clarity.

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

[0137] Figure 3 is a schematic diagram of a thermal control subsystem according to some embodiments of the system.

[0138] Figure 4 is a circuit diagram depicting analog circuitry for thermal control feedback in a thermal control subsystem according to some embodiments of the system.

[0139] Figure 5 is an exemplary screen shot depicting a graphical user interface (GUI) for controlling an electrical signal generation subsystem and a thermal control subsystem according to some embodiments of the system.

[0140] Figure 6 is a schematic diagram of a system for operating a microfluidic device according to some embodiments of the system. Figure 6 The system shown in includes an optical train with 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.

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

[0142] FIG. 8A to FIG. 8C is a diagram showing how to use structured light to compensate for light vignetting. Fig. 8A Shown is how the light intensity measured at the sample plane varies across the field of view. Figure 8B An inverse function for controlling the intensity of light output from the light modulation subsystem is shown. Figure 8C It shows that when, for example Figure 8B The inverse function shown in FIG. 1 is used to control the light intensity measured at the sample plane when the light intensity output from the light source will otherwise produce Fig. 8A The pattern of light intensity is shown in .

[0143] Fig. 9 is a schematic diagram of an impedance measurement circuit according to some embodiments of the system.

[0144] Fig.10 and Fig.11are side and perspective views of a cryovial according to some embodiments of the system.

[0145] Fig.12 is a perspective view of a pair of cryovalves according to some embodiments of the system. As shown, the cryovalves are positioned sideways on a receptacle that holds a microfluidic device.

[0146] Fig.13 yes Fig.12 A perspective view of the various components of the cryovalve is shown in FIG.

[0147] Fig.14 is a perspective view of a cryovalve according to some embodiments of the system.

[0148] Fig.15 and Fig.16 yes Fig.14 A top perspective view and a bottom perspective view of the cover of the freezing valve are shown in FIG.

[0149] Fig.17 yes Fig.14 A perspective view of the bottom portion of the freezing valve is shown in FIG.

[0150] Fig.18 yes Fig.17 A perspective view of the housing of the bottom portion of the freezing valve is shown in FIG.

[0151] Fig.19 yes Fig.14 A perspective view of a radiator with a freeze valve shown.

[0152] Fig. 20 and Fig.21 yes Fig.14 Top and side views of the sleeve of the cryovial are shown in FIG.

[0153] Fig. 22 is a schematic diagram of a system for operating a microfluidic device according to some embodiments of the system. Fig. 22 The system shown in includes an optical system with 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.

[0154] Fig.23 is a schematic diagram of two light emitting diode (LED) arrays according to some embodiments of the system.

[0155] Fig.24 is a schematic diagram of a light pipe / optical integrator according to some embodiments of the system.

[0156] Fig.25 is a schematic diagram of a light source according to some embodiments of the system.

[0157] Fig.26is a schematic diagram of a multi-input light pipe / optical integrator according to some embodiments of the system.

[0158] Fig. 27 Some embodiments of a split lid for a system for operating a microfluidic device are shown.

[0159] Fig.28 Other embodiments of split covers for systems for operating a microfluidic device are shown.

[0160] Fig.29 Yet another embodiment of a split cover for a system for operating a microfluidic device is shown.

[0161] Fig. 30A , Fig. 30B and Fig. 30C Some embodiments of removing a portion of a split cover in a system for operating a microfluidic device are shown.

[0162] Fig.31A , Fig.31B and Fig.31C Some embodiments of methods for adding a fluid sample to a microfluidic device are shown.

[0163] Fig.32 and Fig.33 Front and perspective views of cryovalves in other embodiments of the system are depicted.

[0164] Fig.34 and Fig.35 A perspective view of a cryovalve in yet another embodiment of the system is shown.

[0165] Fig.36 Perspective views of some embodiments of a split cover including a sensor are shown with the split cover in a closed position.

[0166] Fig.37 A perspective view of some embodiments of a split cover including a sensor is shown, with the split cover in an open position.

[0167] Fig.38 Some embodiments of unassembled components of a sensor that may be used with a split cover are shown.

[0168] Fig.39 Some embodiments of assembly components of sensors that may be used with a split cover are shown.

[0169] Fig.40 A side view of some embodiments of a split cover including a sensor is shown with the split cover in a closed position.

[0170] Fig.41Perspective views of some embodiments of optical switches that may be used in sensors are shown.

[0171] Fig.42 and Fig.43 A top view of some embodiments of a sensor is shown in an actuated position.

[0172] Fig.44 The cell / bead distribution in the microfluidic device of some exemplary embodiments is shown. DETAILED DESCRIPTION

[0173] This specification illustrates exemplary embodiments and applications of the present disclosure. However, the present disclosure is not limited to these exemplary embodiments and applications, nor is it limited to the mode shown in this article or the mode in which the exemplary embodiments and applications run. Moreover, the accompanying drawings may show simplified or partial views, and the element sizes in the accompanying drawings may be exaggerated or may not be proportional. In addition, when using "on ... ", "attached to", "connected to", "coupled to" or similar words in this article, an element (e.g., material, layer, substrate, etc.) can be "on another element", "attached to another element", "connected to another element" or "coupled to another element", regardless of whether the one element is directly on another element, attached, connected or coupled to the other element, or there are one or more intermediate elements between the one element and the other element. In addition, unless the context otherwise indicates, if provided, direction (e.g., above, below, top, bottom, side, up, down, below ... below, above ... above, upper, lower, horizontal, vertical, "x", "y", "z", etc.) is relative and is provided only as an example, for the convenience of explanation and discussion and not as a limitation. In addition, where a series of elements (e.g., elements a, b, c) are shown, these illustrations are intended to include any one of the listed elements themselves, any combination of less than all of the listed elements, and / or a combination of all of the listed elements. The division of parts in the specification is only for ease of viewing and does not limit any combination of the elements discussed.

[0174] As used herein, "substantially" means sufficient to achieve the intended purpose. The term "substantially" therefore allows for small, insignificant variations in absolute or perfect conditions, dimensions, measurements, results, etc., such as would be expected by one of ordinary skill in the art but which do not significantly affect overall performance. When used with respect to a numerical value or a parameter or feature that can be expressed as a numerical value, "substantially" means within ten percent.

[0175] The term "plurality" means more than one. As used herein, the term "plurality" may be 2, 3, 4, 5, 6, 7, 8, 9, 10 or more.

[0176] As used herein: μm means micrometer, μm 3 pL stands for cubic micrometer, pL stands for picoliter, and μL (or uL) stands for microliter.

[0177] As used herein, a "microfluidic device" or "microfluidic apparatus" is a device comprising one or more discrete microfluidic circuits and at least one port, the microfluidic circuits being configured to hold a fluid, each microfluidic circuit comprising fluidically interconnected circuit elements (which include, but are not limited to, regions, flow paths, channels, chambers, and / or pens), at least one port being configured to allow fluid (and optionally micro-objects suspended in the fluid) to flow into and / or out of the microfluidic device. Typically, the microfluidic circuit of a microfluidic device will include a flow region and at least one chamber, the flow region may include a microfluidic channel, and the microfluidic circuit will hold a fluid volume of less than about 1 mL (e.g., less than about 750, 500, 250, 200, 150, 100, 75, 50, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, or 2 μL). In some 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 can be configured to have a first end fluidically connected to a first port (e.g., an inlet) in the microfluidic device and a second end fluidically connected to a second port (e.g., an outlet) in the microfluidic device. In some embodiments, the microfluidic device can have more than two ports, for example, 3, 4, 5, 6 or more ports; a typical example can have, for example, two inlets and two outlets for two microfluidic circuits fluidically connected to the same microfluidic device.

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

[0179] As used herein, "microfluidic channel" or "flow channel" refers to the flow area of ​​a microfluidic device having a length that is significantly longer than the horizontal and vertical dimensions. The length of a channel is generally defined by the flow path of the channel. In the case of a straight channel, the length will be the "longitudinal axis" of the channel. The "horizontal dimension" or "width" of a channel is the horizontal dimension observed in a cross-section oriented perpendicular to the longitudinal axis of the channel (or, if the channel is curved, perpendicular to the axis tangent to the flow path of the channel at the plane of the cross section). The "vertical dimension" or "height" of a channel is the vertical dimension observed in a cross-section oriented perpendicular to the longitudinal axis of the channel (or, if the channel is curved, perpendicular to the axis tangent to the flow path of the channel at the plane of the cross section).

[0180] For example, the flow channel can be at least 5 times the length of the horizontal or vertical dimension, for example, 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 1000 times the length, at least 5000 times the length, or longer. In some embodiments, the length of the flow channel is about 100,000 microns to about 500,000 microns, including any value therebetween. In some embodiments, the horizontal dimension is about 100 microns to about 1000 microns (e.g., about 150 to about 500 microns), and the vertical dimension is about 25 microns to about 200 microns (e.g., from about 40 to about 150 microns). It should be noted that the flow channel can have a variety of different spatial configurations in the microfluidic device and is therefore not limited to perfectly linear elements. For example, the flow channel can be or may include one or more portions having the following configurations: curves, bends, spirals, tilts, dips, bifurcations (e.g., multiple different flow paths) and any combination thereof. In addition, the flow channel can have different cross-sectional areas (widening and contraction) along its path to provide a desired fluid flow therein. The flow channel can include a valve, which can be any type known in the microfluidics field. Examples of microfluidic channels including valves are disclosed in U.S. Patents 6,408,878 and 9,227,200, all of which are incorporated herein by reference in their entirety.

[0181] The direction of fluid flow through a flow region (e.g., a channel) or other circuit element (e.g., a chamber) determines the "upstream" and "downstream" orientation of the flow region or circuit element. Thus, an inlet will be located at an upstream position, while an outlet will generally be located at a downstream position. Those skilled in the art will understand that the designation of "inlet" or "outlet" can be changed by reversing the flow within the device or by opening one or more alternative holes.

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

[0183] As used herein, "structured light" is projected light that is modulated to provide one or more lighting effects. A first lighting effect can be projected light that illuminates a portion of a device surface without illuminating (or at least minimizing its illumination of) adjacent portions of the surface, such as a projected light pattern used to activate DEP forces within a DEP substrate as described more fully below. When a structured light pattern is used to activate DEP forces, the intensity (e.g., a change in the duty cycle of a structured light modulator such as a DMD) can be used to change the light power applied to the light-activated DEP actuator, thereby changing 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., drops at the edges of the illuminated area). Structured light is typically generated by a structured light modulator (such as a digital mirror device (DMD), a micro-gate array system (MSA), a liquid crystal display (LCD), etc.). Illuminating a small area of ​​a surface, such as a selected area of ​​interest, with structured light improves the signal-to-noise ratio (SNR) by reducing stray / scattered light by illuminating only the selected area of ​​interest, thereby reducing the darkness of the image. An important aspect of structured light is that it can change rapidly over time. The light pattern from a structured light modulator (e.g., DMD) can be used to automatically focus on difficult targets such as clean mirrors or surfaces that are far from the focus. Using a clean mirror, multiple self-test features, such as measurements of modulation transfer function and field curvature / field tilt, can be replicated without the need for more expensive Shack-Hartmann sensors. In another use of structured light patterns, instead of a camera, a simple power meter can be used to measure the spatial power distribution at the sample surface. Structured light patterns can also be used as reference features for alignment of optical modules / system components, and as manual readouts for manual focus. Another lighting effect that may be achieved by using structured light patterns is the selective curing, such as solidification, of hydrogels within a microfluidic device.

[0184] As used herein, the term "micro-object" generally refers to any microscopic object that can be isolated 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 matrices, Luminex TMBeads, etc.), magnetic beads, microrods, microfilaments, quantum dots, etc.; biological micro-objects, such as cells, biological organelles, vesicles or complexes, synthetic vesicles, liposomes (e.g., synthetic or derived from membrane products), lipid nanorafts, etc.; or combinations of inanimate micro-objects and biological micro-objects (e.g., micro-beads attached to cells, micro-beads coated with liposomes, magnetic beads coated with liposomes, etc.). Beads may include covalently or non-covalently attached parts / molecules, such as fluorescent markers, proteins (including receptor molecules), carbohydrates, antigens, small molecule signaling parts, or other chemical / biological species that can be used in the assay. In some variations, the beads / solid matrix including parts / molecules can be capture beads, for example, which are configured to selectively or non-selectively bind to molecules including small molecules, peptides, proteins or nucleic acids that exist in close proximity. In a non-limiting example, the capture beads may include nucleic acid sequences that are configured to bind nucleic acids having specific nucleic acid sequences, or the nucleic acid sequences of the capture beads may be configured to bind nucleic acid groups having related nucleic acid sequences. Any type of binding can be understood to be selective. When binding of structurally different but physicochemically similar molecules is performed, capture beads comprising moieties / molecules can bind non-selectively, for example, size exclusion beads or zeolites configured to capture molecules of selected size or charge. Lipid nanorafts have been described, for example, in Ritchie et al. (2009) "Reconstitution of Membrane Proteins in Phospholipid Bilayer Nanodiscs", Methods Enzymol, 464: 211-231.

[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, fish cells, etc.), prokaryotic cells, bacterial cells, fungal cells, protozoan cells, etc., cells dissociated from tissues (such as muscle, cartilage, fat, skin, liver, lung, neural tissue, etc.), immune cells (such as T cells, B cells, natural killer cells, macrophages, etc.), embryos (e.g., zygotes), oocytes, egg cells, sperm cells, hybridomas, cultured cells, cells from cell lines, cancer cells, infected cells, transfected and / or transformed cells, reporter cells, etc. Mammalian cells can be from, for example, humans, mice, rats, horses, goats, sheep, cattle, primates, etc.

[0186] A colony of biological cells is "clonal" if all living cells in the colony that are capable of reproduction are daughter cells derived from a single mother cell. In certain embodiments, all daughter cells in a clonal colony are derived from a single mother cell through no more than 10 divisions. In other embodiments, all daughter cells in a clonal colony are derived from a single mother cell through no more than 14 divisions. In other embodiments, all daughter cells in a clonal colony are derived from a single mother cell through no more than 17 divisions. In other embodiments, all daughter cells in a clonal colony are derived from a single mother cell through no more than 20 divisions. The term "clonal cell" refers to cells of the same clonal colony.

[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.

[0188] The phrase "flow of a medium" refers to the overall movement of a fluid medium primarily due to any mechanism other than diffusion, and may encompass perfusion. For example, the flow of a medium may involve the movement of a fluid medium from one point to another due to a pressure difference between the points. Such flow may include continuous flow, pulsed flow, periodic flow, random flow, intermittent flow, or reciprocating flow of a liquid, or any combination thereof. When one fluid medium flows into another fluid medium, turbulence and mixing of the medium may occur. Flow may include pulling a solution through a microfluidic channel and out of a microfluidic channel (e.g., suction) or pushing a fluid into and through a microfluidic channel (e.g., perfusion).

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

[0190] As used herein with respect to different regions within a microfluidic device, the phrase "fluidically connected" means that when the different regions are substantially filled with a liquid (such as a fluid medium), the fluid in each region is connected to form a single body of fluid. This does not mean that the fluids (or fluid media) in the different regions are necessarily identical in composition. Instead, the fluids in different fluidically connected regions of a microfluidic device may have different compositions (e.g., different concentrations of solutes, such as proteins, carbohydrates, ions, or other molecules), which are constantly changing due to the movement of the solutes along their respective downward concentration gradients and / or due to the flow of the fluids through the microfluidic device.

[0191] As used herein, a "flow path" refers to one or more fluidically connected loop elements (e.g., channels, regions, chambers, etc.) that define the trajectory of a medium flow and experience the trajectory of the medium flow. Thus, a flow path is an example of an affected region of a microfluidic device. Other loop elements (e.g., unaffected regions) may be fluidically connected to loop elements that include a flow path but do not experience the medium flow in the flow path.

[0192] As used herein, "isolated micro-objects" limit micro-objects to a defined area within a microfluidic device. For example, a defined area can be a chamber. As used herein, a "chamber" is a region within a microfluidic device (e.g., a loop element) that allows one or more micro-objects to be isolated from other micro-objects within the microfluidic device. Examples of chambers include micropores, which can be regions etched from a substrate (e.g., a planar substrate), such as described in U.S. Patent Application No. 2013 / 0130232 (Weibel et al.) and U.S. Patent Application No. 2013 / 0204076 (Han et al.), or can be regions formed in a multilayer device, such as the microfluidic device described in WO2010 / 040851 (Dimov et al.) or U.S. Patent Application No. 2012 / 0009671 (Hansen et al.). Other examples of chambers include valved chambers, such as described in WO2004 / 089810 (McBride et al.) and U.S. Patent Application Publication No. 2012 / 0015347 (Singhal et al.). Still other examples of chambers include chambers 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; U.S. Patent Application Publication No. 2011 / 0053151 (Hansen et al.); and U.S. Patent Application Publication No. 2006 / 0154361 (Wikswo et al.). Still other examples of chambers include the isolation enclosures described herein. In certain embodiments, the chamber can be configured to hold a fluid volume of approximately 100pL to 1nL, 100pL to 2nL, 100pL to 5nL, 250pL to 2nL, 250pL to 5nL, 250pL to 10nL, 500pL to 5nL, 500pL to 10nL, 500pL to 15nL, 750pL to 10nL, 750pL to 15nL, 750pL to 20nL, 1 to 10nL, 1 to 15nL, 1 to 20nL, 1 to 25nL, or 1 to 50nL. In other embodiments, the chamber can be configured to hold a fluid volume of approximately 20nL to 200nL, 100 to 200nL, 100 to 300nL, 100 to 400nL, 100 to 500nL, 200 to 300nL, 200 to 400nL, 200 to 500nL, 200 to 600nL, 200 to 700nL, 250 to 400nL, 250 to 500nL, 250 to 600nL, or 250 to 750nL.

[0193] As used herein, "enclosure" specifically refers to placing a micro-object within an isolation fence within a microfluidic device. The force used to enclose the micro-object can be any suitable force described herein, such as dielectrophoresis (DEP) force (e.g., optically actuated dielectrophoresis force (OEP)), gravity, magnetic force, locally actuated fluid flow or tilt. In some embodiments, enclosing multiple micro-objects can reposition substantially all of the micro-objects. In some other embodiments, a selected number of multiple micro-objects can be enclosed, while the rest of the multiple micro-objects may not be enclosed. In some embodiments, when enclosing selected micro-objects, DEP forces (e.g., optically actuated DEP forces or magnetic forces) can be used to reposition the selected micro-objects. Typically, the micro-objects can be introduced into a flow region (e.g., a microfluidic channel) of a microfluidic device and then introduced into the chamber by enclosure.

[0194] As used herein, "unwinding" refers to relocating a micro-object from an isolation fence to a new position in a flow area (e.g., a microfluidic channel) of a microfluidic device. The force used to unwind a micro-object can be any suitable force described herein, such as dielectrophoretic force (e.g., optically actuated dielectrophoretic force), gravity, magnetic force, locally actuated fluid flow or tilt. In some embodiments, unwinding a plurality of micro-objects can reposition substantially all of the micro-objects. In some other embodiments, a plurality of micro-objects of a selected number can be unwinded, while the remainder of a plurality of micro-objects may not be unwinded. In some embodiments, when unwinding a selected micro-object, a DEP force (e.g., optically actuated DEP force or magnetic force) can be used to reposition the selected micro-object.

[0195] As used herein, "output" may include, consist of, or consist essentially of relocating a micro-object from a location (e.g., a flow region, a microfluidic channel, a chamber, etc.) within a microfluidic device to a location (such as a well plate, a tube, or other receiving container) outside the microfluidic device. In some embodiments, outputting a micro-object includes extracting (e.g., micropipette) a volume of a medium containing a micro-object from within the microfluidic device and placing the volume of the medium in or on a location outside the microfluidic device. In some related embodiments, access to (e.g., micropipette) an interior region of the microfluidic device is facilitated by disassembling the microfluidic device (e.g., removing an upper layer (such as a lid or cover) of the microfluidic device from a lower layer (such as a substrate) of the microfluidic device before extracting the volume of the medium. In other embodiments, outputting a micro-object includes causing a volume of a fluid containing a micro-object to flow through a flow region (including, for example, a microfluidic channel) of the microfluidic device, flowing out through an outlet of the microfluidic device, and placing the volume of the medium in or on a location outside the microfluidic device. In such embodiments, micro-objects within a microfluidic channel can be exported for further processing without the need to disassemble (e.g., remove the cover of the device) or insert a tool into an interior area of ​​the microfluidic device to remove the micro-objects. "Exporting" may also include repositioning a micro-object from within a chamber that may include an isolation fence to a new location within a flow area (such as a microfluidic channel), as described above with respect to "unenclosing." The chamber is oriented relative to the plane of the microfluidic channel so that the chamber is laterally open from the microfluidic channel, as described herein with respect to the isolation fence, allowing easy export of micro-objects that have been positioned or repositioned (e.g., unenclosed from the chamber) to be disposed within the microfluidic channel.

[0196] The microfluidic device may include a "reached" area and an "unreached" area. As used herein, the "reached" area includes one or more fluid interconnected circuit elements of a microfluidic circuit, each of which is subjected to a medium flow when the fluid is flowing through the microfluidic circuit. The circuit elements of the affected area may include, for example, regions, channels, and all or part of chambers. As used herein, the "unreached" area includes one or more fluid interconnected circuit elements of a microfluidic circuit, each of which is substantially not subjected to a fluid flux when the fluid is flowing through the microfluidic circuit. As long as the fluid connection is configured to enable diffusion, but there is substantially no medium flow between the affected area and the unreached area, the unreached area can be fluidically connected to the affected area. The microfluidic device can therefore be configured to substantially isolate the unreached area from the medium flow in the affected area, while enabling substantially only the diffusion fluid communication between the affected area and the unreached area. For example, the flow channel of a microfluidic device is an example of a reached area, and the isolation area of ​​a microfluidic device (described in further detail below) is an example of an unreached area.

[0197] As used herein, an "unswept" rate of flow of a fluid medium refers to a flow rate that is sufficient to allow components of a second fluid medium in an isolation region of an isolation fence to diffuse into a first fluid medium in a flow region and / or components of a first fluid medium to diffuse into a second fluid medium in an isolation region; and further wherein the first medium does not substantially flow into the isolation region.

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

[0199] In certain embodiments, the support includes a receptacle capable of interacting with a microfluidic device, such as an optically actuated microfluidic device. Figure 1A and Figure 1B The support member 100 in the embodiment includes an exemplary socket 106. However, the shape and function of the socket 106 need not be exactly as Figure 1A and 1BAs shown. For example, the socket can include a cover. In addition, the socket 106 can be adjusted as needed to match the size and type of microfluidic device 110 with which the socket 106 is intended to interact. Various microfluidic devices 110 are known in the art, including devices 110 having optically actuated structures (such as optoelectronic tweezers (OET) structures and / or photoelectrowetting (OEW) structures). Examples of suitable OET structures are described in the following U.S. patent documents, each of which is incorporated herein by reference in its entirety as if fully set forth. These U.S. patents are: U.S. Patent No. RERE44,711 (Wu et al.) (originally published as U.S. Patent No. 7,612,355) and U.S. Patent No. 7,956,339 (Ohta et al.). Examples of OEW structures are described in the following U.S. patent documents, each of which is incorporated herein by reference in its entirety as if fully set forth. These U.S. patents are: U.S. Patent No. 6,958,132 (Chiou et al.) and U.S. Patent Publication No. 2012 / 0024708 (Chiou et al.), both of which are incorporated herein by reference in their entirety as if fully set forth. Another example of an optically actuated microfluidic device includes an OET / OEW combination configuration, examples of which are described in the following U.S. patent documents: U.S. Patent Publication Nos. 20150306598 (Khandros et al.) and 20150306599 (Khandros et al.) and their corresponding PCT Publications WO2015 / 164846 and WO2015 / 164847, both of which are incorporated herein by reference in their entirety as if fully set forth.

[0200] Figure 1A and Figure 1B The support member 100 shown in FIG. 1 further includes a base 102 and a cover 104 (in Figure 1B The support member 100 further includes a plurality of connections: a first fluid input / output 112, a communication connection 114, a power connection 116, and a second fluid input / output 118. The first fluid input / output 112 and the second fluid input / output 118 are configured to deliver cooling fluid to a cooling block ( Figure 3 The first fluid input / output 112 and the second fluid input / output 118 are input or output depending on the direction of the fluid flowing through the support 100. The first fluid input / output 112 and the second fluid input / output 118 are coupled to the cooling block via a first fluid connector 142 and a second fluid connector fluid 144 disposed in the support 100. The communication connection 114 is configured to connect the support 100 to other components of the system for operating the microfluidic device to be shown below. The power connection 116 is configured to provide energy (e.g., electricity) to the support 110.

[0201] In certain embodiments, the support 100 may include an integrated electrical signal generation subsystem. The electrical signal generation subsystem 138 may be configured to apply a bias voltage to 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 such a bias voltage is always applied when the microfluidic device 110 is held by the support 100. Instead, in most cases, the bias voltage will be applied only intermittently, for example, as 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.

[0202] like Figure 2 As shown, the electrical signal generating subsystem 138 will typically include a waveform generator 202. The electrical signal generating subsystem 1 may also include a sensing module 208 (e.g., an oscilloscope) and / or a waveform amplification circuit 204 configured to amplify the waveform received from the waveform generator 202. If the sensing module 208 is present, the sensing module 208 is configured to measure the waveform provided to the microfluidic device 110 held by the support 100. In some embodiments, the sensing module 208 measures the waveform at a location close to the microfluidic device 110 (and the distal end of the waveform generator 20), thereby ensuring a higher accuracy measurement of the waveform actually applied to the microfluidic device 110. For example, data obtained from the measurement of the sensing module 208 is provided as feedback to the waveform generator 202, and the waveform generator 202 can be configured to adjust its output based on such feedback. An example of a suitable combination of a waveform generator 202 and a sensing module 208 is a RED PITAYA TM .

[0203] In some embodiments, the support 100 may include a thermal control subsystem. The thermal control subsystem 140 may be configured to regulate the temperature of the microfluidic device 110 held by the support 100. Figure 3As shown, the thermal control subsystem 140 may include a proximal assembly of a Peltier thermoelectric device 304 and a cooling unit 312. The Peltier thermoelectric device 304 may have a first surface 306 configured to engage with at least one surface of the microfluidic device 110. For example, the cooling unit may include a cooling block 322. A second surface 308 of the Peltier thermoelectric device 304 (e.g., a surface 308 opposite to the first surface 306) may be configured to engage with a surface of such a cooling block 322. All or part of the cooling block 322 (e.g., a portion engaged with the Peltier thermoelectric device 304) may be made of a material having high thermal conductivity. For example, the material may be a metal, such as aluminum. The cooling block 322 may be connected to a fluid path 324, which is configured to circulate a fluid cooled between a fluid cooling device 326 and the cooling block 322. The fluid path 324 may include the fluid input / output 112, 118 and fluid connectors 142, 144 shown in conjunction with FIG. 1. The Peltier thermoelectric device 304 and the cooling block 322 may be mounted on the support 100 .

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

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

[0206] Figure 4The analog circuit 400 shown includes a resistor 402, a thermistor 406, and an analog input 404. The analog input is operably coupled to the electrical signal generating subsystem 138 (e.g., its sensing module 208) and provides a signal thereto that can be used to calculate the temperature of the microfluidic device 110. The thermistor 406 is configured such that when the temperature of the thermistor 406 decreases, its resistance can decrease in a known manner, and when the temperature of the thermistor 406 increases, its resistance can increase in a known manner. The analog circuit 400 is connected to a power supply (not shown) that is configured to deliver a bias voltage to the electrode 408. In a specific embodiment, the resistor 402 can have a resistance of about 10,000 ohms and the thermistor 406 can have a resistance of about 10,000 ohms at 25°C. The power supply (e.g., a DC power supply) can provide a bias voltage of about 5V. The analog circuit 400 is exemplary, and other systems can be used to provide temperature values ​​for feedback to the thermoelectric power module 302.

[0207] In some embodiments, the support 100 also includes a controller 136 (e.g., a microprocessor). The controller 136 can be used to sense and / or control the electrical signal generation subsystem 138. In addition, to the extent that 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, for example, ARDUINO NANO TM ARDUINO TM The controller 136 may be configured to connect to an external controller (not shown) such as a computer or other computing device via a plug / connector 134. In some embodiments, the external controller may include a graphical user interface (GUI) configured to sense and / or control the electrical signal generation subsystem 138 and / or the thermal control subsystem 140. Figure 5 An exemplary GUI 500 configured to control the electrical signal generation subsystem 138 and the thermal control subsystem 140 is shown in FIG.

[0208] In some 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 into the PCB 132. Similarly, to the extent that the support 100 includes a controller 136 or a thermal control subsystem 140, the controller 136 and / or the thermoelectric power module 302 may be mounted on and electrically integrated into the PCB 132.

[0209] Therefore, if Figure 1A and 1BAs shown, the exemplary support 100 may 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 into a PCB 132, thereby forming a printed circuit board assembly (PCBA) 130. As discussed above, the socket 106 may be designed to hold (or "consume") a microfluidic device 110 including an optically actuated microfluidic device.

[0210] In some embodiments, the electrical signal generating subsystem 138 may include a RED PITAYA TM Waveform generator 202 / sensing module 208 and amplifier by RED PITAYA TM The waveform generator 202 generates a waveform and transmits the amplified waveform (voltage) 206 to the waveform amplification circuit 204 of the microfluidic device 110. Figure 1B RED PITAYA TM Units 202, 208 and waveform amplification circuit 204 can be electrically integrated into PCB 132 as electrical signal generation subsystem 138. In addition, RED PITAYA TM Units 202, 208 can be configured to measure the amplified voltage at the microfluidic device 110 and then adjust their own output voltage as needed so that the measured voltage at the microfluidic device 110 is the desired value. For example, the amplification circuit 204 can have a +6.5V to -6.5V power supply generated by a pair of DC-DC converters mounted on the PCB 132, generating a signal of up to 13Vpp at the microfluidic device 110.

[0211] In some embodiments, the support member 100 includes a thermal control subsystem 140 ( Figure 3 ), which has a Peltier thermoelectric device 304, a POLOLU TM Thermoelectric Power Supply (not shown) and ARDUINO NANO TM Controller 136. Feedback for thermal control subsystem 140 may be an analog voltage divider circuit 400 ( Figure 4), which includes a resistor 402 (e.g., resistance 10kOhm + / - 0.1%, temperature coefficient + / - 0.02ppm / °C) and a negative temperature coefficient thermistor 406 (nominal resistance 10kOhm + / - 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., an on-board PID control loop algorithm) to drive the steering signal pin and the pulse width modulation signal pin on the thermoelectric power module 302, thereby actuating the thermal electronic system 140. The liquid cooling unit 326 can be configured to pump fluid through a cooling path 324 (e.g., fluid input / output 112, 118 and fluid connections 142, 144) that is partially located in the support 100 and partially on the periphery of the support 100.

[0212] In some embodiments, support member 100 includes a plurality of components that allow RED PITAYA TM The serial port 114 and Plink tool for the unit to communicate with an external computer. The serial port 114 can also allow the controller 136 to communicate with the external computer. Alternatively, a separate serial port (not shown) can be used to allow the controller 136 to communicate with the external computer. In other embodiments, the support member 100 may include a wireless communication device configured to facilitate wireless communication between components of the support member 100 (e.g., the controller 136 and / or the electrical signal generating subsystem 138) and the external computer, which may include a portable computing device (such as a cellular phone, PDA) or other handheld device. A GUI on an external computer (e.g., such as Figure 5 ) can be configured for various functions, including but not limited to plotting temperature and waveform data, performing scaling calculations for output voltage adjustment, and updating controller 136 and RED PITAYA TM Devices 202, 208.

[0213] In certain embodiments, the support 100 may further include or be connected to an inductance / capacitance / resistance (LCR) meter configured to measure characteristics of the contents (eg, fluid contents) of the microfluidic device 110 .

[0214] For example, the LCR meter can be configured to measure the complex impedance of the system, in particular the complex impedance of the fluid as it enters the microfluidic device 110, is within the microfluidic device 110, and / or exits the microfluidic device 110. In some embodiments, the LCR meter can be connected to and / or integrated into a fluid line that delivers fluid to or removes fluid from the microfluidic device 110. In other embodiments, the LCR meter can be connected to or an integral part of the electrical signal generating subsystem 138. Thus, in some embodiments, the RED PITAYA in the support 100 TM The waveform generator 202 and the sensing module 208 can be configured to function as an LCR meter. In certain embodiments, the electrodes of the microfluidic device 110 configured for use with the electrical signal generation subsystem 138 can also be configured for use with an LCR meter. Measuring the impedance of the system can determine various system characteristics and changes therein, such as the height of a fluid loop within the microfluidic device 110, changes in the salt content of the fluid in the microfluidic device 110 (which may be related to the state of the biological micro-target therein), and the movement of a specific volume of fluid (having different impedances) through the microfluidic device 110.

[0215] In certain embodiments, the impedance of the measuring system can be used to accurately (i.e., close to the true value) and precisely (i.e., repeatably) detect a change from a first fluid in the system (i.e., the 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 a saline solution (e.g., phosphate buffered saline or "PBS"), or vice versa. Alternatively, the first fluid can be a saline solution (e.g., PBS) and the second fluid can be a cell culture medium having an impedance that is detectably different from the saline solution, or vice versa. In further alternatives, the first fluid can be a first cell culture medium and the second fluid can be a second cell culture medium having an impedance that is detectably different from the first cell culture medium. Fig. 9is a schematic diagram showing an impedance measurement circuit 900 for detecting the impedance of a system. The circuit 900 includes an output 902 from a waveform generator 202 of an electrical signal generating subsystem 138 and two inputs 904, 906 of a sensing module 208 of the electrical signal generating subsystem 138. The circuit 900 also includes a microfluidic device 110 (connected via a socket 106 of a support 100) and a shunt resistor 908. The shunt resistor 908 can be selected so that the LCR is accurate enough to measure impedance in the range of 0 to about 5,000 ohms (e.g., a range of 0 to about 4000, 0 to about 3000, 0 to about 2500, 0 to about 2000, 0 to about 1500, or 0 to about 1000 ohms). In the circuit 900, the microfluidic device 110 acts as a measuring unit, and a substrate (e.g., a semiconductor device) and a cover (e.g., having an indium tin oxide (ITO) layer) of the microfluidic device 110 serve as electrodes. In some embodiments, the output 902 of the circuit 900 may be from a RED PITAYA TM The waveform generator 202 of the device, the inputs 904, 906 can be derived from the microfluidic device 110 and are provided by RED PITAYA TM The sensing module 208 of the device receives. In some specific embodiments, the shunt resistor 908 can be a 50 ohm resistor. In these embodiments, the electrical signal generation subsystem 138 can be switched between "optical actuation mode" and "LCR mode". In addition, when in LCR mode, the electrical signal generation subsystem 138 can be connected to a computer running a MATLAB script.

[0216] Thus, one of the systems provides a method for determining the flow volume (V flow). For example, the microfluidic device 110 is initially filled with a first fluid associated with a first impedance (e.g., DI, which is associated with an impedance of about 450 ohms). Then, a second fluid associated with a second impedance detectably different from the first impedance (e.g., PBS, which is associated with an impedance of about 160 ohms) flows into and through the microfluidic device 110. For example, the second fluid can flow into the microfluidic device 110 through a port that can be used as a fluid inlet port or a fluid outlet port. As the second fluid flows into and through the microfluidic device 110, the system continuously measures the complex impedance of the microfluidic device 110. As described above, in order to measure the complex impedance of the microfluidic device 110 at a specific point in time, the system applies a voltage potential to the microfluidic device 110, and concomitantly, receives a signal from the microfluidic device 110 for calculating 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 specific frequency can be selected based on the characteristics 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 flows 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 the flow volume (V flow Starting from the point at which the system begins pumping the second fluid into the microfluidic device 110, the volume of the second fluid required to switch the complex impedance of the microfluidic device 110 from the first impedance to the second impedance may include (1) the flow volume (V flow ), (2) the volume of the fluid outlet port of the microfluidic device, and (3) the flow volume of the conduit that delivers the second fluid from the pump to the microfluidic device 110. Because the flow of the second fluid through the conduit and the fluid outlet port does not change the complex impedance of the microfluidic device 110, the flow volume of the conduit and the inlet can be easily distinguished from the flow volume of the microfluidic device 110.

[0217] Using the calculated flow volume of the microfluidic device 110, the system also provides a method for reliably outputting one or more micro-targets from the microfluidic device 110 in the form of discrete volumes of fluid. flow), the minimum output volume (V) required to output a microtarget (e.g., a biological cell) located within the flow path can be estimated by calculating the portion of the flow path that separates the microtarget from the fluid outlet port of the microfluidic device 110. ex For example, the total length of the flow path (L tot ) can be determined by tracking the flow path of the microfluidic device 110 from the fluid inlet port to the fluid outlet port. The output length (L) of the flow path can be determined by tracking the flow path of the microfluidic device 110 from the location of the micro-target in the flow path to the fluid output port. ex ). Therefore, the minimum amount of fluid (V) required to output the micro-target from the microfluidic device 110 can be calculated. ex ): V ex =(L ex / L tot )*V flow Alternatively, the total volume of the flow path (V flow-tot ) can be estimated based on the predicted geometry of the flow path (e.g. using a CAD drawing). Output the total volume of the flow path (V ex-tot) The same can be calculated based on the predicted geometry of the flow path. In such an embodiment, the minimum amount of fluid (V) required to be output from the microfluidic device 110 to the microtarget ex ) can be calculated as: V ex =(V ex-tot / V flow-tot )*V flow Regardless of the calculation V ex How can we make a certain volume of fluid flow through a ex The microtarget is output from the microfluidic device 110 through the fluid outlet port of the microfluidic device 110. To ensure reliable output, the fluid volume can be equal to C*V ex A certain volume of fluid (V ex-rel ) flows out of the microtarget from the microfluidic device 110, where C is a scaling factor equal to about 1.1 or greater (e.g., about 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, or greater). In some methods, the remaining volume (V res , which is equal to V ex (or V ex-rel ) is discarded before being output from the microfluidic device 110. ex For example, V ex (or V ex-rel) can be equal to 1.0 μL, and the leading volume of 0.5 μL can be discarded, resulting in the microtarget being output with a final volume Vres of 0.5 L. In this way, the microtarget can be output in small but dispersed fluid volumes. Depending on the implementation of the method, V ex 、V ex-rel or V res The volume of the fluid containing the micro-target (i.e., V ex 、V ex-rel or V res ) is output through an output conduit having a finite internal volume before reaching a collection container. Therefore, the calculations used in the method can be adjusted to account for the known or estimated volume of the output conduit. For example, the internal volume of the output conduit may be 5.0 μL. In this case, a V of 1.0 μL ex (or V ex-rel ) will be adjusted to 6.0 μL and the discarded lead volume of 0.5 μL will be adjusted to 5.5 μL, resulting in a V of 0.5 μL res Remain unchanged.

[0218] In certain embodiments, the support 100 includes one or more valves coupled to the support 100, the one or more valves being configured to limit (e.g., stop) fluid movement within a microfluidic device 110 coupled to the support 100. Although the flow of fluid into and out of the microfluidic device 110 can be controlled, for example, via a pump, movement of the fluid lines connecting the pump to the microfluidic device 110 may cause undesirable movement (e.g., drift and / or oscillation) of the fluid within the microfluidic device 110 even when the pump is turned off. Such movement, in turn, may disrupt processes occurring within the microfluidic device 110, such as detecting and / or selecting micro-objects (e.g., for counting, characterization, and / or movement between channels and chambers) or assays performed within the microfluidic device 110. One or more valves located in the support 100 can reduce or prevent such undesirable movement of fluid within the microfluidic device 110. Suitable valves have substantially no internal dead space (i.e., space within the valve that allows easy access to the fluid when the fluid flows through the valve, but little fluid flux). In certain embodiments, at least one of the one or more valves is a thermally controlled flow controller, such as a cryogenic valve. Fig.10 and Fig.11A thermal control flow controller 1000 for use with a support 100 according to one embodiment of the system is shown. The flow controller 1000 includes a temperature regulating device 1004, a thermal interface 1006, and a flow segment (hidden) of a fluid line 1008. The temperature regulating device 1004 may include one or more Peltier thermoelectric devices (e.g., a stack of two, three, four, five or more Peltier devices). The thermal interface 1006 may be made of a material with high thermal conductivity that is resistant to thermal damage, such as a metal (e.g., copper). The thermal interface 1006 may surround the flow portion of the fluid line 1008. For example, the thermal interface 1006 may be a sleeve or other object that completely surrounds the flow segment of the fluid line 1008, or the thermal interface 1006 may have a grooved surface that accommodates the flow segment of the fluid line 1008 in its grooves. The fluid in the fluid line 1008 may be a liquid that freezes into a solid at a temperature achievable by the flow controller 1000. The thermal interface 1006 is disposed near the temperature adjustment device 1004 , and preferably contacts a thermally conductive surface thereof to increase the efficiency of the flow controller 1000 .

[0219] In some embodiments, the thermal control flow controller 1000 may include a heat sink 1002, which may be made of one or more materials having high thermal conductivity (and low thermal capacitance), such as aluminum. Alternatively, the flow controller 1000 may be configured to rest on and / or be secured to the heat sink 1002. Additionally, the flow controller 1000 may include an insulating material 1010, which may be configured to prevent moisture, which may occur when moisture condenses on the thermal interface 1006 and / or the temperature regulating device 1004, from interfering with the function of the flow controller 1000. The flow controller 1000 may also include a cover 1012. The cover 1012 or another device (e.g., a clamp) may be configured to hold the thermal interface 1006 against the temperature regulating device 1004, and thereby, for example, improve the efficiency of the flow controller 1000.

[0220] Fig.12 1 shows a socket 106 and a pair of valves according to another embodiment, each valve being a thermally controlled flow controller 1000. The flow controllers 1000 are disposed directly upstream and downstream of the socket 106. Fig.12As shown, each flow controller 1000 includes a heat sink 1002 and a housing 1014. Each housing 1014 contains a temperature regulating device 1004, a thermal interface 1006, and a flow section of a fluid line 1008. It can be seen that the fluid line 1008 leaves the flow controller 1000 and enters the socket 106. The housing 1014 can be made of a material with low thermal conductivity and / or low air permeability. For example, the material can be a polymer such as PVC. Each housing 1014 can have a volume of at least twice (e.g., 2 to 10 times, 2 to 7 times, 2 to 5 times, 2 to 4 times, or 2 to 3 times) the volume of the respective temperature regulating device 1004 contained therein. The housing can be configured to prevent moisture from interfering with the function of the flow controller 1000, which may occur when moisture condenses on the respective temperature regulating device 1004 and / or the thermal interface 1006. Fig.12 Also shown is a secondary heat sink 1020 upon which the flow controller 1000 may be mounted. The secondary heat sink 1020 is configured to absorb heat from the heat sink 1002 of the flow controller 1000.

[0221] Fig.13 The heat sink 1002 and housing 1014 of the thermal control flow controller 1000 are shown, and Fig.12 The lower side of the housing 1014 is similar to that shown in FIG. Fig.13 , a groove 1016 configured to accommodate a fluid line 1008 (not shown) and / or at least a portion of the thermal interface 1006 is shown. The groove 1016 may be further configured to hold the thermal interface 1006 (not shown) against the temperature regulating device 1004 (e.g., one or more (e.g., stacked) Peltier thermoelectric devices, not shown).

[0222] Fig.14 The exterior of a thermal control flow controller 1000 according to yet another embodiment is shown. As shown, the flow controller 1000 includes a cover 1030, a bottom portion 1040, and a heat sink 1002. The cover 1030 defines a corresponding plurality of indicator openings 1034, 1036, which are configured to allow an indicator (e.g., LED) to be observed from an external position of the cover 1030. The indicator can be configured to indicate whether the flow controller 1000 is open or closed and / or whether the flow section of the fluid line 1008 is in an open configuration (i.e., not frozen) or in a closed configuration (i.e., frozen). In addition, the cover 1030 can define a fastener opening 1032, which is configured to accommodate fasteners (e.g., screws) for assembly of the flow controller 1000. The bottom portion 1040 defines a plurality of fluid line openings 1042, which are configured to allow a fluid line (not shown) to enter the interior bottom portion 1040.

[0223] Fig.15 and16 They are shown respectively Fig.14 The top and bottom of the cover 1030 are shown, and the bottom portion 1040 is not shown. The indicator openings 1034, 1036 and the fastener opening 1032 are also shown. Fig.15 and Fig.16 Shown in. Fig.16 Also shown is a cavity 1038 formed in the underside of the cover 1030 that is configured to hold a PCB (not shown) of the thermal control flow controller 1000. The PCB may include circuitry configured to control one or more thermostats 1004 (not shown) and / or one or more indicators (not shown). The cover 1030 may be made of a low thermal conductivity material such as a polymer (e.g., PVC).

[0224] Fig.17 Shows Fig.14 1002 and the heat sink 1002 are shown in the figure, without the cover 1030. The bottom portion 1040 includes a sleeve 1050 and a housing 1044 configured to hold the sleeve 1050. The bottom portion 1040 also defines a fastener opening 1048, which is configured to receive a fastener (e.g., a screw) for mounting the cover 1030 and the bottom portion 1040 on the heat sink 1002. In addition to holding the sleeve 1050, the housing 1044 also defines a plurality of fluid line openings 1042 ( Fig.18 ), which corresponds to the plurality of fluid line openings 1052 in the sleeve 1050 (as shown Fig.21 The fluid line opening 1042 extends completely through the housing 1044 within a horizontal plane of the housing 1044. Fig.18 1 is a perspective view from below of housing 1044. The perspective view angle shows two sets of corresponding fluid line openings 1042 and two cavities 1046 formed in the underside of housing 1044. Cavities 1046 in each housing 1044 are configured to hold temperature regulating devices 1004 (e.g., each having one or more Peltier thermoelectric devices (e.g., a stack of two or more Peltier thermoelectric devices, not shown) and wiring associated therewith (not shown).

[0225] Fig.19 A heat sink 1002 is shown that optionally defines two cavities 1060, each cavity 1060 being configured to hold a temperature regulating device 1004 (e.g., having one or more (e.g., two or more stacked) Peltier thermoelectric devices). Optionally, the heat sink 1002 is configured to be coupled to a secondary heat sink 1020 or a support 100, which can serve as a secondary heat sink.

[0226] Fig. 20 and Fig.21A sleeve 1050 is shown that is configured to accommodate 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 a groove configured to accommodate the flow segments of the fluid lines 1008. Therefore, the sleeve 1050 is an embodiment of the thermally conductive interface 1006. Therefore, the sleeve 1050 helps to keep the flow segments of the fluid lines 1008 near the temperature regulating device 1004 (not shown). The sleeve 1050 can be made of a high thermal conductivity (and low thermal capacitance) material such as a metal (e.g., copper). Fig.21 The side view in FIG. 1 shows an opening 1052 of the fluid line 1008 defined by the sleeve 1050. As shown, the fluid line opening 1052 passes completely through the sleeve 1050 in the horizontal plane of the sleeve 1050. The fluid line opening 1052 is substantially aligned with the housing 1044 (e.g., Fig.18 ) in the corresponding fluid line opening 1042, so that when the sleeve 1050 is disposed in the housing 1044 (as shown Fig.17 ), the fluid line 1008 can pass through the housing 1044 and the sleeve 1050. In addition, when the sleeve 1050 is disposed in the housing 1044 (as shown), the fluid line 1008 can pass through the housing 1044 and the sleeve 1050. Fig.17 ), the sleeve 1050 is placed in contact with the tops of two temperature regulating devices 1004 (eg, each of which may include one or more (eg, two or more stacked) Peltier thermoelectric devices, not shown).

[0227] In some embodiments, the thermal control flow controller 1000 further includes a thermistor (not shown). The thermistor is configured to monitor the temperature of the sleeve and / or the temperature regulating device 1004 (or its surface). The monitored temperature can provide feedback to indicate the open or closed state of the flow controller 1000.

[0228] In certain embodiments, as described above, the thermal control flow controller 1000 further includes a printed circuit board (PCB, not shown) or is operably coupled to a PCB. The PCB can be configured to interact with the thermistor. The PCB can also be configured to regulate the current (e.g., DC) delivered to the temperature regulating device 1004. In addition, the PCB can be configured to reduce the current delivered to the temperature regulating device 1004.

[0229] The above-described thermal control flow controller 1000 is robust and substantially eliminates dead space (compared to other fluid valves) where bacteria or other debris may accumulate and / or grow. In addition, the flow controller 1000 reduces microbial contamination associated with other types of valves. In addition, the flow controller 1000 limits the movement of fluid in a microfluidic device (e.g., microfluidic device 110) connected thereto, which movement would otherwise result in bending of fluid lines connected to the inlet and outlet of the microfluidic device. In order to optimize the system to minimize fluid movement within the microfluidic device, in practice, one or more flow controllers 1000 should be arranged as close as possible to the inlet and outlet of the microfluidic device.

[0230] Unfortunately, the thermal control flow controller 1000 has several limitations. In some configurations, the thermal control flow controller may take a long time to cool to the desired temperature and freeze the fluid line, thereby preventing accurate control of the flow of fluid into and out of the microfluidic device. In some cases, cooling to the desired temperature may take about 45 to about 90 seconds. Similarly, over a long period of time, the thermal control flow controller will accumulate moisture and form ice, thereby increasing the time required to thaw the fluid in the fluid line and reopen the valve. In some cases, it may be difficult to accurately control the temperature because the thermistor is not located near the fluid line. In addition, the thermal control flow controller contains many components required to connect the fluid line to the Peltier device.

[0231] In other embodiments, the thermal control flow controller may include one or more cryo valves that are not subject to these limitations. Figure 32 to Figure 33 Examples of these thermally controlled flow controllers are depicted in . Fig.32 (vertical section), the thermal control flow controller 2000 includes a base 2016 (or substrate) that can be mounted to a heat sink (not shown), which can be a secondary heat sink 1020 or a support 100. In some configurations, the base 2016 itself can be configured as a heat sink without being mounted to a separate heat sink.

[0232] The base 2016 can be connected to the pipe 2012. The pipe 2012 surrounds some other components and captures heat and conducts it to the base 2016, which can dissipate the heat when the base 2016 is configured as a heat sink, or conduct the heat to a separate heat sink (e.g., the secondary heat sink 1020 or the support 100). The base 2016 can be connected using any connector (including Fig.32 2010) is connected to the pipe 2012. Other connectors can be used, for example, pins, clamps, etc.

[0233] The top of the substrate 2016 and the bottom of the tube 2012 are each configured to abut or be adjacent to the Peltier device 2004. The Peltier device 2004 may include a 2-layer Peltier stack, as shown; alternatively, the Peltier device 2004 may include a 3-layer, 4-layer, or more layer stack. Fig.32 Two Peltier devices are shown, but additional Peltier devices may be used. The hot side of each Peltier device 2004 is positioned adjacent to or near the tube 2012 and the substrate 2016, respectively, so that heat can be conducted away from the Peltier device 2004. In some configurations, the substrate 2016 and the tube 2012 may be configured with one or more indentations 2009 that help stabilize the Peltier device 2004 after the thermal control flow controller 2000 is assembled.

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

[0235] In some embodiments, the thermal interface 2014 also includes a central portion to which a thermal sensor (e.g., a thermistor) can be coupled. The central portion can include a hole (e.g., 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 in one or more openings 2011.

[0236] The various components of the thermal control flow controller 2000 may be enclosed within the cover 2022, such as Fig.33 As shown (another vertical cross section, where the controller 2000 is relative to Fig.32 90° about the z-axis). The cover 2022 can be made of any material with low thermal conductivity (e.g., plastic). The thermal control flow controller 2000 can also include a barrier material located in any desired cavity of the controller 2000 (e.g., within the cover 2022). Fig.33 In the configuration shown, the barrier material 2024 can be inserted around the thermal interface 2014 and any gaps between the base 2016 and the conduit 2012 and any other gaps between components in 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 in any internal portion of the controller 2000. In some embodiments, the barrier material 2024 can include a polymer, such as polyurethane, etc. In some embodiments, the barrier material can include a spray foam or foam cutouts made of expanding foam (e.g., polyurethane foam).

[0237] like Fig.33 As shown, the thermal control flow controller 2000 may also include a guide 2020. The guides may be located on both sides of the thermal interface 2014 and may assist the user in feeding a fluid line (not shown) through the thermal interface 2014. The guide 2020 may be made of any material with low thermal conductivity (e.g., plastic). The guide 2020 may be part of a cover 2022 included with the thermal control flow controller 2000.

[0238] The pipe 2012 of the thermal control flow controller 2000 includes a bridge-like design. It is capable of conducting heat from the hot sides of the two sandwiched Peltier devices to the same heat sink. Therefore, the ability of the thermal control flow controller 2000 to transfer heat away from the Peltier device is improved relative to the thermal control flow controller 1000.

[0239] exist Figure 34 to Figure 35Other embodiments of the thermal control flow controller are depicted in . In these embodiments, the thermal control flow controller 3000 includes a base 3016 (or substrate) that can be mounted to a heat sink (not shown), such as the secondary heat sink 1020 or the support 100. In some configurations, the base 3016 itself can be configured as a heat sink without being mounted to a separate heat sink.

[0240] The base 3016 includes an upper surface, a portion of which has been configured to mate with other components of the controller 3000. The upper mating portion 3012 of the base 3016 can be configured to mate with and abut the bottom of the Peltier device 3004 and / or mate with the bottom of the cover 3022. Fig.34 There are three Peltier stacks in the Peltier device 3004 shown in FIG, but fewer (e.g., 2) or more (e.g., 4 or more) Peltiers may be included. In some cases, only a single Peltier may be used in the Peltier device 3004. Each tie of the Peltier stack increases the absolute temperature difference between the hot and cold surfaces of the Peltier device, thereby reducing the total heat flux that the Peltier device stack needs to maintain. When operating the thermal control flow controller 3000, the liquid in the fluid line is cooled (and sometimes supercooled) to a temperature sufficient to nucleate ice. Although this result can be achieved using a single-joined Peltier device, it can be more easily achieved using a multi-joined structure.

[0241] The Peltier device 3004 is configured to have a thermally conductive interface 3014 (or cold head) resting on a joint structure (e.g., the top surface of). In some configurations, the thermally conductive interface 3014 can be configured to have similar dimensions to the topmost Peltier of the multi-joint structure. The thermally conductive interface 3014 can include one or more (e.g., two, three, four, six, eight) openings 3011, which can be used to surround corresponding one or more fluid lines (not shown), which are used to input and / or remove fluids from one or more microfluidic devices, as described herein. In a preferred embodiment, the thermally conductive interface 3014 includes two openings 3011, which are configured to surround an inlet and outlet fluid line pair leading to a single microfluidic device 110. In other embodiments, the thermally conductive interface 3014 includes two openings 3011, which are configured to surround each of the two inlet (or outlet) fluid lines 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 each of two microfluidic devices 110, or it can controllably freeze / thaw each of four inlet (or outlet) fluid lines leading to four separate microfluidic devices 110, and so on.

[0242] In some embodiments, the thermal interface 3014 also includes a central portion to which a thermal sensor 3015 (e.g., a thermistor) can be coupled. The central portion can include a hole (e.g., central hole 3013) in which the thermal sensor 3015 can be located. The thermal sensor 3015 is used to measure the temperature of the fluid line located in one or more openings 3011.

[0243] The various components of the thermal control flow controller 3000 may be enclosed by a cover 3022, such as Fig.35 (perspective view of a vertical cross section). The cover 3022 can be made of any material with low thermal conductivity (e.g., plastic). The thermal control flow controller 3000 can also include a barrier material located in any desired cavity of the controller 3000 (e.g., within the cover 3022). Fig.35 In the configuration shown, a barrier material (not shown) may be inserted around the thermal interface 3014 and any gaps between the Peltier stack 3004 and the cover 3022, as well as any other gaps between components in the controller 3000. The barrier material prevents or reduces the ability for 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, etc. In some embodiments, the barrier material may include a spray foam or foam cutouts made from any expanding foam (e.g., polyurethane foam).

[0244] like Fig.35 As shown, the thermal control flow controller 3000 can include a guide 3020. The guide 3020 can be located on both sides of the thermal interface 3014 and assist the user in feeding a fluid line (not shown) through the thermal interface 3014. The guide 3020 can be made of any material with low thermal conductivity (e.g., plastic). The guide 3020 can be part of a cover 3022 included in the thermal control flow controller 3000.

[0245] The thermal control flow controllers 2000 and 3000 may have a thermal interface (or cold head) having a diameter of about 9 mm. 2 To about 25mm 2 , or about 10mm 2 To about 20mm 2 , or about 13mm 2 To about 18mm 2 In some embodiments, the thermal interface may have a contact surface of about 20 mm (e.g., a surface that contacts a Peltier device). 3 To about 60mm 3 , or about 25mm 3 To about 50mm 3 , or about 30mm 3To about 40mm 3 volume. This small thermal mass, coupled with a relatively large surface area for contacting the Peltier device, can reduce the time required to cool (or subcool) the fluid lines and the fluid flowing through the fluid lines. In certain embodiments, the thermally controlled flow controllers 2000 and 3000 can achieve freezing of fluid within a flow segment of one or more fluid lines in a time of 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 a range from about 20 seconds to about 35 seconds, from about 20 seconds to about 30 seconds, or from about 23 seconds to about 27 seconds). In certain related embodiments, the thermally controlled flow controllers 2000 and 3000 can achieve thawing of frozen fluid within a flow segment of one or more fluid pipelines in a time of 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 a range from about 25 seconds to about 40 seconds, from about 25 seconds to about 35 seconds, or from about 28 seconds to about 32 seconds).

[0246] The thermal control flow controllers 2000 and 3000 also contain openings for fluid lines to the microfluidic devices. The associated guides in these devices allow blind routing of the fluid lines through the cold head, making them easy to assemble.

[0247] The thermal control flow controllers 2000 and 3000 also contain a barrier material. The barrier material acts as a moisture barrier and keeps the Peltier device (and therefore the controllers 2000 and 3000) operational for extended periods of time without the accumulation of ice that would degrade performance.

[0248] In some embodiments, the support 100 may also include or interact with an O2 and CO2 source configured to maintain culture conditions. In some embodiments, the support 100 may also include or interact with a humidity monitor / regulator.

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

[0250] In some 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 can be, for example, a projector (e.g., a video projector or a digital projector). An example of a suitable light modulation subsystem is from ANDOR TECHNOLOGIES TM MOSAIC TM In other embodiments, the light modulation subsystem 634 may include a micro shutter array system (MSA), which may provide improved contrast. In other embodiments, the light modulation subsystem 634 may include a scanning laser device. In some embodiments, the light modulation subsystem 634 is capable of emitting structured and unstructured light.

[0251] In some embodiments, the support 100 and the light modulation subsystem 634 are each individually configured to be mounted on a microscope, such as a standard research-grade optical microscope or a fluorescence microscope. For example, the support 100 can be configured as a mounting platform for a microscope. The light modulation subsystem 634 can be configured to be mounted on a port of a microscope.

[0252] Thus, in some embodiments, the system can be used in a method of converting an optical microscope into a microscope configured for operating a microfluidic device 110. The method can include the steps of mounting a system including a support 100 (e.g., as described herein) and a light modulation subsystem 634 (e.g., as described herein) on a suitable microscope. The support 100 can be mounted to a platform of the optical microscope, and the light modulation subsystem 634 can be mounted to a port of the optical microscope. In some 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).

[0253] In other embodiments, the support 100 and the light modulation subsystem 634 described herein can be an integral component 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).

[0254] In certain related embodiments, the system provides a microscope configured to operate a microfluidic device 110. The microscope may 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 may be configured to (1) receive structured light from the light modulation subsystem 634 and focus the structured light on at least a first region in 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 a detector 602. When the device 110 is held by the support 100, the optical system may be further configured to receive unstructured light from a second light source 622 and focus the unstructured light on at least a second region of the microfluidic device 110. In certain embodiments, the first region and the second region of the microfluidic device 110 may be overlapping regions. For example, the first region may be a subset of the second region.

[0255] In some embodiments, the microscope of the system may further include one or more detectors 602. The detector 602 may include, but is 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. If there are at least two detectors 602, one detector 602 may be, for example, a fast frame rate camera, and the other detector 602 may be a high sensitivity camera. The microscope may further include an eyepiece configured to be visible to the user. In addition, the optical system may be configured to receive light reflected and / or emitted 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 further include different tube lenses for different detectors 602, so that the final magnification of each detector 602 may be different.

[0256] In some embodiments, the light modulation subsystem 634 of the microscope of the 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. In addition, the DMD, LCD, LCOS, FLCOS, and / or 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), which may provide improved contrast. In some embodiments, the microscope of the 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.

[0257] In some embodiments, the system 600 / microscope of the 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 the light modulation subsystem 634 to form modulated structured light 652 for optically driven electrical activation and / or fluorescence excitation. The second light source 622 can be used to provide background illumination for bright field or dark field imaging (e.g., using unstructured light 654). An example of such a configuration is shown in FIG. Figure 6 6. A first light source 632 is shown providing structured light 650 to a light modulation subsystem 634, which provides modified structured light 652 to the optical system of the microscope. A second light source 622 is shown providing unstructured light 654 to the optical system via a beam splitter 624. The modified structured light 652 from the light modulation subsystem 632 and the unstructured light 654 from the second light source 622 travel together through the optical system to a beam splitter 606, where the light 652, 654 is reflected downwardly to a sample plane 610 by an objective 608 (which may be a lens). Reflected and / or emitted light 662, 664 from the sample plane 610 then passes through the objective 608, travels back through the beam splitter 606 to the dichroic filter 604. The light 662, 664 may be modulated to reflect the structured light 652 and the unstructured light 654, respectively, from the sample plane 610. Alternatively, light 662, 664 may originate at or below sample plane 610. Only a portion of light 662, 664 that reaches dichroic filter 604 passes through filter 604 and reaches detector 602. Depending on how the system is used, beam splitter 606 may be replaced with a dichroic filter (e.g., to detect fluorescent emissions originating from sample plane 610 or below sample plane 610).

[0258] like Figure 6As shown, the second light source 432 emits blue light. The blue light reflected from the sample plane 610 is able to pass through the dichroic filter 604 and reach the detector 602. In contrast, the structured light from the light modulation subsystem 634 is reflected from the sample plane 610, but does not pass through the dichroic filter 604. In this example, the dichroic filter 604 filters out visible light with wavelengths longer than 495nm. If the light emitted from the light modulation subsystem 634 does not include any wavelengths shorter than 495nm, this filtering will only be completed for the light from the light modulation subsystem 634 (as shown). In practice, if the light from the light modulation subsystem 634 includes wavelengths shorter than 495nm (such as blue light wavelengths), some of the light from the light modulation subsystem 634 will pass through the filter 604 to the detector 602. In this case, the filter 604 is used to change the balance between the amount of light reaching the detector 602 from the first light source 632 to the second light source 622. This is beneficial if the first light source 632 is significantly stronger than the second light source 632.

[0259] To achieve the same goal, i.e., to change the balance between the amount of light reaching the detector 602 from the first light source 632 and the second light source 622, Figure 6 An alternative to the arrangement shown is to have a second light source 622 emitting red light and a filter 604 that filters out visible light having wavelengths shorter than 650 nm.

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

[0261] In some embodiments, the first light source 632 can emit a broad spectrum of wavelengths (e.g., "white" light). The first light source 632 can emit, for example, at least one wavelength suitable for exciting a phosphor. The first light source 632 can be powerful enough so that the structured light emitted by the light modulation subsystem 634 can activate photophoresis in the optically actuated microfluidic device 110. In some embodiments, the first light source 632 can include, for example, those high-intensity discharge arc lamps including metal halides, ceramic discharges, sodium, mercury, and / or xenon. In other embodiments, the first light source 632 can include one or more LEDs (e.g., an LED array, such as 4 LEDs in a 2x2 array or 9 LEDs in a 3x3 array). The LEDs can include broad-spectrum "white" light LEDs (e.g., UHP-T-LED-white light by PRIZMATIX) or various narrow-band wavelength LEDs (e.g., emitting wavelengths of about 380nm, 480nm, or 560nm). In other embodiments, the first light source 632 can include a laser configured to emit light of a selectable wavelength (e.g., for OET and / or fluorescence).

[0262] In some embodiments, the second light source 622 is suitable for bright field illumination. Therefore, the second light source 622 may include one or more LEDs (e.g., an LED array, such as 4 LEDs of a 2x2 array or 9 LEDs of a 3x3 array). The LED may be configured to emit white (i.e., wide spectrum) light, blue light, red light, etc. In some embodiments, the second light source 622 may emit light with a wavelength of 495nm or shorter. For example, the second light source 622 may emit light with a wavelength of approximately 480nm, approximately 450nm, or approximately 380nm. In other embodiments, the second light source 622 may emit light with a wavelength of 650nm or longer. For example, the second light source 622 is capable of emitting light with a wavelength of approximately 750nm. In other embodiments, the second light source 622 may emit light with a wavelength of approximately 560nm.

[0263] In some embodiments, the optical system of the microscope of the system includes a dichroic filter 604 that at least partially filters out visible light having a wavelength longer than 495 nm. In other embodiments, the optical system of the microscope of the system includes a dichroic filter 604 that at least partially filters out visible light having a wavelength shorter than 650 nm (or less than 620 nm). More generally, the optical system may 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 may be located near the detector 602 (along the optical system). Alternatively, the optical system may include one or more dichroic filters 604 that are 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. This balance may be used to ensure that structured light does not dominate unstructured light at the detector 602 (or in an image obtained by the detector 602 ).

[0264] In some embodiments, the optical system of the microscope of the system may include an objective lens 608 configured to focus structured and unstructured light on the microfluidic device 110, the objective lens being selected from 100x, 60x, 50x, 20x, 10x, 5x, 4x, or 2x. These magnifications are listed for illustration, not limitation. The objective lens may have any magnification.

[0265] The microscope of the system may include any support 100 described herein. Thus, for example, the support 100 may include an integrated electrical signal generation subsystem 138 configured to at least intermittently establish 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 may include a thermal control subsystem 140 configured to regulate the temperature of the microfluidic device 110 when the device 110 is held by the support 100.

[0266] Any system or microscope 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).

[0267] Figure 7 A structured light path 700 in an optical system is shown according to some embodiments of the system. Figure 7 The structured optical path 700 shown in FIG. 1 starts with a DMD 702 including a glass cover 704 (e.g., a 20 mm glass plate). The DMD 702 may be similar to Figure 6 702 modifies light from a light source (not shown) to form structured light 708. The structured light 708 is then focused on a tube lens 706 toward an objective lens 710 (which may be a lens). The objective lens 710 in turn focuses the structured light 708 onto a cover 712 (e.g., a cover glass). The cover 712 may be a cover of 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.

[0268] Figure 7 Also shown is an imaging optical path 750 in an optical system according to some embodiments of the system. Figure 7 The imaging optical path 750 shown in FIG. 1 starts from a sample plane 752, which may coincide with the cover 712 of the microfluidic device 110. The sample plane 752 may be similar to Figure 6 750 is a perspective view of the sample plane 610 shown in FIG. 750. Thus, light 758 in the imaging light path 750 may be reflected from the sample plane 752. Alternatively, the light 758 may have passed through the sample plane 752. From the sample plane 752, the light 758 is focused by the objective lens 754 and the achromatic tube lens 756 toward the camera plane 760. The camera plane 760 may coincide with a detector (not shown), similar to Figure 6Detector 602 is shown. In this manner, imaging optical path 750 can be used to visualize a sample or a portion thereof disposed at a sample plane 752 (eg, contained within microfluidic device 110).

[0269] Fig. 22 A system 600 is shown, having a configuration similar to Figure 6 The optical system shown. Fig. 22 In the illustrated system 600, the second light source 622 and the beam splitter 624 are disposed in the main light path between the sample plane 610 and the detector 602, rather than as in Figure 6 662, 664. In such an embodiment, the size and shape of the second light source is designed and configured so that it does not interact with the reflected and / or emitted light 662, 664. In addition, the beam splitter 624 can only be used as a filter to modify the unstructured light 654 from the second light source 622 without changing the direction of the unstructured light 654. In other embodiments, the system 600 may not include the beam splitter 624.

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

[0271] Fig.23 Two LED arrays are shown that can be used as light sources in the system 600 described herein. The first LED array 1102 includes four LEDs in a 2×2 array. The second LED array 1104 includes 9 LEDs in a 3×3 array. The square array produces a higher light intensity per unit area than the non-square array. The LEDs in the array can have the same color / wavelength (e.g., ultraviolet, 380nm, 480nm, or 560nm). Alternatively, various subsets in the LED array can have different colors / wavelengths. In addition, the LEDs can emit narrowband wavelengths (e.g., 450nm wavelength) by themselves, but when excited with narrowband wavelengths, the LEDs can be coated with phosphor materials to emit white light.

[0272] Fig.24 A light pipe (or optical integrator) 1112 is shown, which may be configured to receive and transmit signals from, for example, Fig.23The light source of one of the LED arrays 1102, 1104 shown. The light pipe 1112, also known as a "non-imaging collection optical device", is configured to propagate light from one end thereof (i.e., the input aperture) to the other end thereof (i.e., the output aperture), and the light emitted from the output aperture has a substantially uniform intensity (i.e., the flux of light passing through a first area of ​​defined dimensions at the plane of the output aperture is substantially the same as the flux of light passing through any other area at the plane of the output aperture having the same defined dimensions). The main body wall of the light pipe 1112 can be constructed of clear glass or clear plastic. The light pipe 1112 can be obtained from, for example, EDMOND OPTICS.

[0273] Fig.25 A light source 1122 is shown that includes a plurality of 3×3 LED arrays 1104 coupled to a surface 1124. The surface 1124 can be an LED board. The light source 1122 can be disposed within the system such that it is movable relative to an aperture configured to receive light emitted from the light source 1122. For example, the system can include a light pipe / optical integrator 1112, the input aperture 1112 of which can be configured to receive light emitted from one of the plurality of LED arrays 1104 coupled to the surface 1124. Thus, different LED arrays 1104 can be used as the light source (e.g., via the light pipe / optical integrator 1112), depending on the relative positions of the surface 1124 of the light source 1122 and the light pipe / optical integrator 1112.

[0274] Fig.26 A multi-input light pipe / optical integrator 1132 is shown. The multi-input light pipe 1132 has multiple (e.g., 3) input apertures, each associated with a light propagation path and a respective light source 1134, 1136, 1138, and a fewer (e.g., 2) dichroic filters 1140, 1142. Each dichroic filter 1140, 1142 is configured to reflect light from a corresponding light source 1136, 1138. Fig.26The illustrated multi-input light pipe 1132 has a first light source 1134, a second light source 1136, and a third light source 1138, any of which may be an LED array (e.g., a 2x2 or 3x3 array of LEDs). The first light source 1134 may be an LED array that emits light at approximately 380 nm. The second light source 1136 may be an LED array that emits light at approximately 480 nm. The third light source 1138 may be an LED array that emits light at approximately 560 nm. Thus, the wavelength of light emitted from the multi-input light pipe 1132 may be controlled by selectively activating the first light source 1134, the second light source 1136, and the third light source 1138. The multi-input light pipe 1132 is configured such that light from any one of the light sources 1134, 1136, 1138, or any combination thereof, entering the corresponding input aperture will have a substantially uniform intensity when emitted from the output aperture. The body wall of the multi-input light pipe 1132 may be constructed of clear glass or clear plastic.

[0275] In some embodiments, the microscope of the 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 photoactuated electrical activation, phosphor excitation, and bright field illumination. In this arrangement, structured illumination can be used to compensate for optical vignetting or any other arbitrary non-uniformity in the illumination. Optical vignetting is the appearance of illumination 804 toward the edges of the field of view 802 (e.g., Fig. 8A ). The light intensity of a single light source can be measured pixel by pixel, and the resulting information is used to generate an inverse optical vignetting function 814 (e.g. Figure 8B ). The inverse optical vignetting function 814 may then be used to adjust the light output from the light modulation subsystem, thereby producing a uniform illumination field 824 (e.g., Figure 8C )

[0276] The system also provides a method for manipulating a micro-target in an optically actuated microfluidic device 110 using light. The method includes placing the optically actuated microfluidic device 110 on a support 100 of any of the systems or microscopes described herein, placing a micro-target on or in the optically actuated microfluidic device 110, focusing structured light from the light modulation subsystem 634 onto a first area on the surface of the optically actuated microfluidic device 110, and moving the focused structured light to a second area on the surface of the optically actuated microfluidic device 110. If the micro-target is located near the first area, moving the focused light can cause a directional movement of the micro-object. The focused structured light can be used, for example, to create a grating around the micro-target. Alternatively, the focused structured light can be used to at least partially contact a fluid droplet containing the micro-target.

[0277] In another embodiment of the method of using light to manipulate micro-targets in an optically actuated microfluidic device 110, the light pattern is spatially fixed and the optically actuated microfluidic device 110 moves relative to the light pattern. For example, the optically actuated microfluidic device 110 can be moved using a motorized or mechanical microscope stage, which 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-target (e.g., a biological cell or a solution droplet optionally containing a micro-target of interest) on a manipulable stage.

[0278] In other embodiments, the system for operating the microfluidic device can be configured with an inlet for directly (e.g., manually or automatically) introducing a fluid sample into the microfluidic device. In the above-described embodiment, the fluid sample is introduced (and removed) by the first fluid input / output pipeline 112 and the second fluid input / output pipeline 118. The internal volume of the microfluidic device can be limited to, for example, 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 approximately 10 to approximately 50 microliters, approximately 10 to approximately 40 microliters, approximately 10 to approximately 30 microliters, approximately 5 to approximately 25 microliters, approximately 5 to approximately 20 microliters, approximately 5 to approximately 15 microliters, approximately 2 to approximately 10 microliters, or approximately 2 to approximately 5 microliters). In some cases, only about half of this fluid amount (for example, about 25 microlitres or less, about 20 microlitres or less, about 15 microlitres or less, about 10 microlitres or less, or about 2 to about 10 microlitres, or about 1 to about 5 microlitres) usually flows through microfluidic device, because the other half of fluid is kept relatively static by microfluidic device to analyze. Fluid sample that flows into (and flows out of) microfluidic device by first fluid input / output pipeline or second fluid input / output pipeline forms relatively discrete fluid packet ideally, because only limited amount of fluid can be inserted in microfluidic device at any given time. However, the length of fluid line between pump and microfluidic device for first and second fluid input / output pipeline can be very long (for example, about 50cm, about 75cm, about 100cm, about 125cm, about 150cm or longer) and has the internal volume far greater than about 5 microlitres. Therefore, very small fluid sample at the beginning can be thinned or dispersed as moving through fluid line before being introduced in microfluidic device. Furthermore, as the sample becomes dispersed, micro-objects (eg, cells or beads) within the sample may become unevenly distributed in the fluid sample, resulting in uneven loading of micro-objects between channels within the microfluidic device.

[0279] Figure 27 to Figure 3The exemplary embodiment shown in 1 reduces or prevents the dispersion of fluid samples and the related non-uniform distribution of micro-objects in the fluid samples, because the sample can be directly introduced into the microfluidic device. In these embodiments, the microfluidic device is maintained by a socket as a part of a support. The socket includes a cover, which can be divided into 2 (or more) parts. One of these parts can be separated from the other parts so that the part no longer covers (or contacts) the microfluidic device, thereby allowing the fluid sample to be directly introduced into the microfluidic device without the need to flow the sample through the fluid line. At the same time, the other parts of the cover remain in place, thereby maintaining the microfluidic device in the socket.

[0280] like Fig. 27 As shown, in these embodiments, the system for operating a microfluidic device includes a support 1200 substantially similar to the support 100, a socket 1206 substantially similar to the socket 106, a first fluid input / output line 1212 substantially similar to the first fluid input / output line 112, and a second fluid input / output line 1218 substantially similar to the second fluid input / output line 118. In certain embodiments, the socket 1206 includes a surface 1203 configured to support a microfluidic device 1210 and a cover 1204 configured to secure the microfluidic device 1210 within the socket 1206. The surface 1203 of the socket 1206 can include a substantially flat area that engages a corresponding substantially flat bottom surface of the microfluidic device 1210. The resulting interface can operably couple the microfluidic device 1210 to the socket 1206, for example, to establish a functional interconnection, such as an electrical connection. Alternatively or additionally, the socket 1206 may include features (e.g., pins, grooves) extending from or into the surface 1203. These features may engage with the microfluidic device 1210 to 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, for example, an electrical connection. The cover 1204 may engage with the top surface of the microfluidic device 1210. The resulting interface may operably couple the microfluidic device 12 with one or both of the first fluid input / output line 1212 and the second fluid input / output line 1218. In some embodiments, the cover 1204 may be connected to the surface 1203, for example, by a hinge or the like. In some related embodiments, the cover 1204 may include a latch (or other fixing mechanism, such as a screw, a pin, a clamp, etc.) configured to hold the cover 1204 in a closed position. Thus, the latch can facilitate forming an interface between the cover 1204 and the top surface of the microfluidic device 1210 .

[0281] One or both of the first fluid input / output line 1212 and the second fluid input / output line 1218 can be connected to a pump at one end and to a fluid port (not shown) included in the cover 1204 at the other end. The fluid port can engage both the end of the fluid line and the inlet / outlet of the microfluidic device 1210, thereby forming a fluid connection between the fluid line and the inlet / outlet. Alternatively or additionally, one of the first fluid input / output line 1212 or the second fluid input / output line 1218 can be connected to the fluid port at one end and to a container at the other end, such as a waste container or a container for holding a sample (e.g., a sample to be introduced into the microfluidic device 1210 or a sample that has been output from the microfluidic device 1210). Optionally, the fluid port includes a seal, a compression fitting, etc. to ensure a leak-proof connection between its corresponding fluid line and the microfluidic device.

[0282] In some embodiments, the cover 1204 includes two parts: a first part 1204A and a second part 1204B. Fig. 27 As shown, the second portion 1204B of the cover can be separated from the first portion 1204A. This allows the second portion 1204B to be separated from the first portion 1204A. Fig. 27 1204B) to an open position that allows access to an inlet / outlet on the microfluidic device (e.g., an inlet / outlet located on the upper surface of the microfluidic device 1210). An example of an open position of the second portion 1204B of the cover is shown in FIG. Fig.28 and Fig.29 The second portion 1204B can be moved to any number of open positions away from the microfluidic device 1210, not just Fig.28 and Fig.29 4B is in the open position. When the second portion 1204B is in the open position, the first portion 1204A of the cover remains in place, so that the microfluidic device 1210 remains on the surface of the support 1200 even if the second portion 1204B is moved. While the second portion 1204B of the cover is in the open position, the first fluid input / output line 1212 can be connected to the fluid port on the first portion of the cover 1204A, thereby remaining in place (e.g., maintaining the fluid connection between the first fluid input / output line 1212 and the corresponding inlet / outlet of the microfluidic device 1210). The second fluid input / output line 1218, which can be connected to the fluid port on the second portion 1204B of the cover, moves with the second portion 1204B, thereby decoupling the fluid connection between the second fluid input / output line 1218 and the corresponding inlet / outlet of the fluidic device 1210.

[0283] In some embodiments, when the second portion 1204B of the cover is in the open position, the insert can be placed in the position previously occupied by the second portion 1204B of the cover when it is in its closed position. In some configurations, the insert can be shaped substantially similar to the second portion 1204B of the cover. However, in other configurations, the insert can be shaped differently. The insert can provide a variety of functions. The first function is to prevent contamination that may potentially be caused by exposing the inlet / outlet of the microfluidic device 1210, which is covered when the second portion 1204B of the cover is in the closed position. In some embodiments, the insert includes a fluid inlet (e.g., a well) through which a fluid sample can be introduced directly into the microfluidic device 1210. The fluid inlet of the insert is positioned to engage with the inlet / outlet of the microfluidic device 1210, which engages with the fluid port 1222 of the second fluid input / output line 1218 when the second portion 1204B of the cover is in the closed position. An example of an insert is Fig.28 The insert 1207 shown in FIG. 1 includes a custom fluid well for inserting a fluid sample. Another example of an insert is Fig.29 1209, which includes a fluid trap that has not yet been customized. As can be seen, the fluid trap of insert 1207 is larger than the fluid trap of insert 1209 and includes a funnel-shaped design. Regardless of its exact shape, the fluid trap can be configured to hold 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 above endpoints, such as about 5 microliters to about 25 microliters) of a fluid sample.

[0284] In some configurations, you can use FIG. 30A to FIG. 30C In these embodiments, the second portion 1204B of the cover includes a latch 1205 and a hinge 1225, which can be as shown in FIG. Fig. 30A The latch 1205 is configured to releasably hold the second portion 1204B of the cover in a closed position. The latch 1205 may be configured as shown in FIG. Fig. 30B 1204B. This action releases the second portion 1204B of the cover from its closed position covering the microfluidic device 1210. Of course, the latch 1205 and its actuation can have any other number of configurations, and fixing mechanisms other than latches, such as clamps, friction locks, screws, magnets, etc., can replace the latch 1205. Once the second portion 1204B of the cover has been released from its position on the microfluidic device 1210, it can be moved to any desired position by rotating the second portion 1204B about the hinge 1225, including Fig. 30C The location shown in . Fig. 30C The position shown in FIG. 1 is rotated about 180° from the closed position, but any rotation angle 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 1204B of the cover can be rotated at least about 60°, about 75°, about 90°, about 105°, about 120°, about 135°, about 150°, or more to achieve the open position of the second portion 1204B. Fig. 30C As shown, an insert such as insert 1209 may be placed in the location where the second portion 1204B of the cover was previously located.

[0285] The insert (including insert 1207 or 1209) can be configured to be operably coupled to the socket 1206 and / or the microfluidic device 1210 so that the second fluid inlet / outlet of the fluid medium flowing into the microfluidic device can be reliably achieved. For example, the insert can be configured to engage with the first portion 1204B of the cover. The insert typically includes features that can be used to (i) secure the insert and / or remove the insert from the socket 1206 and / or the microfluidic device 1210, and / or (ii) align the insert with the microfluidic device 1210. One such feature is a retaining mechanism 1215 (such as a retaining mechanism 1215) that helps hold the second portion 1204B of the cover in place. Fig.31B In some configurations, the retaining mechanism includes one or more magnets oriented to form an attractive interaction with one or more corresponding magnets in matching locations on the first portion 1204A of the cover and / or on the surface of the microfluidic device 1210. Fig.31B , a single magnet is shown interacting with a corresponding magnet (not shown) on the first portion 1204A of the cover. Another possible feature of the insert is an alignment feature that helps align the insert to the correct position above the microfluidic device so that a second fluid input / output line of the microfluidic device 1210 is operably connected to the fluid inlet of the insert. The alignment feature may include, for example, one or more pins 1217 that fit within matching holes 1213 in the underside and optionally extend through the insert. However, registration features may be used in place of pins for this alignment function. Similar retaining and / or alignment features can facilitate proper positioning and / or alignment of the second portion 1204B of the cover with the first portion 1204A of the cover, such as Fig.31B (including corresponding retaining mechanisms 1216 (e.g., magnets) and alignment features 1214 (e.g., holes for adapter pins 1217)) and as shown elsewhere in this document.

[0286] With the insert in place, e.g. Fig.31A1207, a small fluid sample can be introduced into the microfluidic device 1210 for analysis. In some configurations, such a small fluid sample can be introduced manually, for example, using a pipette / micropipette 1220, such as Fig.31C In an alternative embodiment, a small fluid sample can be automatically introduced, for example, using a pipette / micropipette 1220. A fluid inlet (e.g., Fig.31B 1219) to introduce a small fluid sample into the microfluidic device 1210. In these configurations, the first input / output fluid line 1212 remains engaged with the fluid port of the first portion 1204A of the cover and is fluidically connected to the inlet / outlet of the microfluidic device 1210. Since the first input / output fluid line 1212 also remains connected to the pump, the first input / output fluid line 1212 can ensure that the pump can pull (using suction or other force) at least a portion of the fluid sample from the fluid inlet of the insert (e.g., the well 1219) into the microfluidic device 1210 and through the microfluidic device 1210. This action maintains the desired rate of fluid flow in the microfluidic device and allows the microfluidic device 1210 to analyze all or part of the fluid sample. The suction (or other force) can be sufficient to pull a preselected volume of sample fluid into the microfluidic device 1210. For example, the preselected volume can be equal to + / - about 100% of the flow volume within the microfluidic device, wherein the flow volume is the volume of the microfluidic device that experiences flow when the medium flows through the microfluidic device (i.e., the swept area as described in U.S. Pat. No. 10,010,882). In certain embodiments, the preselected volume can be about 1 microliter to about 25 microliters (e.g., about 1.5 microliters to about 20 microliters, about 2 microliters to about 15 microliters, about 2.5 microliters to about 10 microliters, about 3 microliters to about 7 microliters, or any range bounded by two of the above endpoints) of the fluid sample, and the suction is stopped after the preselected volume of the fluid sample is achieved.

[0287] In some embodiments, it may be helpful to know whether the second portion of the split cover is in place, whether the insert 1207 is in place, or whether neither the second portion of the split cover nor the insert is in place over the microfluidic device. In these embodiments, the system may be modified with a sensor to detect whether the second portion of the split cover or the insert is present over the microfluidic device. Figure 36 to Figure 43 Some embodiments of the sensor are depicted in .

[0288] like Fig.36 As shown, the system for operating the microfluidic device 1210 may include a support 1200, a socket 1206 including a substrate 1201, a split cover 1204 having a first portion 1204A and a second portion 1204B, and a sensor 1300. The second portion 1204B of the split cover may include a latch 1205, such as Fig.36 as shown, and can be moved to the open position, as Fig.37 As shown. The cover 1204 may also 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 cover with the first portion 1204A of the cover. The base 1201 of the microfluidic device 1210 and the socket 1206 may be located on a support 1200, which may include a substrate 1390, such as a printed circuit board (PCB).

[0289] exist Fig.38 13. An exemplary sensor 1300 is depicted in FIG. The sensor 1300 may include a sensor cover 1302, a magnetic assembly 1304, an extension 1310, a housing 1312, and a connector 1314. The sensor cover 1302 operates to protect and insulate some components of the sensor 1300. The magnetic 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 sensing the presence of a cover or insert. The first housing 1306 insulates and protects the magnets 1308. In some embodiments, the first housing 1306 includes a bottom portion that includes one or more openings to allow each magnet 1308 to contact an upper surface of the extension 1310. In some embodiments, the first housing may be engaged with the second housing 1312 via a fastening mechanism (e.g., an opening in the first housing 1306 that mates with a post in the second housing 1310, a bolt, a clamp, glue, etc., and any combination thereof). The first end of each extension 1310 is configured to be connected to the second housing 1312, for example, via an opening (such as Fig.38 The second opposite end of each extension is configured to controllably extend downward through an opening in the bottom of the housing 1312. Connectors 1314 (e.g., screws, as shown) may be used to attach the various components of the sensor 1300 together.

[0290] The various components of the sensor are Fig.39 The sensor cover 1302 is transparent so that the rest of the components can be seen. Fig.39 Also shown is how the sensor 1300 may be attached to the socket 1206, for example, at a peripheral or angular location.

[0291] exist Fig.40 A side view of the sensor 1300 attached to the base 1201 of the socket 1206 and in operation can be seen in FIG. The figure shows the split cover 1204 attached to the base 1201 of the socket 1206, which rests on the base plate 1390 included in the support 1200. Fig.40 As shown, the sensor 1300 is located at the interface between the edge of the split cover 1204 and the base 1201. The second part 1204B of the split cover 1204 has been equipped with an actuator 1355 (e.g., a screw, a pin, etc.) that contacts one of the extensions 1310 and forces the extension 1310 downward when the second part 1204B is in the closed position.

[0292] like Fig.40 As shown at the bottom of , the end of the extension 1310 is forced downward so that it interrupts the optical switch by preventing light from a first element of the optical switch (e.g., an LED) from reaching a second element of the optical switch (e.g., a phototransistor). Fig.41 An optical switch 1365 is depicted in FIG. 1 , with the remainder of the system not depicted except for a substrate 1390 on which the optical switch 1365 is located. Fig.41 Two optical switches 1365 are depicted, corresponding to Fig.38 1310. However, depending on the desired functionality of the sensor, the sensor may include a single optical switch 1365 and a single extension 1310, or three or more optical switches 1365 and corresponding extensions 1310. The optical switches may be connected to a circuit that is part of the electrical signal generating subsystem of the support 1200. Each optical switch 1365 is positioned below a single extension 1310. When the corresponding extension 1310 is forced downward, it interrupts the optical signal of the optical switch 1365 and signals the presence of the actuator 1355.

[0293] like Fig.42 and Fig.43 As shown, the split cover 1204 and the inserts 1207, 1209 can each be configured to include an actuator, but in different positions. The split cover 1204 can be configured with the actuator in a first position 1361 so as to be located above the first of the extensions 1310. The insert 1207 can be configured with the actuator in a second position 1362 so as to be located above the second of the extensions 1310. Of course, the actual position of the actuator can be changed, as long as it is known which position is associated with the split cover 1204 and which position is associated with the insert 1207. A similar configuration of optical switches and extensions can also be used to determine whether the insert 1207 or the insert 1209 is located above the microfluidic device.

[0294] In the presence of sensor 1300, the system can detect the presence of the removable portion of the split cover, the insert, or even when neither is present. When the second portion 1204B of the split cover 1204 is located above the microfluidic device 1210, the actuator in the removable portion 1204B can be located, for example, in a first position 1361 and can force the lower extension 1310 in the optical sensor 1365 downward and interrupt the signal between the associated optical switches. When the inserts 1207, 1209 are located above the microfluidic device 1210, the actuator in the insert can be located, for example, in a second position 1362 and can force the lower extension 1310 in the optical sensor 1365 downward and interrupt the signal between the associated optical switches. When neither the second portion 1204B of the split cover nor the insert is present, the extension 1310 is not forced downward and the signal between the two optical switches is not disturbed. The magnet 1308 holds the extension 1310 in an upward position (i.e., a position that does not interrupt the associated optical switches). In the absence of the magnet, the extension will be in the down position, interrupting the optical switch. The magnetic force from the magnet is strong enough to hold the extension in this up position when the split cover is open and no insert is present, but weak enough to be overcome by the actuators on the cover and insert. Thus, the extension 1310 can be made of any magnetic material that works in this manner with the magnet 1308.

[0295] Other types of sensing and interrupting mechanisms can be used to indicate the presence of the second portion of the split cover or the insert. Examples of these sensing and interrupting mechanisms include magnetic proximity switches, mechanical switches, conductive contact switches, and the like.

[0296] These embodiments allow small fluid samples to be introduced directly into the microfluidic device 1210 without being diluted or dispersed. In these embodiments, samples containing a small number of precious cells (e.g., 200,000 or less) can be introduced, typically with a small volume (e.g., 200 microliters, 150 microliters, 100 microliters, 50 microliters or less). Such fluid samples cannot usually be analyzed and / or recovered using conventional techniques (e.g., fluorescence activated cell sorters or microfluidic chips that only use flow to sort cells) without significant loss of material.

[0297] Example

[0298] Example 1: Introducing samples into a microfluidic chip using a split cap

[0299] Plasma cells were isolated from mice and used system (Berkeley Lights, Inc.) and loaded it into the OptoSelect TMTo test the effect of well introduction on cell density and distribution within the channels of a microfluidic chip, (i) (ii) using a small volume introduction method on a system with a split cover mount The system uses a small volume introduction method, or (iii) an insert having a split cover mount in an open configuration and a well having fluid connections to the inlet / outlet of the microfluidic chip. Plasma cells were loaded onto the OptoSelect system using a well-introduction method. TM 11k and 14k chips. The small volume introduction method involves pulling a discrete approximately 5 microliter cell sample into the microfluidic chip. The cell sample is followed by a 7.5 microliter 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 introduction method involves manually pipetting approximately 3.5 microliters of cell sample into the well of the insert of the split cover (open configuration) and using negative pressure to pull the cell sample into the microfluidic chip. After loading, the fluid flow is stopped, the cells in each channel of the microfluidic chip are counted, and the introduction density and coefficient of variation (CV) under each condition are determined.

[0300] Fig.44 The sum of import density and the sum of import CV for each chip type broken down by import type and placement type are shown. The color shows details about the import type. The shape shows details about the placement type. Details are shown for the device ID. The view is filtered on the sum of import CV to keep only non-null values. Fig.44 As shown, the well introduction method is more efficient in OptoSelect compared to the small volume introduction method. TM The low volume approach yielded higher average lead density in both 11k and 14k chips (4.8x10^6 and 4.5x10^6 respectively), with the split cover mount in the closed position. The average inlet density on the system was 2.8x10^6 and 2.1x10^6 respectively, with a standard placement cover. The OptoSelect 1000 system produced an average import density of 2.7x10^6 and 2.4x10^6, respectively. In addition, compared with the small volume import method, for well import, OptoSelect 1000 TM The average CV of the 11k and 14k chips was significantly reduced (8% and 10%, respectively), and the small volume introduction method was more effective in the case of a split cover arrangement in the closed position. The system produced an average CV of 26% and 30% respectively, with the standard placement cover yielded an average CV of 26% and 27% on the systems, respectively.

[0301] Thus, the well introduction method using the split cover mount produces surprising improvements in cell loading. For any embodiment of the system / microscope disclosed herein having a split cover mount, it is expected that the same Fig.44 Results are similar to those shown in .

[0302] Although specific 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 present invention, and it will be apparent to those skilled in the art that various changes and modifications (such as the dimensions of the various components) may be made without departing from the scope of the disclosed invention, which is limited only by the appended claims and their equivalents. Accordingly, the specification and drawings are to be regarded as illustrative rather than restrictive.

Claims

1. A system for operating a microfluidic device, the system comprising: a first surface configured to engage and be operably coupled to a microfluidic device; as well as A cover configured to hold the microfluidic device on a first surface, the cover comprising: a first cover portion having a first fluid line connected to a first fluid port, the first fluid port being configured to operably couple with a first fluid inlet / outlet of the microfluidic device and to allow fluid medium to flow into and / or out of the first fluid inlet / outlet of the microfluidic device; and a second cover portion having a second fluid line connected to a second fluid port, the second fluid port being configured to be operably coupled to a second fluid inlet / outlet of the microfluidic device and to allow fluid medium to flow into and / or out of the second fluid inlet / outlet of the microfluidic device, wherein the second cover portion is separable from the first cover portion and is movable between a closed position and an open position, in which the second fluid port of the second cover portion is operably coupled to the second fluid inlet / outlet of the microfluidic device, and in which a portion of the microfluidic device containing the second fluid inlet / outlet is exposed, Wherein, when the second cover portion is in the open position, the first cover portion holds the microfluidic device on the first surface.

2. The system according to claim 1, wherein: When the second cover portion is in the open position, the first fluid port of the first cover portion remains operably coupled to the first fluid inlet / outlet of the microfluidic device.

3. The system according to claim 2, wherein: The first fluid port of the first cover portion is connected to a pump configured to remove fluid from the microfluidic device.

4. The system according to claim 1, wherein: The cover also includes a hinge configured to move the second portion of the cover between an open position and a closed position.

5. The system according to claim 1, wherein: The cover also includes a latch configured to releasably retain the second cover portion in a closed position.

6. The system according to any one of claims 1 to 5, further comprising: The insert is configured to operably couple with the second fluid inlet / outlet of the microfluidic device and allow the fluid medium to flow into the second fluid inlet / outlet of the microfluidic device when the second cover portion is in the open position.

7. The system according to claim 6, wherein: The insert is configured to engage with the first cover portion.

8. The system according to claim 6, wherein: The insert includes a fluid well configured to be in fluid communication with a second fluid inlet / outlet of the microfluidic device.

9. The system according to claim 8, wherein: The fluid trap is configured to hold 25 microliters or less of a fluid sample.

10. The system according to claim 8, wherein: The fluid trap is configured to hold fluid samples ranging from 5 microliters to 15 microliters.

11. The system according to claim 1, wherein: The first surface includes a support.

12. The system according to claim 11, wherein: The support includes a receptacle configured to receive and engage the microfluidic device.

13. The system of claim 1, further comprising: The electrical signal generating subsystem is configured to apply a bias voltage to a pair of electrodes in the microfluidic device when the microfluidic device is operably coupled to the first surface or support.

14. The system of claim 1, further comprising: A thermal control subsystem is configured to regulate the temperature of the microfluidic device when the microfluidic device is operably coupled to the first surface or support.

15. The system of claim 1, wherein: The support also includes a microprocessor that controls one or both of the electrical signal generation subsystem and the thermoelectric power module.

16. The system of claim 15, wherein: The support member includes a printed circuit board PCB, and wherein at least one of the electrical signal generating subsystem, the thermoelectric power module and the microprocessor is mounted on and / or integrated with the PCB.

17. The system of claim 1, further comprising: The light modulation subsystem is configured to emit structured light onto the microfluidic device when the microfluidic device is operably coupled to the first surface or support.

18. The system of claim 17, wherein: The first surface, support, and / or light modulation subsystem are configured to be mounted on a light microscope.

19. The system of claim 17, wherein: The first surface, the support and / or the light modulation subsystem are integral components of the light microscope.

20. The system of claim 1, further comprising: At least one flow controller is operably coupled to one or both of the first fluid line and the second fluid line.

21. The system of claim 20, wherein: The at least one flow controller includes a first thermally controlled flow controller operably coupled to the first fluid line and / or the second fluid line to selectively allow fluid to flow through the first fluid line and / or the second fluid line.

22. A microscope configured to operate a microfluidic device, the microscope include: The system of claim 11, configured to hold and operably couple to a microfluidic device; a light modulation subsystem configured to emit structured light; and Optical system, Wherein, when the microfluidic device is held by and operably coupled to the support, the optical system is configured to: (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 the unstructured light source onto at least a second region of the microfluidic device, and (3) Capturing reflected and / or emitted light from the microfluidic device and directing the captured light to a detector.

23. A method for analyzing a fluid sample, the method comprising: connecting the microfluidic device to a system for operating the microfluidic device, wherein the system comprises: a first surface configured to engage and be operably coupled to a microfluidic device, and A cover configured to hold the microfluidic device on a first surface, the cover comprising: a first cover portion having a first fluid line connected to a first fluid port, the first fluid port being configured to operably couple with a first fluid inlet / outlet of the microfluidic device and to allow fluid medium to flow into and / or out of the first fluid inlet / outlet of the microfluidic device, and a second cover portion having a second fluid line connected to a second fluid port, the second fluid port being configured to operably couple with a second fluid inlet / outlet of the microfluidic device and to allow fluid medium to flow into and / or out of the second fluid inlet / outlet of the microfluidic device, wherein the second cover portion is separable from the first cover portion and is movable between a closed position, in which a second fluid port of the second cover portion 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 containing the second fluid inlet / outlet is exposed; moving the second cover portion from a closed position to an open position, thereby exposing a second fluid inlet / outlet of the microfluidic device; providing a fluid sample in fluid communication with a second fluid inlet / outlet of the microfluidic device; applying suction to the first fluid line to draw at least a portion of the fluid sample into the microfluidic device; and The at least a portion of the fluid sample drawn into the microfluidic device is processed.

24. The method according to claim 23, further comprising: placing an insert in a position previously occupied by the second cover portion in the closed position, the insert comprising a fluid well configured to be in fluid communication with a second fluid inlet / outlet of the microfluidic device, Wherein, providing the fluid sample comprises: introducing the fluid sample into a fluid well of the insert.

25. The method according to claim 23 or 24, wherein: The system is a system according to claim 1.

26. The method according to claim 23 or 24, wherein: The system is a microscope according to claim 22.

27. The method according to claim 23 or 24, wherein: Sufficient suction is applied to pull a preselected volume of fluid sample between 2 microliters and 10 microliters into the microfluidic chip, at which point suction is stopped.

28. The method according to claim 23 or 24, wherein: The fluid sample includes micro-objects.

29. The method according to claim 28, wherein: The micro-objects include biological micro-objects.

30. The method according to claim 23 or 24, wherein: The microfluidic device includes (i) a flow region having a plurality of microfluidic channels and (ii) a plurality of chambers, wherein each chamber of the plurality of chambers is fluidly connected to one of the plurality of microfluidic channels.

31. The method according to claim 30, wherein: The method produces an introduced cell density of at least 4x10^6.

32. The method of claim 30, wherein: The method described yielded a coefficient of variation CV of less than 20% for the introduced cell density in different passages.

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