Particle classification and sorting system and method

Abstract

In some examples, a method of treating particles in a particle stream is provided. The method comprises: delivering a microfluidic flow from a microfluidic aperture into a flow environment, the microfluidic flow comprising a particle flow comprising a plurality of particles; directing interrogation electromagnetic radiation at the particles in the microfluidic flow and monitoring responsive emissions from the radiated particles; then directing sorting electromagnetic radiation at at least some of the particles in the microfluidic flow to sort the particles into at least two populations according to the monitored responsive emissions of the particles; the microfluidic flow includes a continuous phase flow of liquid.

Description

1. Field of the Technology

[0001] The present disclosure relates to the classification and sorting of particles such as asymmetric biological cells. 2. Background Art

[0002] The classification of particles with different properties is useful for many subsequent processes. For example, classifying sperm cells into X and Y populations enables downstream separation or sorting of these two populations. One type of sperm cell may be more suitable for certain types of animal breeding. For example, bovine X sperm cells are more suitable for inseminating dairy cows to produce a milk-producing population dominated by female offspring.

[0003] There are some difficulties in the classification and sorting of bovine sperm cells, including low sorting efficiency and slow sorting speed. Low sorting efficiency results in a low percentage of the desired cells (e.g., X sperm cells) in the collection container (compared to the total number of cells introduced into the classification and sorting system). Poor sorting efficiency may be caused by many factors, including poor orientation of the cells being classified, inaccurate classification techniques, inefficient sorting techniques, and associated processes negatively affecting cell motility. Slow sorting speed prolongs the time that biological cells are outside of optimal storage conditions and may therefore also affect cell motility.

[0004] In this specification, the citation of patent specifications, other external documents, or other information sources is generally for the purpose of providing context for discussing the features of the inventions disclosed herein. Unless otherwise specifically stated, such citations to external documents should not be construed as an admission that such documents or such information sources are prior art in any jurisdiction or form part of the common general knowledge in the art.

[0005] The object of the present invention is to provide an improved method for classifying and / or sorting particles with distinguishable properties, or at least to provide the public with a useful option for a method of classifying and / or sorting particles. 3. Summary of the Invention

[0006] In some examples, a method of processing particles in a particle stream is provided. The method includes: conveying a microfluidic stream from a microfluidic orifice into a flow environment, the microfluidic stream including a particle stream containing a plurality of particles; directing interrogation electromagnetic radiation to the particles in the microfluidic stream and monitoring the response emission from the irradiated particles; subsequently directing sorting electromagnetic radiation to at least some of the particles in the microfluidic stream so as to sort the particles into at least two populations based on the monitored response emission of the particles; wherein the microfluidic stream includes a continuous-phase flow of a liquid.

[0007] In an example, the particle stream may be surrounded by a sheath flow.

[0008] In an example, the flow environment includes one or more of the following: a microchannel, which optionally includes a substantially transparent material; a substrate exposed to a fluid environment; a liquid fluid environment; a gaseous fluid environment. The flow environment can include a gaseous fluid environment and be at least partially surrounded by a gaseous sheath that is substantially parallel to the microfluidic flow and moves relative to the gaseous environment.

[0009] In an example, the sorting includes one or more of the following operations: directing the subsequent sorting electromagnetic radiation to particles in one of the populations in the microfluidic flow to apply radiation pressure on the particles; directing the subsequent sorting electromagnetic radiation to particles in one of the populations in the microfluidic flow to ablate the particles; directing the subsequent sorting electromagnetic radiation to particles that are biological cells, where the subsequent sorting electromagnetic radiation is configured to transfer energy to selected cells, the energy being lower than a predetermined ablation threshold equivalent to rupturing the cell membranes and higher than a predetermined pretreatment threshold equivalent to the cells not surviving after a freezing and thawing process.

[0010] In an example, the interrogation electromagnetic radiation and the sorting electromagnetic radiation are directed through a common optical objective.

[0011] In an example, the interrogation electromagnetic radiation and the sorting electromagnetic radiation are directed through the common optical objective at an angle relative to each other.

[0012] In an example, the angle between the interrogation electromagnetic radiation and the sorting electromagnetic radiation directed through the common optical objective is adjusted to define the distance between the beams between the focus of the interrogation electromagnetic radiation and the focus of the sorting electromagnetic radiation within the microfluidic flow.

[0013] In an example, the microfluidic flow is delivered from a flow control device having a microfluidic orifice, the flow control device being shaped to define a region above the microfluidic orifice through which the interrogation electromagnetic radiation and / or the sorting electromagnetic radiation are directed. The objective optical component can be at least partially positioned within the region.

[0014] In an example, the interrogation electromagnetic radiation propagates as an interrogation beam that intersects the microfluidic flow at an excitation distance from the microfluidic orifice, the excitation distance being between 25 μm and 1000 μm.

[0015] In an example, the excitation distance is less than 400 μm and greater than one of the following: 25 μm, 50 μm, 100 μm.

[0016] In an example, the interrogating electromagnetic radiation and the subsequent sorting electromagnetic radiation propagate as respective interrogating beams and respective sorting beams, the respective interrogating beam and the respective sorting beam intersect the microfluidic flow, and the distance between the separated beams includes one or more of the following: at least 10 μm; between 10 μm and 400 μm.

[0017] In an example, the interrogating electromagnetic radiation and / or the sorting electromagnetic radiation is controlled to propagate as a respective beam that is equal to or wider than the microfluidic flow when intersecting the microfluidic flow.

[0018] In an example, monitoring the response emission from the irradiated particles includes using the outputs from a plurality of sensors arranged around the microfluidic flow, the plurality of sensors being arranged to capture response emissions from different directions.

[0019] In an example, the output from the sensors is normalized to compensate for differences in one or more of the following properties of the response emissions captured from the respective sensors: amplitude; phase; propagation delay; refractive effect when entering the flowing environment from the microfluidic flow.

[0020] In an example, the method includes adjusting the cross-section of the microfluidic orifice.

[0021] In an example, the flow rate of the microfluidic flow is from 5 m / s to 20 m / s.

[0022] In some examples, a method of processing particles in a particle stream is provided. The method includes: conveying a microfluidic flow from a microfluidic orifice into a flowing environment, the microfluidic flow including a particle stream comprising a plurality of particles; directing interrogating electromagnetic radiation through a common optical objective to the particles in the microfluidic flow and monitoring the response emission from the irradiated particles; and subsequently directing sorting electromagnetic radiation through the common optical objective lens to at least some of the particles in the microfluidic flow so as to sort the particles into at least two populations based on the monitored response emission of the particles.

[0023] In an example, the particle stream can be surrounded by a sheath flow.

[0024] In some examples, the interrogating electromagnetic radiation and the sorting electromagnetic radiation are directed through the common optical objective at an angle relative to each other.

[0025] In some examples, the angle between the interrogating electromagnetic radiation and the sorting electromagnetic radiation directed through the common optical objective is adjusted so as to define the distance between the foci of the interrogating electromagnetic radiation and the sorting electromagnetic radiation within the microfluidic flow.

[0026] In some examples, the microfluidic flow is delivered from a flow control device having the microfluidic aperture, the flow control device being shaped to define a region above the microfluidic aperture through which the interrogation electromagnetic radiation and / or the sorting electromagnetic radiation is directed.

[0027] In some examples, the objective optical component is at least partially positioned within the region.

[0028] In some examples, the interrogation electromagnetic radiation propagates as an interrogation beam that intersects the microfluidic flow at an excitation distance from the microfluidic aperture, the excitation distance being between 25 μm and 1000 μm.

[0029] In some examples, the excitation distance is less than 400 μm and greater than one of: 25 μm, 50 μm, 100 μm.

[0030] In some examples, the interrogation electromagnetic radiation and the subsequent sorting electromagnetic radiation propagate as a respective interrogation beam and a respective sorting beam that intersect the microfluidic flow and are separated by a beam separation distance that includes one or more of: at least 10 μm; between 10 μm and 400 μm.

[0031] In some examples, the interrogation electromagnetic radiation and / or the sorting electromagnetic radiation is controlled to propagate as a sorting beam that is equal to or wider than the microfluidic flow when intersecting the microfluidic flow.

[0032] In some examples, monitoring the response emission from the irradiated particles includes using the output from a plurality of sensors arranged around the microfluidic flow, the plurality of sensors using a photoelectric array of detectors arranged to capture the response emission from different directions.

[0033] In some examples, the output from the sensors is normalized to compensate for differences in one or more of the following properties of the response emissions captured from the respective sensors: amplitude; phase; propagation delay; refractive effect when entering the flow environment from the microfluidic flow.

[0034] In some examples, the method includes adjusting the cross-section of the microfluidic aperture.

[0035] In some examples, the flow rate of the microfluidic flow is from 5 m / s to 20 m / s.

[0036] In some examples, the microfluidic flow includes a continuous phase flow of a liquid.

[0037] In some examples, the flow environment includes one or more of the following: a microchannel, which optionally includes a substantially transparent material; a substrate exposed to a fluid environment; a liquid fluid environment; a gaseous fluid environment.

[0038] In some examples, the flow environment includes a gaseous fluid environment and is at least partially surrounded by a gaseous sheath that is substantially parallel to the microfluidic flow and moves relative to the gaseous environment.

[0039] In some examples, the sorting includes one or more of the following operations: directing a subsequent sorting electromagnetic radiation to particles in one of the populations in the microfluidic flow to apply a radiation pressure on the particles; directing the subsequent sorting electromagnetic radiation to particles in one of the populations in the microfluidic flow to ablate the particles; directing the subsequent sorting electromagnetic radiation to particles that are biological cells, wherein the subsequent sorting electromagnetic radiation is configured to transfer energy to selected cells, the energy being lower than a predetermined ablation threshold equivalent to rupturing the cell membranes and higher than a predetermined pretreatment threshold equivalent to the cells not surviving after a freezing and thawing process.

[0040] In some examples, a method of processing particles in a particle stream is provided. The method includes: conveying a microfluidic flow from a microfluidic orifice to a flow environment, the microfluidic flow including a particle stream comprising a plurality of particles; directing a probing beam to the particles in the microfluidic flow and monitoring a response emission from the irradiated particles; subsequently sorting the particles into at least two populations based on the monitored response emission of the particles; wherein the excitation distance between the orifice and the probing beam is less than 1000 um.

[0041] In an example, the particle stream can be surrounded by a sheath flow.

[0042] In some examples, the excitation distance is less than 400 um.

[0043] In some examples, the excitation distance is greater than one of the following: 25 um, 50 um, 100 um.

[0044] In some examples, the flow rate of the microfluidic flow is from 5 m / s to 20 m / s.

[0045] In some examples, sorting the particles includes directing sorting electromagnetic radiation to at least some of the particles in the microfluidic flow so as to sort the particles into the at least two populations.

[0046] In some examples, the sorting includes one or more of the following operations: directing the subsequent sorting electromagnetic radiation to particles in one of the populations in the microfluidic flow to apply radiation pressure on the particles; directing the subsequent sorting electromagnetic radiation to particles in one of the populations in the microfluidic flow to ablate the particles; directing the subsequent sorting electromagnetic radiation to particles that are biological cells, wherein the subsequent sorting electromagnetic radiation is configured to transfer energy to selected cells, the energy being lower than a predetermined ablation threshold equivalent to rupturing the cell membranes and higher than a predetermined pretreatment threshold equivalent to the cells not surviving after a freezing and thawing process.

[0047] In some examples, the interrogation electromagnetic radiation and the sorting electromagnetic radiation are directed through a common optical objective.

[0048] In some examples, the interrogation electromagnetic radiation and the sorting electromagnetic radiation are directed through the common optical objective at an angle relative to each other.

[0049] In some examples, the angle between the interrogation electromagnetic radiation and the sorting electromagnetic radiation directed through the common optical objective is adjusted to define an interbeam distance between the focus of the interrogation electromagnetic radiation within the microfluidic flow and the focus of the sorting electromagnetic radiation within the microfluidic flow.

[0050] In some examples, the microfluidic flow is delivered from a flow control device having a microfluidic orifice, the flow control device being shaped to define a region above the microfluidic orifice through which the interrogation electromagnetic radiation and / or the sorting electromagnetic radiation are directed.

[0051] In some examples, the objective optics are at least partially positioned within the region.

[0052] In some examples, the interrogation electromagnetic radiation and the subsequent sorting electromagnetic radiation propagate as a respective interrogation beam and a respective sorting beam, the respective interrogation beam and the respective sorting beam intersecting the microfluidic flow and separated by an interbeam distance, the interbeam distance including one or more of the following: at least 10 μm; between 10 μm and 400 μm.

[0053] In some examples, the interrogation electromagnetic radiation and / or the sorting electromagnetic radiation are controlled to propagate as a sorting beam that is equal to or wider than the microfluidic flow when intersecting the microfluidic flow.

[0054] In some examples, the microfluidic flow includes a continuous phase flow of a liquid.

[0055] In some examples, the flow environment includes one or more of the following: a microchannel, which optionally includes a substantially transparent material; a substrate exposed to a fluid environment; a liquid fluid environment; a gaseous fluid environment.

[0056] In some examples, the flow environment includes a gaseous fluid environment and is at least partially surrounded by a gaseous sheath that is substantially parallel to the microfluidic flow and moves relative to the gaseous environment.

[0057] In some examples, monitoring the response emissions from these irradiated particles includes using the outputs from a plurality of sensors arranged around the microfluidic flow, the plurality of sensors using a photoelectric array with detectors arranged to capture response emissions from different directions.

[0058] In some examples, the output from the sensors is normalized to compensate for differences in one or more of the following properties of the response emissions captured from the respective sensors: amplitude; phase; propagation delay; refractive effects when entering the flow environment from the microfluidic flow.

[0059] In some examples, the method includes adjusting the cross-section of the microfluidic orifice.

[0060] In some examples, a corresponding apparatus is provided.

[0061] In an example, a method of processing particles in a particle stream is provided. The method includes: conveying a microfluidic flow from a microfluidic orifice into a flow environment, the microfluidic flow including a particle stream and a surrounding sheath flow, the particle stream comprising a plurality of particles; directing interrogating electromagnetic radiation to the particles in the microfluidic flow and monitoring response emissions from the irradiated particles; sorting the particles into at least two populations based on the monitored response emissions of the particles; wherein the microfluidic orifice has a cross-section that extends more along one axis than along a perpendicular axis.

[0062] In an example, the microfluidic flow includes one or more of the following: a continuous-phase flow of a liquid; a dispersed flow of droplets.

[0063] In an example, the flow environment includes one or more of the following: a microchannel; a substrate exposed to a fluid environment; a liquid fluid environment; a gaseous fluid environment.

[0064] In an example, the flow environment includes a gaseous fluid environment and is at least partially surrounded by a gaseous sheath that is substantially parallel to the microfluidic flow and moves relative to the gaseous environment.

[0065] In an example, the sorting includes one or more of the following operations: directing subsequent sorting electromagnetic radiation to particles in one of the populations in the microfluidic flow to apply radiation pressure on the particles; directing subsequent sorting electromagnetic radiation to particles in one of the populations in the microfluidic flow to ablate the particles; applying an electrostatic force to droplets of the microfluidic flow surrounding particles in one of the populations.

[0066] In an example, the interrogation electromagnetic radiation and the subsequent sorting electromagnetic radiation propagate through a common objective lens.

[0067] In an example, the interrogation electromagnetic radiation and the subsequent sorting electromagnetic radiation propagate as a respective interrogation beam and a respective sorting beam, the respective interrogation beam and the respective sorting beam intersect the microfluidic flow, and a beam separation distance is provided between the beams, the beam separation distance including one or more of the following: at least 10 μm; between 10 μm and 400 μm.

[0068] In an example, the sorting electromagnetic radiation is controlled to propagate as a sorting beam that intersects the microfluidic flow to provide an elliptical or circular intensity pattern within the microfluidic flow.

[0069] In an example, the sorting beam is wider than the microfluidic flow.

[0070] In an example, the interrogation electromagnetic radiation is controlled to propagate as an ultraviolet interrogation beam or an infrared interrogation beam.

[0071] In an example, the interrogation electromagnetic radiation propagates as an interrogation beam that intersects the microfluidic flow at an excitation distance from the microfluidic pore, the excitation distance being between 25 μm and 1000 μm.

[0072] In an example, the interrogation electromagnetic radiation is controlled to propagate as an interrogation beam that intersects the microfluidic flow to provide an elliptical or circular intensity pattern within the microfluidic flow.

[0073] In an example, the interrogation electromagnetic radiation is controlled to propagate as an ultraviolet interrogation beam or an infrared interrogation beam.

[0074] In an example, the particles are biological cells, and the subsequent sorting electromagnetic radiation is configured to transfer energy to selected cells, the energy being lower than a predetermined ablation threshold corresponding to rupturing the cell membrane and higher than a predetermined pretreatment threshold corresponding to the cells not surviving after a freezing and thawing process.

[0075] In an example, the microfluidic pore has a cross-section that is one of the following shapes: elliptical; rectangular; parallelogram; trapezoidal; polygonal; square.

[0076] In an example, a ratio of an extent of the cross-section of the microfluidic pore along one axis to an extent of the cross-section of the microfluidic pore along a perpendicular axis is between 1:100 and 2:3.

[0077] In an example, the ratio is between 1:50 and 1:10.

[0078] In an example, the microfluidic pore has an adjustable cross-section.

[0079] In an example, a size and / or shape of the cross-section of the microfluidic pore is adjustable.

[0080] In an example, the cross-section is automatically adjusted in response to a performance metric associated with the microfluidic flow.

[0081] In an example, a cleaning mode is characterized by a maximum cross-sectional area of the microfluidic pore and a cleaning flow of a liquid through the microfluidic pore, optionally, wherein the cleaning flow has a flow rate higher than the microfluidic flow.

[0082] In an example, the method includes: after directing the probing electromagnetic radiation, directing focused electromagnetic radiation to the sheath flow to vaporize a portion of the microfluidic flow.

[0083] In an example, the focused electromagnetic radiation is controlled in response to monitored responses of the particles.

[0084] In an example, monitoring the response emissions from the irradiated particles includes using outputs from a plurality of sensors arranged around the microfluidic flow.

[0085] In an example, the plurality of sensors are implemented as a photoelectric array having detectors arranged to capture response emissions from different directions.

[0086] In an example, the output from the sensors is adjusted based on the positions of the sensors.

[0087] In an example, the output from the sensors is normalized to compensate for differences in one or more of the following properties of the response emissions captured from the respective sensors: amplitude; phase; propagation delay; refractive effects when entering the flow environment from the microfluidic flow.

[0088] In an example, the plurality of sensors are arranged in a plane perpendicular to a longitudinal axis of the microfluidic flow, and wherein the plurality of sensors are arranged in an arc around the microfluidic flow or in a substantially straight line adjacent to the microfluidic flow.

[0089] In an example, these outputs from these sensors corresponding to the response emissions from the particles are integrated to generate a signal for classifying the particles.

[0090] In an example, the sheath extends parallel to the microfluidic flow and at least partially surrounds the microfluidic flow and the fluid environment.

[0091] In an example, the sheath includes one or more of the following: a gaseous flow that moves relative to the fluid environment and through which the probing electromagnetic radiation is directed; a transparent solid material through which the probing electromagnetic radiation is directed.

[0092] In an example, the microfluidic pore is defined in a flow control device for generating the microfluidic flow.

[0093] In an example, a method for processing particles in a particle stream is provided, the method including: conveying a microfluidic flow from a microfluidic pore into a flow environment, the microfluidic flow including a particle stream and a surrounding sheath flow, the particle stream containing a plurality of particles; directing probing electromagnetic radiation to the particles in the microfluidic flow and monitoring the response emissions from the irradiated particles; sorting the particles into at least two populations based on the monitored response emissions of the particles; after directing the probing electromagnetic radiation, directing focused electromagnetic radiation to the sheath flow to vaporize a portion of the microfluidic flow.

[0094] In an example, the microfluidic flow includes one or more of the following: a continuous phase flow of a liquid; a dispersed flow of droplets.

[0095] In an example, the flow environment includes one or more of the following: a microchannel; a substrate exposed to a fluid environment; a liquid fluid environment; a gaseous fluid environment.

[0096] In an example, the flow environment includes a gaseous fluid environment and is at least partially surrounded by a gaseous sheath that is substantially parallel to the microfluidic flow and moves relative to the gaseous environment.

[0097] In an example, the sorting includes one or more of the following operations: directing subsequent sorting electromagnetic radiation to the particles in one of the populations in the microfluidic flow to apply radiation pressure on the particles; directing subsequent sorting electromagnetic radiation to the particles in one of the populations in the microfluidic flow to ablate the particles; applying an electrostatic force to the droplets of the microfluidic flow surrounding the particles in one of the populations.

[0098] In an example, the probing electromagnetic radiation and the subsequent sorting electromagnetic radiation propagate through a common objective lens.

[0099] In an example, the interrogating electromagnetic radiation and the subsequent sorting electromagnetic radiation propagate as a respective interrogating beam and a respective sorting beam, the respective interrogating beam and the respective sorting beam intersect the microfluidic flow, and a distance between the beams is separated, the distance between the beams including one or more of the following: at least 10 μm; between 10 μm and 400 μm.

[0100] In an example, the sorting electromagnetic radiation is controlled to propagate as a sorting beam that intersects the microfluidic flow to provide an elliptical or circular intensity pattern within the microfluidic flow.

[0101] In an example, the sorting beam is wider than the microfluidic flow.

[0102] In an example, the interrogating electromagnetic radiation is controlled to propagate as an ultraviolet interrogating beam or an infrared interrogating beam.

[0103] In an example, the interrogating electromagnetic radiation propagates as an interrogating beam that intersects the microfluidic flow at an excitation distance from the microfluidic pore, the excitation distance being between 25 μm and 1000 μm.

[0104] In an example, the interrogating electromagnetic radiation is controlled to propagate as an interrogating beam that intersects the microfluidic flow to provide an elliptical or circular intensity pattern within the microfluidic flow.

[0105] In an example, the interrogating electromagnetic radiation is controlled to propagate as an ultraviolet interrogating beam or an infrared interrogating beam.

[0106] In an example, the particle is a biological cell, and the subsequent sorting electromagnetic radiation is configured to transfer energy to a selected cell, the energy being below a predetermined ablation threshold equivalent to rupturing the cell membrane and above a predetermined pretreatment threshold equivalent to the cell becoming non-viable after freezing and thawing processes.

[0107] In an example, the microfluidic pore has a cross-section that extends more along one axis than along a perpendicular axis.

[0108] In an example, the microfluidic pore has a cross-section that is one of the following shapes: elliptical; rectangular; parallelogram; trapezoidal; polygonal.

[0109] In an example, a ratio of the extension of the cross-section of the microfluidic pore along the one axis to the extension of the cross-section of the microfluidic pore along the perpendicular axis is between 1:100 and 2:3.

[0110] In an example, the ratio is between 1:50 and 1:10.

[0111] In an example, the microfluidic pore has an adjustable cross-section.

[0112] In an example, the size and / or shape of the cross-section of the microfluidic pore is adjustable.

[0113] In an example, the cross-section is automatically adjusted in response to a metric associated with the microfluidic flow.

[0114] In an example, a cleaning mode is characterized by a maximum cross-sectional area of the microfluidic pore and a cleaning flow of liquid through the microfluidic pore, optionally wherein the cleaning flow has a flow rate higher than the microfluidic flow.

[0115] In an example, the focused electromagnetic radiation is emitted in response to a monitored response of the particles.

[0116] In an example, monitoring the response emission from the irradiated particles includes using outputs from a plurality of sensors arranged around the microfluidic flow.

[0117] In an example, the plurality of sensors are implemented as a photoelectric array having detectors arranged to capture response emissions from different directions.

[0118] In an example, the output from the sensors is adjusted according to the position of the sensors.

[0119] In an example, the output from the sensors is normalized to compensate for differences in one or more of the following properties of the response emissions captured from the respective sensors: amplitude; phase; propagation delay; refractive effect when entering the flow environment from the microfluidic flow.

[0120] In an example, the plurality of sensors are arranged in a plane perpendicular to the longitudinal axis of the microfluidic flow, and wherein the plurality of sensors are arranged in an arc around the microfluidic flow or in a substantially straight line adjacent to the microfluidic flow.

[0121] In an example, the outputs from the sensors corresponding to the response emissions from the particles are integrated to generate a signal for classifying the particles.

[0122] In an example, a sheath extends parallel to the microfluidic flow and at least partially surrounds the microfluidic flow and the fluid environment.

[0123] In an example, the sheath includes one or more of the following: a gaseous flow that moves relative to the fluid environment and through which the interrogating electromagnetic radiation is directed; a transparent solid material through which the interrogating electromagnetic radiation is directed.

[0124] In an example, the microfluidic pore is defined in a flow control device for generating the microfluidic flow.

[0125] In an example, a method for processing particles in a particle stream is provided, the method comprising: conveying a microfluidic stream from a microfluidic orifice into a flow environment, the microfluidic stream comprising a particle stream and a surrounding sheath flow, the particle stream comprising a plurality of particles; directing interrogation electromagnetic radiation to the particles in the microfluidic stream and monitoring response emissions from the irradiated particles; sorting the particles into at least two populations based on the monitored response emissions of the particles; wherein monitoring response emissions from the irradiated particles comprises using outputs from a plurality of sensors arranged around the microfluidic stream.

[0126] In an example, the microfluidic stream comprises one or more of the following: a continuous phase flow of a liquid; a dispersed flow of droplets.

[0127] In an example, the flow environment comprises one or more of the following: a microchannel; a substrate exposed to a fluid environment; a liquid fluid environment; a gaseous fluid environment.

[0128] In an example, the flow environment comprises a gaseous fluid environment and is at least partially surrounded by a gaseous sheath that is substantially parallel to the microfluidic stream and moves relative to the gaseous environment.

[0129] In an example, the sorting comprises one or more of the following operations: directing subsequent sorting electromagnetic radiation to particles in one of the populations in the microfluidic stream to apply radiation pressure on the particles; directing subsequent sorting electromagnetic radiation to particles in one of the populations in the microfluidic stream to ablate the particles; applying an electrostatic force to droplets of the microfluidic stream surrounding particles in one of the populations.

[0130] In an example, the interrogation electromagnetic radiation and the subsequent sorting electromagnetic radiation propagate through a common objective lens.

[0131] In an example, the interrogation electromagnetic radiation and the subsequent sorting electromagnetic radiation propagate as a respective interrogation beam and a respective sorting beam, the respective interrogation beam and the respective sorting beam intersect the microfluidic stream, and a beam separation distance is provided between the beams, the beam separation distance comprising one or more of the following: at least 10 μm; between 10 μm and 400 μm.

[0132] In an example, the sorting electromagnetic radiation is controlled to propagate as a sorting beam that intersects the microfluidic stream to provide an elliptical or circular intensity pattern within the microfluidic stream.

[0133] In an example, the sorting beam is wider than the microfluidic stream.

[0134] In an example, the interrogation electromagnetic radiation is controlled to propagate as an ultraviolet interrogation beam or an infrared interrogation beam.

[0135] In an example, the interrogating electromagnetic radiation propagates as an interrogating light beam that intersects the microfluidic flow at an excitation distance from the microfluidic pore, the excitation distance being between 25 μm and 1000 μm.

[0136] In an example, the interrogating electromagnetic radiation is controlled to propagate as an interrogating light beam that intersects the microfluidic flow to provide an elliptical or circular intensity pattern within the microfluidic flow.

[0137] In an example, the interrogating electromagnetic radiation is controlled to propagate as an ultraviolet interrogating light beam or an infrared interrogating light beam.

[0138] In an example, the particle is a biological cell, and the subsequent sorting electromagnetic radiation is configured to transfer energy to selected cells, the energy being below a predetermined ablation threshold equivalent to rupturing the cell membrane and above a predetermined pre-treatment threshold equivalent to the cell becoming non-viable after freezing and thawing processes.

[0139] In an example, the microfluidic pore has a cross-section that extends more along one axis than along the perpendicular axis.

[0140] In an example, the microfluidic pore has a cross-section that is one of the following shapes: elliptical; rectangular; parallelogram; trapezoidal; polygonal.

[0141] In an example, the ratio of the extension of the cross-section of the microfluidic pore along the one axis to the extension of the cross-section of the microfluidic pore along the perpendicular axis is between 1:100 and 2:3.

[0142] In an example, the ratio is between 1:50 and 1:10.

[0143] In an example, the microfluidic pore has an adjustable cross-section.

[0144] In an example, the size and / or shape of the cross-section of the microfluidic pore is adjustable.

[0145] In an example, the cross-section is automatically adjusted in response to a metric associated with the microfluidic flow.

[0146] In an example, the method includes a cleaning mode characterized by the maximum cross-sectional area of the microfluidic pore and a cleaning flow of liquid through the microfluidic pore, optionally, where the cleaning flow has a flow rate higher than the microfluidic flow.

[0147] In an example, the focused electromagnetic radiation is controlled based on the monitored response of the particles.

[0148] In an example, the method includes: after directing the probing electromagnetic radiation, directing the focused electromagnetic radiation to the sheath flow to vaporize a portion of the microfluidic flow.

[0149] In an example, the plurality of sensors are implemented as an optoelectronic array having detectors arranged to capture response emissions from different directions.

[0150] In an example, the output from the adjusted output is scaled according to the position of the sensor.

[0151] In an example, the output from the sensors is normalized to compensate for differences in one or more of the following properties of the response emissions captured from the respective sensors: amplitude; phase; propagation delay; refractive effect when entering the flow environment from the microfluidic flow.

[0152] In an example, the plurality of sensors are arranged in a plane perpendicular to the longitudinal axis of the microfluidic flow, and wherein the plurality of sensors are arranged in an arc around the microfluidic flow or in a substantially straight line adjacent to the microfluidic flow.

[0153] In an example, the outputs from the sensors corresponding to the response emissions from the particles are integrated to generate a signal for classifying the particles.

[0154] In an example, the sheath extends parallel to the microfluidic flow and at least partially surrounds the microfluidic flow and the fluid environment.

[0155] In an example, the sheath includes one or more of the following: a gaseous flow moving relative to the fluid environment through which the probing electromagnetic radiation is directed; a transparent solid material through which the probing electromagnetic radiation is directed.

[0156] In an example, the microfluidic orifice is defined in a flow control device for generating the microfluidic flow.

[0157] In an example, a method of processing particles in a particle stream is provided. The method includes: delivering a microfluidic flow from a microfluidic orifice to a flow environment, the microfluidic flow including a particle stream and a surrounding sheath flow, the particle stream including a plurality of particles; directing probing electromagnetic radiation to the particles in the microfluidic flow and monitoring response emissions from the irradiated particles; sorting the particles into at least two populations based on the monitored response emissions of the particles; generating a sheath including a gaseous flow moving relative to the fluid environment, extending substantially parallel to the microfluidic flow, and at least partially surrounding the microfluidic flow and the fluid environment.

[0158] In an example, the microfluidic flow includes one or more of the following: a continuous phase flow of a liquid; a dispersed flow of droplets.

[0159] In an example, the flow environment includes one or more of the following: a microchannel; a substrate exposed to a fluid environment; a liquid fluid environment; a gaseous fluid environment.

[0160] In an example, the flow environment includes a gaseous fluid environment and is at least partially surrounded by a gaseous sheath that is substantially parallel to the microfluidic flow and moves relative to the gaseous environment.

[0161] In an example, the sorting includes one or more of the following operations: directing subsequent sorting electromagnetic radiation to particles in one of the populations in the microfluidic flow to ablate the particles; directing subsequent sorting electromagnetic radiation to particles in one of the populations in the microfluidic flow to apply radiation pressure on the particles; applying an electrostatic force to droplets of the microfluidic flow surrounding particles in one of the populations.

[0162] In an example, the interrogation electromagnetic radiation and the subsequent sorting electromagnetic radiation propagate through a common objective lens.

[0163] In an example, the interrogation electromagnetic radiation and the subsequent sorting electromagnetic radiation propagate as a respective interrogation beam and a respective sorting beam, the respective interrogation beam and the respective sorting beam intersect the microfluidic flow, and a beam separation distance is separated, the beam separation distance including one or more of the following: at least 10 μm; between 10 μm and 400 μm.

[0164] In an example, the sorting electromagnetic radiation is controlled to propagate as a sorting beam that intersects the microfluidic flow to provide an elliptical or circular intensity pattern within the microfluidic flow.

[0165] In an example, the sorting beam is wider than the microfluidic flow.

[0166] In an example, the interrogation electromagnetic radiation is controlled to propagate as an ultraviolet interrogation beam or an infrared interrogation beam.

[0167] In an example, the interrogation electromagnetic radiation propagates as an interrogation beam that intersects the microfluidic flow at an excitation distance from the microfluidic orifice, the excitation distance being between 25 μm and 1000 μm.

[0168] In an example, the interrogation electromagnetic radiation is controlled to propagate as an interrogation beam that intersects the microfluidic flow to provide an elliptical or circular intensity pattern within the microfluidic flow.

[0169] In an example, the interrogation electromagnetic radiation is controlled to propagate as an ultraviolet interrogation beam or an infrared interrogation beam.

[0170] In an example, the particle is a biological cell, and the subsequent sorting electromagnetic radiation is configured to deliver energy to a selected cell, the energy being below a predetermined ablation threshold equivalent to rupturing the cell membrane and above a predetermined pre-treatment threshold equivalent to the cell becoming immotile after freezing and thawing.

[0171] In an example, the microfluidic pore has a cross-section that extends more along one axis than along the perpendicular axis.

[0172] In an example, the microfluidic pore has a cross-section that is one of the following shapes: oval; rectangular; parallelogram; trapezoid; polygon.

[0173] In an example, the ratio of the extension of the cross-section of the microfluidic pore along the one axis to the extension of the cross-section of the microfluidic pore along the perpendicular axis is between 1:100 and 2:3.

[0174] In an example, the ratio is between 1:50 and 1:10.

[0175] In an example, the microfluidic pore has an adjustable cross-section.

[0176] In an example, the size and / or shape of the cross-section of the microfluidic pore is adjustable.

[0177] In an example, the cross-section is automatically adjusted in response to a metric associated with the microfluidic flow.

[0178] In an example, the cleaning mode is characterized by the maximum cross-sectional area of the microfluidic pore and a cleaning flow of liquid through the microfluidic pore, the cleaning flow having a flow rate higher than the microfluidic flow.

[0179] In an example, the focused electromagnetic radiation is emitted in control based on the monitored response of the particles.

[0180] In an example, monitoring the response emission from the irradiated particles includes using the output from a plurality of sensors arranged around the microfluidic flow.

[0181] In an example, the plurality of sensors are implemented as a photoelectric array having detectors arranged to capture response emissions from different directions.

[0182] In an example, the output from the sensors is adjusted based on the position of the sensors.

[0183] In an example, the output from the sensors is normalized to compensate for differences in one or more of the following properties of the response emissions captured from the respective sensors: amplitude; phase; propagation delay; refractive effect when entering the flow environment from the microfluidic flow.

[0184] In an example, the plurality of sensors are arranged in a plane perpendicular to the longitudinal axis of the microfluidic flow, and wherein the plurality of sensors are arranged in an arc around the microfluidic flow or in a substantially straight line adjacent to the microfluidic flow.

[0185] In an example, the outputs from these sensors corresponding to the response emissions from the particles are integrated to generate a signal for classifying the particles.

[0186] In an example, the method includes: after guiding the probing electromagnetic radiation, guiding focused electromagnetic radiation to the sheath flow to vaporize a portion of the microfluidic flow.

[0187] In an example, the probing electromagnetic radiation is guided through the sheath.

[0188] In an example, the microfluidic orifice is defined in a flow control device for generating the microfluidic flow.

[0189] In an example, there is provided an apparatus for processing particles in a particle stream. The apparatus includes: means for: conveying a microfluidic flow from a microfluidic orifice to a flow environment, the microfluidic flow including a particle stream and a surrounding sheath flow, the particle stream containing a plurality of particles; means for: guiding probing electromagnetic radiation to the particles in the microfluidic flow and monitoring the response emissions from the irradiated particles; means for: sorting the particles into at least two populations based on the monitored response emissions of the particles; wherein: the microfluidic orifice has a cross-section that extends more along one axis than along a perpendicular axis; and / or the means for monitoring the response emissions from the irradiated particles uses the outputs from sensors arranged around the microfluidic flow; and / or the apparatus includes means for: after guiding the probing electromagnetic radiation, guiding focused electromagnetic radiation to the sheath flow to vaporize a portion of the microfluidic flow; and / or the apparatus includes means for: generating a sheath that includes a gaseous flow moving relative to the fluid environment, extending substantially parallel to the microfluidic flow, and at least partially surrounding the microfluidic flow and the fluid environment.

[0190] In an example, the microfluidic flow includes one or more of the following: a continuous phase flow of a liquid; a dispersed flow of droplets.

[0191] In an example, the flow environment includes one or more of the following: a microchannel; a substrate exposed to a fluid environment; a liquid fluid environment; a gaseous fluid environment.

[0192] In an example, the flow environment includes a gaseous fluid environment and is at least partially surrounded by a gaseous sheath that is substantially parallel to the microfluidic flow and moves relative to the gaseous environment.

[0193] In an example, the component for sorting comprises one or more of the following: a component for directing subsequent sorting electromagnetic radiation to particles in one of the populations in the microfluidic flow to apply radiation pressure on the particles; a component for directing subsequent sorting electromagnetic radiation to particles in one of the populations in the microfluidic flow to ablate the particles; a component for applying an electrostatic force to droplets of the microfluidic flow surrounding particles in one of the populations.

[0194] In an example, the interrogation electromagnetic radiation and the subsequent sorting electromagnetic radiation are arranged to propagate through a common objective lens.

[0195] In an example, the interrogation electromagnetic radiation and the subsequent sorting electromagnetic radiation are arranged to propagate as a respective interrogation beam and a respective sorting beam, the respective interrogation beam and the respective sorting beam intersect the microfluidic flow, and a beam separation distance is separated, the beam separation distance comprising one or more of the following: at least 10 μm; between 10 μm and 400 μm.

[0196] In an example, the sorting electromagnetic radiation is arranged to propagate as a sorting beam, the sorting beam intersects the microfluidic flow to provide an elliptical or circular intensity pattern within the microfluidic flow.

[0197] In an example, the sorting beam is wider than the microfluidic flow.

[0198] In an example, the interrogation electromagnetic radiation is arranged to propagate as an ultraviolet interrogation beam or an infrared interrogation beam.

[0199] In an example, the interrogation electromagnetic radiation is arranged to propagate as an interrogation beam, the interrogation beam intersects the microfluidic flow at an excitation distance from the microfluidic pore, the excitation distance being between 25 μm and 1000 μm.

[0200] In an example, the interrogation electromagnetic radiation is arranged to propagate as an interrogation beam, the interrogation beam intersects the microfluidic flow to provide an elliptical or circular intensity pattern within the microfluidic flow.

[0201] In an example, the interrogation electromagnetic radiation is arranged to propagate as an ultraviolet interrogation beam or an infrared interrogation beam.

[0202] In an example, the particle is a biological cell, and the subsequent sorting electromagnetic radiation is configured to transfer energy to a selected cell, the energy being lower than a predetermined ablation threshold equivalent to rupturing the cell membrane and higher than a predetermined pretreatment threshold equivalent to the cell not surviving after a freezing and thawing process.

[0203] In an example, the microfluidic pore has a cross-section that is one of the following shapes: oval; rectangular; parallelogram; trapezoid; polygon.

[0204] In an example, the ratio of the extension of the cross-section of the microfluidic pore along one axis to the extension of the cross-section of the microfluidic pore along the perpendicular axis is between 1:100 and 2:3.

[0205] In an example, the ratio is between 1:50 and 1:10.

[0206] In an example, the microfluidic pore has an adjustable cross-section.

[0207] In an example, the size and / or shape of the cross-section of the microfluidic pore is adjustable.

[0208] In an example, the device is configured to automatically adjust the cross-section in response to a performance metric associated with the microfluidic flow.

[0209] In an example, the device is configured to operate in a cleaning mode, which is characterized by the maximum cross-sectional area of the microfluidic pore and a cleaning flow of liquid through the microfluidic pore, optionally, wherein the cleaning flow has a flow rate higher than the microfluidic flow.

[0210] In an example, the focused electromagnetic radiation is emitted in response to the monitored response of the particles.

[0211] In an example, the plurality of sensors are implemented as a photoelectric array with detectors, and the detectors are arranged to capture response emissions from different directions.

[0212] In an example, the output from the sensors is adjusted according to the position of the sensors.

[0213] In an example, the output from the sensors is arranged to be normalized to compensate for differences in one or more of the following properties of the response emissions captured from the respective sensors: amplitude; phase; propagation delay; refractive effect when entering the flow environment from the microfluidic flow.

[0214] In an example, the plurality of sensors are arranged in a plane perpendicular to the longitudinal axis of the microfluidic flow, and wherein the plurality of sensors are arranged in an arc around the microfluidic flow or in a substantially straight line adjacent to the microfluidic flow.

[0215] In an example, the outputs from the sensors corresponding to the response emissions from the particles are integrated to generate a signal for classifying the particles.

[0216] In an example, the device includes a second sheath extending parallel to the microfluidic flow and at least partially surrounding the microfluidic flow and the fluid environment.

[0217] In an example, the second sheath comprises one or more of: a gaseous flow moving relative to the fluid environment, the interrogating electromagnetic radiation being directed through the flow; a transparent solid material, the interrogating electromagnetic radiation being directed through the transparent solid material.

[0218] In an example, the microfluidic well is defined in a flow control device for generating the microfluidic flow.

[0219] Aspects of the present invention may also be broadly described as consisting of the parts, elements and features mentioned or indicated individually or collectively in the specification of the present application, and in any or all combinations of two or more of the parts, elements or features, and where the specific technical elements mentioned herein have known equivalents in the art to which the invention relates, such known equivalents are deemed to be incorporated herein as if set forth individually. 4. Description of the drawings

[0220] The present invention will now be described by way of example only and with reference to the accompanying drawings, in which:

[0221] Figure 1 is a schematic diagram of a system for classifying and sorting particles according to some examples;

[0222] Figure 2 is a schematic diagram of a portion of a system for classifying and sorting particles according to some examples;

[0223] Figure 3 illustrates a longitudinal cross-section of a flow control device for use with a system for classifying and sorting particles according to some examples;

[0224] Figure 4 Illustrated Figure 3 a transverse cross-section of a flow control device;

[0225] Figure 5 illustrates an aperture of a delivery tube for use with a system for classifying and sorting particles according to some examples;

[0226] Figure 6 The use of surface tension to improve orientation of particles according to examples is illustrated;

[0227] Figure 7 illustrates an aperture of a delivery tube for use with a system for classifying and sorting particles according to some examples;

[0228] Figure 8Illustrates an adjustable aperture of a delivery tube for use with a system for classifying and sorting particles according to some examples;

[0229] Figure 9 Illustrates a side view of an adjustable aperture of a delivery tube for use with a system for classifying and sorting particles according to some examples; and

[0230] Figures 10a to 10c Illustrates the intensity profile of a light beam for interrogating or sorting particles according to some examples.

[0231] Figure 11 Illustrates the intensity region of a light beam focused at a nominal focus X according to an example;

[0232] Figure 12 Illustrates the intensity region of a light beam focused off-center of a microfluidic flow according to an example;

[0233] Figure 13 Illustrates light beam separation vs. flow rate according to an example;

[0234] Figure 14 Illustrates a sheath including a discrete sheath member for an objective lens according to an example;

[0235] Figure 15 Illustrates an embodiment of a vaporization device according to an example;

[0236] Figure 16a and Figure 16b Illustrates droplet formation adjustment according to some examples; and

[0237] Figures 17a and 17b illustrate a detection device according to some examples.

[0238] Figure 18 Illustrates an example of the present invention, which shows a flared aperture.

[0239] Figure 19 Illustrates an example showing a tapered microfluidic delivery tube;

[0240] Figure 20a Illustrates an example of a flow control device shaped to define a region in the upstream direction of an aperture through which at least a portion of an interrogation beam and / or a sorting beam is directed and / or in which an objective lens is at least partially located;

[0241] Figure 20b Illustrates an example showing a stable microfluidic flow exiting an aperture;

[0242] Figure 21a and 21bIllustrates the influence of the excitation distance on the particle discrimination resolution;

[0243] Figures 22a to 22c Illustrates an example where the interrogation and sorting beams propagate at an angle to each other and towards a common objective lens;

[0244] Figure 23 Illustrates an example of using an arrangement of optical components to provide angled interrogation and sorting beams; and

[0245] Figures 24a to 24c Illustrates an example of a beam splitter arrangement for providing angled interrogation and sorting beams. 5. Detailed Description

[0246] In the claims as well as in the above specification, all transitional phrases such as "comprising", "including", "carrying", "having", "containing", "involving", "holding", "consisting of", etc. shall be understood to be open-ended, i.e., meaning including but not limited to. Only the transitional phrases "consisting of" and "consisting essentially of" shall be respectively closed or semi-closed transitional phrases.

[0247] As used herein, the term "about" refers to a reasonable deviation amount that modifies a term such that the final result is not significantly changed. For example, when applied to a value, the term shall be understood to include a deviation of + / - 5% of that value.

[0248] All definitions as defined and used herein shall be understood to take precedence over dictionary definitions, definitions in incorporated documents by reference, and / or the ordinary meaning of the defined terms.

[0249] Unless explicitly indicated to the contrary, the indefinite articles "a" and "an" as used in this specification and claims shall be understood to mean "at least one / kind".

[0250] The terms "can" and "may" are used interchangeably in this disclosure and indicate that the recited element, component, structure, function, functionality, object, advantage, operation, step, process, device, system, equipment, result, or illustration can be used, included, or produced, or otherwise represents the proposition indicated in the statement for a particular example using (or referring to) the term.

[0251] As used in this specification and the claims, the phrase "and / or" shall be understood to mean "either or both" of the elements so combined - i.e., elements that are present jointly in some cases and separate in other cases. Multiple elements listed with "and / or" shall be construed in the same manner, i.e., "one or more" of the elements so combined. In addition to the elements specifically identified in the "and / or" clause, other elements may optionally be present, whether related or unrelated to those specifically identified. Thus, as a non-limiting example, the mention of "A and / or B" when used in conjunction with open-ended language such as "comprising / including", in one example, may refer only to A (optionally including elements other than B); in another example, only to B (optionally including elements other than A); in yet another example, to both A and B (optionally including other elements); and so on.

[0252] As used in this specification and the claims, when referring to a list of one or more elements, the phrase "at least one / at least a single one" shall be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including each and every element specifically listed within the list of elements and at least one of each element, and does not exclude any combination of the elements in the list. In addition to the elements specifically identified within the list of elements referred to by the phrase "at least one / at least a single one", this definition also allows for the optional presence of elements, whether related or unrelated to those specifically identified. Thus, as a non-limiting example, "at least one of A and B" (or equivalently, "at least one of A or B", or equivalently, "at least one of A and / or B"), in one example, may refer to at least one A, optionally including more than one A, where B is absent (and optionally including elements other than B); in another example, may refer to at least one B, optionally including more than one B, where A is absent (and optionally including elements other than A); in yet another example, refers to at least one A, optionally including more than one A, and at least one B, optionally including more than one B (and optionally including other elements); and so on.

[0253] It is intended that references to numerical ranges disclosed herein (e.g., 1 to 10) also include references to all rational numbers within that range (e.g., 1, 1.1, 2, 3, 3.9, 4, 5, 6, 6.5, 7, 8, 9, and 10) and any range of any rational numbers within that range (e.g., 2 to 8, 1.5 to 5.5, and 3.1 to 4.7), and thus, all sub-ranges of all ranges expressly disclosed herein are hereby expressly disclosed. These are merely examples of specific intentions, and all possible numerical combinations between the lowest and highest values recited should be considered to be expressly stated in this application in a similar manner.

[0254] Whenever a range is given in the specification (e.g., a temperature range, a time range, or a composition range), all intermediate ranges and sub - ranges, as well as all individual values included in the given range, are intended to be included in this disclosure.

[0255] Specific details are set forth below, such as specific examples or examples for illustrative purposes and not for limitation. Those skilled in the art will understand that other examples may be employed in addition to these specific details. In some cases, detailed descriptions of well - known methods, nodes, interfaces, circuits, and devices are omitted so as not to obscure the description with unnecessary details. Those skilled in the art will understand that the described functions may be implemented in one or more nodes using hardware circuits (e.g., analog and / or discrete logic gates interconnected to perform a dedicated function, ASICs, PLAs, etc.) and / or using software programs and data in conjunction with one or more digital microprocessors or general - purpose computers. Nodes that communicate using an air interface also have appropriate radio communication circuits. Additionally, where appropriate, the technology may alternatively be regarded as being fully embodied in any form of computer - readable memory, such as solid - state memory, disk, or optical disk containing an appropriate set of computer instructions that will cause a processor to execute the techniques described herein.

[0256] Hardware implementations may include or cover, but are not limited to, digital signal processor (DSP) hardware, reduced - instruction - set processors, hardware (e.g., digital or analog) circuits, including but not limited to one or more application - specific integrated circuits (ASICs) and / or one or more field - programmable gate arrays (FPGAs), and (where appropriate) state machines capable of performing such functions. Memory may be used to store temporary variables, hold and transfer data between processes, non - volatile configuration settings, standard message formats, etc. Any suitable form of volatile memory and non - volatile storage devices may be employed, including random - access memory (RAM) implemented as metal - oxide semiconductor (MOS) or integrated circuit (IC), and storage devices implemented as hard - disk drives and flash memory.

[0257] Some or all of the described apparatus or functionality may be instantiated in a cloud environment such as Docker, Kubernetes, or Spark. This cloud functionality may be instantiated at the network edge, device edge, local premises, or on a remote server coupled via a network such as 4G or 5G. Alternatively, the functionality may be implemented in dedicated hardware.

[0258] The term "restriction" as used herein refers to a constraint on the cross - sectional shape and size of a particle flow in a fluid flow. For example, the diameter of a circular cross - section of a flow may be constrained, or the dimensions of the major and minor axes of an elliptical cross - section flow may be constrained, such that a single narrow trajectory may be produced, where any polar axis of the particles has a minimum deviation from the defined central longitudinal axis of the flow.

[0259] The "orientation" of an asymmetric particle (including a cell) refers to the primary angle of the plane of a representative sample of the particle with respect to an axis that is substantially perpendicular to the flow axis of the particle. In the absence of any feature imposing a directional torque on the particle, it is expected that the orientation of the plane will be randomly distributed and face any angle of approximately 360°. A cell sample on which a directional torque has been imposed by a directional feature will have a non-random angular orientation that preferentially directs the plane of the particle at a specific angle such that a primary angle can be determined or observed.

[0260] "Cell" and "X cell" are referred to herein as examples of specific types of particles that may be desired to be retained within a microfluidic sorting device. When the term "cell" is used herein, "cell" may be replaced by the term "particle", and it is not required that the cell / particle be a living cell. One of ordinary skill in the art will readily understand that reference to an X cell is intended to indicate any other cell or particle having properties suitable for interrogation and sorting according to the present invention. Specifically, an X cell may be replaced herein by any type of particle or cell (including substantially symmetric and asymmetric cells, neurons, red blood cells, labeled cells, viruses, or microbiota, as will be known to one of ordinary skill in the art).

[0261] The term "microfluidic flow" as referred to herein means a liquid flow having at least one geometrically constrained dimension in which surface forces dominate volume forces. In an example, this may include a liquid flow having a submillimeter diameter or other cross-sectional dimension. In an example, the microfluidic flow may be a continuous-phase flow of a liquid, such as an uninterrupted flow of one or more aqueous solutions. This may be a laminar flow having a particle flow including particles and a sheath flow surrounding the particle flow. The microfluidic flow may alternatively or additionally include a dispersed flow of droplets. The microfluidic flow may be associated with one or more performance metrics, such as flow rate, cross-sectional diameter and / or size, distance to droplet formation.

[0262] The term "flow environment" as referred to herein means an environment through which a microfluidic flow can flow. Examples include a microchannel, which may include a material forming an elongated lumen or path through which the microfluidic flow flows, such as glass. The path may be completely surrounded by the material between each end of the path, or the path may have at least one boundary exposing the microfluidic flow to a fluid environment, where the material forming the substrate abuts the other boundaries of the microfluidic flow. In another example, the flow environment may be a fluid environment or volume, which may be substantially static or may itself be flowing. In this example, the microfluidic flow may not abut a material substrate but may be completely surrounded by the fluid environment. The fluid environment may be a liquid (such as an aqueous solution) or a gas (such as air).

[0263] Figure 1Illustrated is a sorting system 100 that includes a preparation station 105 that transports prepared particles to a flow control device 110 that transports the particles into a microfluidic stream 115 for downstream processing. The microfluidic stream 115 can be a laminar flow having a cross-sectional size within a predetermined range and transported within a flow environment. In an example, the flow environment can include a volume of gas (such as air) or a microchannel that completely or partially surrounds the microfluidic channel. One or more illuminators 120 generate interrogation beams, such as infrared (IR) or ultraviolet (UV) illuminators or other radiation devices. The interrogation beam irradiates the particles within the microfluidic stream at an interrogation region 125. The irradiation of the particles causes these particles to emit an irradiation pattern that is detected by one or more detectors 130, such as scattered light or fluorescence. The measured characteristics of the detected irradiation pattern generate one or more signals that are forwarded to an analysis unit 135.

[0264] The analysis unit 135 can include a processor and a memory and is configured to interpret the signals to control a sorting device 140 that processes particles in different populations P1 and P2 based on an analysis of the signals associated with those particles. An example analysis unit 135 is described in International Patent Publication WO2022139597A1, which is incorporated herein by reference.

[0265] If the analysis unit 135 determines, based on an analysis of their respective measurement signals, that a particle has a particular classification, it controls the sorting device 140 to sort the particle. Various sorting methods can be employed to select the subpopulation of particles.

[0266] The illuminator 120 directs interrogation electromagnetic radiation to generate an "interrogation beam" and includes an excitation source adapted to cause particles such as cells (e.g., stained cells) to emit a signal or pattern. In one example, the illuminator includes an infrared (IR) or mid-IR laser 123, more preferably a mid-IR quantum cascade laser (QCL). These lasers have the potential to focus sufficient energy onto a single cell for accurate, high-speed measurements. The QCL offers several advantages over traditional mid-IR sources, such as delivering a very high spectral power density and a very high spatial or angular power density. This allows the QCL to place 10,000,000 times more effective mid-IR power onto a single cell than traditional mid-IR sources. The QCL also enables the detection of cells with lower staining levels or, in some cases, label-free detection of cells. Dyes or labels may be able to alter or damage the cells. Thus, when using label-free detection of cells, the energy of the measurements using mid-IR irradiation is 25 times lower than the energy used in FACS, eliminating photon damage and enabling high-throughput (>10,000 cells / second) capabilities. Mid-IR can include wavelengths from 5 microns to 28 microns.

[0267] In an alternative example, the illuminator 120 generates a UV laser 123. This can be pulsed continuous wave or quasi-continuous wave. The illuminator can be an LED, such as a UV LED. In one example, the illuminator generates a probe beam 123 having a wavelength of 300 nm to 400 nm (where a specific example is 355 nm). In an alternative or additional mode, the beam frequency can range from continuous wave to 100 MHz. In another alternative or additional mode, the beam power is 1 mW to 1 W.

[0268] Focusing optics are included within the system to achieve focusing and / or spatial shaping of the beam.

[0269] The sorting device 140 can include a radiation source (or nudging laser) configured to direct radiation onto the particles to impart at least one of a force and a torque to each particle, thereby causing each particle to undergo at least one of displacement and orientation (or nudging) relative to an axis defined by the direction of the fluid flow. In an example, the system further includes at least one of free space optics, optical fibers, and other waveguides configured to direct the radiation from the radiation source onto the microfluidic fluid flow. In an example, the radiation source of the sorting device 140 includes a laser and can be configured for stroboscopic operation. In the examples described herein, the sorting electromagnetic radiation (also referred to herein as "radiation pressure") emitted or directed from such a radiation source is referred to as the "sorting beam". Some examples can utilize a mid-IR beam as described above as the illuminator.

[0270] A particular implementation of the sorting device can include a radiation source for "nudging" the particles based on a classification (e.g., P1 or P2) determined by the analyzer 135 using the output of the detector 130. In one example, the microfluidic flow is contained within a microchannel, and the nudged particles then travel until they reach a bifurcation point 145. In some examples, the continuous fluid flow can split into two or more branches corresponding to two or more different collection tubes 160. While in Figure 1is shown as having two collection branches, but it should be understood that the stream 115 can be split into three, four, five, or more different collection branches. The number of collection branches can be achieved by using a specific configuration of the sorting device 140, which can include baffles, vanes, and / or bifurcation members 145 disposed within the microfluidic stream 115. This will be more understood when the microchannel is considered as a three-dimensional structure. Additionally, the sorting device 140 can include two or more radiation sources, which are optionally positioned on opposite sides of the microfluidic stream and are individually controllable. These radiation sources "nudge" the particles in multiple directions within the microfluidic stream. In one example, the optionally positioned radiation sources can be a plurality of radiation sources arranged around the circumference of a circular cross-section microfluidic channel, and these circumferentially arranged radiation sources allow for precise control of the particles in any direction. Example sorting devices are described in International Patent Publication WO2020 / 013903A2, which is incorporated herein by reference.

[0271] Those skilled in the art will understand that alternative methods can be employed within the sorting device to achieve the separation of particles with desired characteristics. For example, using electrostatic sorting methods or microbubble-induced particle sorting is known to those skilled in the art, and these methods are designed to accomplish the same task. Here, microbubbles or droplets including the nudged particles can move laterally relative to an axis corresponding to the flow direction and do not necessarily remain entrained within the separated fluid streams. While increasing the power of the radiation sources or similar alternative modifications to the sorting device can increase the initial separation distance between the particle streams, it may be desirable to do so without affecting the viability of, for example, cells. Therefore, using such sorting methods, careful control of the radiation sources is required to ensure that the selected cells are shifted into a different flow path compared to the unselected cells and that the selected cells do not become immotile or non-viable due to the radiation. This shifting effect is preferably achieved by applying electromagnetic radiation (e.g., via a laser) to change the direction of the cells from a first flow path to a different flow path. Then, the particle flow paths containing the selected (P1) cells or unselected (P2) cells can be directed to a first collection container, and the particle streams containing other cell subpopulations can be discarded or collected in a second different collection container.

[0272] In another example, the sorting device 140 includes an electromagnetic radiation source that irradiates the microfluidic flow to effect ablation or damage of selected particles therein. In this example, the ablation-causing sorting electromagnetic radiation from the radiation source so emitted or directed is also referred to as a "sorting beam". The device is particularly useful for removing undesired cells within a larger population. For example, during the generation of a cell population for CAR T cell therapy, there may be certain types of cells that do not exhibit the desired phenotype. The cells in this first population (P1) are disrupted, denatured, or rendered immotile by the sorting device. An alternative population in the microfluidic flow 115 that does not exhibit the desired characteristics (such as desired cells that have not been selected by the analysis unit) remains undisturbed (P2). The sorted or processed cells in the microfluidic flow 115 can then be collected in one or more collection containers 160 for further use. The sorting device 140 thus provides a cell population (P2) enriched in the desired characteristics. When using sperm cells, the desired population can include motile X cells.

[0273] In a specific example, the selected cells are asymmetric cells that exhibit particularly desired characteristics, such as sperm cells, red blood cells, or nerve cells. The specific classification of the selected cells preferably includes sperm cells carrying a Y chromosome (which corresponds to male sperm) or sperm cells carrying an X chromosome (which corresponds to female sperm).

[0274] In some examples, a pulsed beam is used for sorting. In other examples, a continuous wave beam is used for sorting. The amount of energy transmitted to the focus can be described by the peak energy density in J / cm 2 units, which is calculated by dividing the laser pulse energy (joules) by the effective focal spot area (cm 2 ). In one example of achieving particle / cell ablation, the peak energy density is between 0.1 J / cm 2 and 100 J / cm 2 . In another example, the sorting laser is adapted to cause the liquid at the focus to phase change to a gas. In this example, the peak energy density can be between 10 J / cm 2 and 1000 J / cm 2 . If the particle / cell is also within the focal spot region, it is greater than about 0.1 J / cm 2The peak energy density can result in ablation of the particles / cells. Ablation can be considered as the process of transferring energy to the particles sufficient to permanently inactivate the particles. In the context of biological cells, this can include rendering the cells non-viable and unable to perform their normal functions or purposes. For example, sperm cells can be ablated to rapidly induce permanent immotility, or sperm cells can be ablated to "prime" the sperm cells so that they cannot survive downstream processes such as freezing and thawing. In the former, ablation can involve rupturing the cell surface membrane, which disrupts cell integrity. During "priming", the cell surface membrane remains substantially intact, even though motility may be reduced or stopped.

[0275] In some examples, the numerical aperture of the optical component (such as an objective lens for providing the sorting beam) ranges between 0.25 and 0.7. The wavelength of the sorting beam can range between 300 nm and 600 nm, with specific examples being 480 nm to 580 nm and 300 nm to 400 nm. In one example, the sorting beam wavelength is 532 nm + / - 5 nm. Alternatively, the sorting beam wavelength can be 355 nm + / - 5 nm.

[0276] The preparation station 105 can include means for staining a batch of cells (e.g., sperm cells collected from a bull). Various other preparation steps can be performed, such as diluting a batch of sperm samples or adding media components known to those skilled in the art. In an example, the interrogation region 125 can include means for detecting the properties of each of a plurality of particles such that the particles can be identified. To this end, fluorescent dyes (such as fluorescein isothiocyanate (FITC), R-phycoerythrin (PE), allophycocyanin (APC), and peridinin-chlorophyll-protein-based dyes, as well as Alexa Fluor dyes and green fluorescent protein (GFP)) can be deployed to interrogate individual particles as they pass through the interrogation region 125, and the fluorescent dyes can have excitation and emission wavelengths within the UV spectrum (or other regions of the electromagnetic spectrum, assuming the light source and detector are appropriately configured). As an example, where the particles are cells, viability assays (such as calcein-AM and ethidium homodimer-1) can be used to sort viable cells from non-viable cells. Alternatively, the cells can be stained with Hoechst 33342, which is a fluorophore commonly used to stain DNA in mammalian sperm carrying X and Y chromosomes. In these examples, the interrogation region 125 can identify the primary emission wavelength from a given cell, and the sorting device can be used to sort the cells within the microfluidic flow accordingly.

[0277] In one example, the analysis unit 135 can be integrated with the CPU of the computing device, or can be separately integrated with the detector 130 or the sorting device 140. In an example, the analysis unit can be implemented as a dedicated logic circuit, such as an FPGA or an ASIC. In an example, the sorting device can include a radiation source (or a nudge laser) and can be configured to optionally direct the radiation onto each particle to apply at least one of a force and a torque to each particle. The movement of the particle can be at least one of shifting and orienting (or nudging) each particle relative to an axis defined by the direction of the fluid flow along the microfluidic flow. To this end, the sorting device 140 can be configured to direct the radiation onto the particle when the identity of the particle indicates that the particle should be irradiated. For example, directed into separate particle flow streams intended for different collection branches of the microfluidic flow. In an example, the movement of the particle can be achieved by applying a static charge (as in FACS (fluorescence-activated cell sorting)), by buoyancy, by magnetic activation (as in magnetic-activated cell sorting), etc. In an example where the particle is an unwanted cell or particle, the radiation directed onto the particle can be calibrated to ablate the particle to damage, kill, or reduce the function of the particle / cell. In such examples, it may not be necessary to separate the ablated particles from the non-ablated particles.

[0278] The flow control device 110 receives the particle stream 107 from the preparation station, which particle stream contains a solution of cells from a preparation batch, such as an aqueous solution. The flow control device 110 also receives the sheath flow 108, which may also include an aqueous solution. In one example, the flow control device 110 combines the particle stream and the sheath flow to generate a controlled laminar flow containing cells, where the sheath flow extends coaxially around the particle stream, thereby forming a microfluidic flow. The flow rates of the particle stream 107 and the sheath flow 108 can be controlled, and the flow control device 110 can include components arranged to control the particle stream and the sheath flow so as to orient and / or confine these cells within the laminar flow. Since some cells are asymmetric, orienting them in a preferred plane improves their interaction with downstream devices, such as the detector 130 and the sorting device 140. In addition, confining the cells within a narrow flow path increases the likelihood that the downstream irradiator 120 will impinge thereon as expected.

[0279] The flow rates of the particle stream 107 and the sheath stream 108 can be controlled, and the flow control device 110 can include components arranged to control the particle stream and the sheath stream so as to orient and / or confine the cells within a laminar or microfluidic flow. In a particular example of the present disclosure, the sample flow rate of the microfluidic flow is in the range of from about 0.1 μl / min to about 20,000 μl / min, from about 10 μl / min to about 5,000 μl / min. In other examples, the microfluidic flow rate is greater than about 100 μl / min, greater than about 500 μl / min, greater than about 1,000 μl / min, greater than about 2,000 μl / min, greater than about 4,000 μl / min, greater than about 5,000 μl / min, greater than about 6,000 μl / min, greater than about 7,000 μl / min, greater than about 8,000 μl / min, greater than about 9,000 μl / min, greater than about 10,000 μl / min, greater than about 11,000 μl / min, greater than about 12,000 μl / min, greater than about 15,000 μl / min, and / or greater than about 20,000 μl / min.

[0280] In some examples, the flow environment into which and through which the microfluidic flow flows is a volume of gas. This can facilitate higher flow rates compared to using microchannels due to reduced friction with the surface of the microfluidic channel. The higher flow rate reduces the overall sorting and separation time per particle, which in the case of biological cells reduces their degradation prior to being stored more optimally (such as by freezing). The faster flow rate also improves the overall performance of the system, enabling more efficient processing of larger and / or more samples. The reduced surface friction of the microfluidic flow can also improve the accuracy of sorting and / or separation by reducing unwanted internal hydrodynamic forces that can displace the particles in an unexpected manner.

[0281] The effectiveness of the flow control device 110 in orienting and / or confining the cells can improve the efficiency of the overall sorting system 100. An example of a flow control device for improving cell orientation and / or confinement is the delivery tube as described in International Patent Publication WO2020 / 013903, which is incorporated herein by reference. Relative to Figure 3 An example flow control device according to an example is described. Alternatively, other arrangements can be used, for example, the flow control device can be part of a cytometer.

[0282] Methods and apparatuses of some examples of flow control devices are useful in achieving the orientation of non-spherical particles such as sperm cells. In one example, the angle of non-spherical particles can be controlled to achieve a desired angle relative to one or more radiation beams that can be used to interrogate and / or sort the particles. In a particular example, the particles are oriented via an orientation member based on hydrodynamic and / or radiation pressure. This optimizes the interrogation and sorting of the particles, where the absorption and / or emission of radiation can be highly orientation-dependent in asymmetric particles such as sperm cells. Thus, it may be desirable to orient the preferred facets of the asymmetric particles towards the incident radiation beam, which has the largest or smallest surface area or some other property.

[0283] In an example, the microfluidic flow 115 exits the orifice 113 of the flow control device 110 and enters the flow environment. The flow environment can be liquid or gaseous or a combination of these. For the liquid and / or gaseous environment in which the microfluidic flow flows, they can be static or include movement. The microfluidic flow can exit in a downward or gravity-based direction or in a direction at an angle relative to gravity (e.g., vertical or angled substantially upward). In one example, the flow environment is defined by a microfluidic channel, microchannel, or conduit that allows the microfluidic flow to travel from the orifice in a controlled manner and restricts the flow of particles when the particles pass through the interrogation region or station 125 and the sorting devices 140, 143. In some examples, the flow environment can include a liquid having substantially the same liquid viscosity as that in the microfluidic flow. In some examples, the flow environment is a liquid having a higher viscosity than the microfluidic flow. In some examples, the fluid in the flow environment can move in the direction of movement of the microfluidic flow. The rate of this movement can be the same as or different from the rate of the microfluidic flow. In an alternative example, the flow environment includes a gaseous environment. In examples of liquid and / or gaseous flow environments, the microfluidic flow does not need to be defined by a channel or conduit. This method has many advantages and results in reduced friction with the conduit, thereby improving the laminar flow and enabling higher flow rates. The inventors have found that in the case where the orifice engages with the channel or conduit, the orifice size must be aligned with the internal dimensions of the conduit to ensure that the microfluidic flow maintains laminar flow and has minimal turbulence. The conduit size is not always consistent with the orifice size, which limits the range of orifice sizes. By employing a fluid flow environment, this allows for flexibility in the size and shape of the orifice. In particular, using an orifice smaller than a standardized conduit also allows for a reduced proportion of sheath flow compared to the particle flow. This reduction in sheath volume thus increases the selected cell concentration. Omitting the conduit also reduces the refraction of the interrogation beam 123 and, if a conduit is used, reduces the refraction of the sorting beam 143 through the conduit wall, thereby improving the accuracy of interrogation and sorting.

[0284] In one example, apparatuses and methods are provided for separating cells within a microfluidic flow in a gaseous environment. Some examples address effects that occur in the case of interrogation or sorting of particles in a microfluidic flow in a gaseous environment. For example, upon entry into the cylindrical profile of the microfluidic flow, light or other electromagnetic radiation (EMR) may refract due to, for example, a refractive index mismatch between water and air, which may subsequently distort the light beam. The inventors have also found that there is astigmatism from the cylindrical flow profile, which gives two foci, one vertical and one horizontal. This effect causes interference with a light beam entering the flow to interrogate or sort particles.

[0285] Figure 10a An intensity distribution plot is shown that depicts a cross-section of a microfluidic flow generated using a modeling program (e.g., using Python / NumPy). The X and Y axes represent the width and length of the cross-section of the flow. The nominal focus of the light beam is at X = Y = 0 at the center of the circle. It can be observed that there are two foci, where the strongest focus (“horizontal focus”) is offset from the center of the flow. The “vertical focus” remains focused near the center of the flow at and around the nominal focus. The effect of this phenomenon is that the aiming of particles flowing on the Z-axis (out of the page) is disrupted because the interrogation and / or sorting light beams 123, 143 are separated and defocused. Additionally, the emission propagating from the particles after interrogation is disrupted, resulting in low particle identification and sorting efficiency. Figure 10b and Figure 10c An intensity plot is shown as a cross-section through the Z-axis and the Y-axis. Figure 10b An enlarged view of the horizontal focus is shown, and it can be seen that due to the refractive index mismatch, the point-like light beam elongates in the Y direction. Figure 10c Illustrates how the vertical focus of the light beam (at the nominal focus) elongates along the Z-axis. This effect is caused by portions of the light beam intersecting the surface of the microfluidic flow from non-vertical directions and being refracted to cause double focusing. The actual distance between the center of the horizontal focus and the vertical focus depends on the diameter of the flow, the refractive index of the fluid, and the numerical aperture of the objective lens.

[0286] When the light beam enters the microfluidic flow, this unusual effect of the light beam defocusing and elongating in the vertical direction has prompted the inventors to develop optical enhancements that enable the interrogation and / or sorting light beams to be optimally aimed at particles flowing on the Z-axis.

[0287] In one example, a method of applying a radiation beam to particles flowing in a microfluidic flow is provided, wherein the beam passes through a refractive index boundary before entering the microfluidic flow, and wherein the particle flow path is adjusted and / or shifted to offset from the center of the flow path. This has the effect of moving the particles into the higher intensity radiation of the horizontal focus of the incident interrogation or sorting beam. In another example, the present invention includes a method of applying a radiation beam to particles flowing in a microfluidic flow in a gaseous environment, wherein the horizontal focus is adjusted to the center or around the circular cross-section of a substantially cylindrical microfluidic flow. This has the effect of changing the focus and enhancing the radiation pressure intersecting the particles in the flow. Both examples have the effect of improving or eliminating the effects of the refractive index boundary, and thus increase the intensity of the laser power intersecting the particles, thereby achieving enhanced sorting. In the examples provided above, the applied radiation can be optimized for nudging, interrogation, vaporization or ablation. The microfluidic flow of the above examples may be in a gaseous environment or in a solid tube or channel or in any other environment that causes a refractive index boundary of the refracted radiation beam.

[0288] In some examples, using an adjusted beam focus enables the user to minimize the amount of power used to generate the laser and apply to the particles. This can have benefits in minimizing heating of optical components and microfluidic flow, and can reduce the need to displace particles and / or adjust microfluidic flow. Minimizing the power directed into the flow also minimizes collateral damage to other flowing particles, and can enable control over whether cavitation bubbles are formed.

[0289] In one example, the interrogation and / or sorting beams are adjusted to form beam shapes with unequal aspect ratios, such as an elliptical beam shape. This beam shape enables optimization of the energy applied to the particles while spreading the beam over the flow path of confined particles in the microfluidic flow. This approach is intended to improve beam interactions with particles when needed and reduce missed beam interactions with particles when needed. Adjusting the beam shape in this manner may also provide more uniform interactions with a higher proportion of particles compared to a point-like beam that may have high intensity in a small area and very low intensity elsewhere.

[0290] In some examples, the interrogation beam and / or sorting beam has a beam width of 1 micron to 500 microns. In examples that achieve a more restricted flow, the interrogation and / or sorting beam width is 5 microns to 100 microns.

[0291] In addition to this bifocal effect caused by the different refractive indices of the flow environment and the microfluidic flow, the inventors also observed that the focus of a light beam entering the microfluidic flow is misaligned compared to a light beam entering the flow from a fluid of the same refractive index. Figure 11 The intensity region when the beam is focused at the nominal focal point X is shown.Figure 12 Shows the intensity regions when the nominal focus is adjusted to be off - center of the flow cross - section. It can be clearly observed that an off - center focus provides a larger region of uniform intensity within the flow.

[0292] When applied to the action of interrogating or sorting cells within a microfluidic flow, the inventors have found that this lack of beam intensity at the nominal focus means that cells that should interact with the beam may not interact with it. This is especially true when the confinement of the cells is low (i.e., the X - axis or Y - axis spread of the cells throughout the flow is high), because the chance of a particular cell falling outside the optimal intensity region is lower. Therefore, to achieve the desired interaction between the beam and a large proportion of the particles, a higher beam power may be required, which may have a negative impact on some particles. To address this issue, the inventors took the unusual step of intentionally adjusting the focus beyond the nominal focus, such as Figure 11 and Figure 12 shown by moving the focus "X". This adjustment has the effect of increasing the intensity around the center of the flow while also providing a more uniform and widespread distribution of intensity across the entire flow cross - sectional area. A defocused or diffused beam may also or alternatively be used to achieve a similar effect.

[0293] In an example, as an alternative or additional feature of the method as previously described, a method is provided for applying radiation pressure to particles flowing in a microfluidic flow in a gaseous environment, wherein the focus of the radiation pressure is adjusted to be off - center of the cross - section of a substantially cylindrical microfluidic flow. The focus can be at a point within the flow that achieves a maximum intensity region above a threshold. The focus can be adjusted to be offset from the center of the cross - section of the flow in a direction defined by the beam propagation and along an axis defined by the beam trajectory, where the focus is offset by a distance between about 10% and 30% of the flow diameter. In some examples, the offset distance is between about 15% and 25% of the flow diameter. This technique is referred to herein as "nominal focus offset" and has the effect of enhancing the radiation pressure intersecting the particles in the flow. It also has the effect of increasing the intensity of the laser power intersecting the particles, resulting in enhanced interrogation and sorting.

[0294] In some examples, an interrogation metric is measured due to the interaction of an interrogation beam with at least one of a microfluidic flow and one or more particles within the flow. The interrogation metric is transmitted to a control system for processing, and then an output signal adjusts flow parameters based on whether the interrogation metric is above or below a certain threshold. In one example, the output signal causes a change in one or more of positioning, flow rate, confinement, or orientation. In one example, the interrogation metric is one or more of fluorescence pulse width, intensity, and the ability to distinguish particle characteristics or populations.

[0295] In another example, the illuminator 120 and / or the sorting device 140 includes a diffractive optical element (DOE) that diffuses the interrogation beam 123 and / or the sorting beam 143 to achieve a more uniform intensity across the cross-section of the microfluidic flow. In an example, the DOE provides a uniform intensity profile over the distance of the restricted particle flow. In one example, the DOE projects a substantially "top hat" profile of the respective beams 123, 143 within the microfluidic flow.

[0296] In some examples, the focus and / or profile of the interrogation and / or sorting beam can be adjusted dynamically. This may depend on the estimated position of the particle flow within the sheath flow of the microfluidic flow. The area mapping phase shift (AMPS) technique can be used to estimate the particle flow position, and the focus can be set based on this estimated position. For example, the focus can be set at a predetermined distance beyond the estimated distance of the estimated position. This can be achieved using mechanical adjustment of the optical components that provide the interrogation beam 123 or the sorting beam 142. Alternatively, the position of the particle flow can be adjusted to a predetermined positional relationship with the focus. For example, the particle delivery device 110 can be adjusted based on the estimated particle flow position such that the position is adjusted to the desired positional relationship with the focus.

[0297] In another example, the focus of the interrogation and / or sorting beam is adjusted to be offset from the center of the microfluidic flow. In an example, the offset is 15% to 25% of the diameter of the flow to account for refraction.

[0298] To address or at least improve the problems described above related to refraction and double focusing, the inventors surprisingly found that placing the illuminator 120 at a specific distance from the fluid flow enables accurate interrogation and sorting. This "working distance" is measured from the emission side of the final optical component forming the illuminator to the focus of the beam within the microfluidic flow. When using a microfluidic flow in a gaseous environment, it is generally desirable to maximize the working distance to reduce the likelihood of droplets contacting the optics (through which the interrogation and / or sorting beam propagates) or the detectors (at which emissions are detected). In one example, a nominal focus offset is achieved by adjusting the positioning of the lens to account for defocusing and achieve a uniform beam intensity.

[0299] In one example, the illuminator emits an interrogation beam and the sorting device emits a sorting beam, and both beams propagate through a single objective lens (optical component). In another example, the interrogation beam and the sorting beam can pass through separate respective optical components and at least one shared optical component.

[0300] In one example, the working distance is at least 5 mm to 50 mm, and in some examples, the working distance is greater than 10 mm and less than 40 mm. The inventors have calculated that in some examples, the working distance is 10% to 30% of the flow diameter to achieve an optimal offset from the flow.

[0301] In cases where the flow environment includes a gaseous fluid environment, the gaseous fluid includes a gas, and the gas may include one or more gases or gas mixtures selected from the group consisting of: air, nitrogen, carbon dioxide, methane, or a noble gas (such as helium, argon, neon, xenon, or krypton). In certain examples, the gaseous fluid is an inert gas, such as nitrogen or a noble gas. Without being bound by theory, it is believed that these inert gases reduce the oxidation and contamination of the fluid flow, which can lead to an increase in cell viability after downstream processing.

[0302] The gaseous fluid environment can be maintained at a fluid temperature and / or a fluid pressure, which enhances at least one of cell flux, detection accuracy, sorting accuracy, or cell health. Herein, cell health refers to the likelihood of survival, motility, or viability during the sorting process. In an example, the temperature is between 18 °C and 37 °C. Maintaining the temperature above 20 °C can help ensure minimal viscosity to assist in maintaining flow and reducing blockages. In an alternative example, the temperature is maintained below 15 °C. Operating at this temperature can minimize the impact of thermal stress on the cells and can ensure maximization of cell motility and viability after the cells flow through the system.

[0303] To maintain an appropriate working distance according to the above limitations while providing a focus with appropriate power for interrogation / sorting, the numerical aperture of the sorting and / or interrogation beam can be optimized. The inventors have found that the numerical aperture of the objective lens can be between approximately 0.1 and 0.7, or in some cases between 0.2 and 0.4.

[0304] In an example, when the microfluidic flow exits the orifice into the gaseous environment, the width of the beam is between 30 μm and 200 μm. In certain examples, it may be preferred to maintain a flow width between 70 μm and 110 μm. The present inventors have found that these ranges provide a flow that is wide enough to accommodate the particle flow when the particle flow is combined with the sheath flow. These widths also enable accurate sorting by nudging the particles into different flow paths or using an ablation sorting laser to ablate unwanted particles.

[0305] In some examples, the distance from the center point of orifice 113 aligned with the terminal surface of the flow control device to the point where the interrogation beam contacts the microfluidic flow is 25 μm to 1000 μm. This distance is referred to as the "excitation distance", as Figure 2illustrated by arrow ED therein. A shorter excitation distance (e.g., 50 μm to 500 μm) may be preferred to retain confinement of the flow within the flow environment.

[0306] In some examples described herein, steps are taken to adjust the focus or other beam properties to account for the cylindrical form of the flow. In these cases, the excitation distance should be long enough to allow the flow surface to become smooth. After excitation from the aperture, the flow tapers and stabilizes as Figure 20b shown. The inventors have found that attempting to interrogate at an excitation distance too close to the aperture results in a poor cellular emission signal. This is thought to be due to too little interrogation radiation reaching the cells, or too little emission radiation reaching the detector. This may be due to refraction of the beam through the non-stable flow. In some examples, the interrogation beam comprises a focused beam forming a conical shape 2001 as Figure 20a shown. If the excitation distance ED is too small, there is a risk that the cone will be interrupted by the lower surface of the flow control device 2005 as Figure 20a shown. In some examples, it is also desirable to provide a distance to allow the flow to stabilize its cross-sectional shape and form a circular cross-section. These features can help to achieve accurate focusing of the beam onto particles within the flow and also minimize any unpredictable refraction effects as the beam enters or exits the flow.

[0307] The lower surface of the flow control device 2005 may be shaped to define a region above the aperture through which the interrogation beam and / or sorting beam may be directed. This avoids clipping of portions of the radiation cone 2001, which improves interrogation performance. Similarly, the objective lens may be at least partially positioned within this region to allow the interrogation beam to be focused closer to the aperture. Figure 20a The region shown is in the form of a triangular recess extending above the line of the aperture. However, other shapes and configurations may alternatively be used for these purposes. This region above the aperture may be defined by the external shape of the flow control device, which may include a recess, bevel, or notch in the terminal surface that houses the aperture.

[0308] However, as described in more detail below, some examples of the present invention utilize the proximity of the interrogation beam contact point to the aperture, i.e., minimizing the excitation distance. This can provide benefits in terms of reducing divergence of the flow and limiting losses of the sample flow. Figure 21a Experimental results are shown for different excitation distances from the aperture to the focus of the interrogation beam. As Figure 21aAs shown, the discrimination resolution is calculated by computing the separation between two fluorescence emission peaks. The discrimination resolution refers to the ability to discriminate between two cell populations and can be related to the amount of overlap between the two peaks, where more overlap corresponds to lower discrimination resolution because cells cannot be discriminated within the overlap. In this example, the peaks correspond to X and Y sperm cells stained with a DNA-specific stain. It can be observed that the fluorescence intensity decreases at lower excitation distances, which is understood to be the result of clipping of the light beam or emission on the lower surface of the flow focusing device. This results in relatively more overlap between the peaks.

[0309] On the other hand, taking an excitation distance of 100 μm as a baseline (100%), it can also be observed that at higher excitation distances, the discrimination resolution decreases. Thus, in the example, an optimal discrimination resolution can be obtained within a range of excitation distances that is not too small and not too large.

[0310] The X cell selection metric also indicates that higher excitation distances result in a decrease in the number of X cells selected from a combination of X and Y cells. In cases where it is desired to select and sort X cells from Y cells, this effect on X cell selection will have a negative impact on the sorting efficiency, throughput, and purity of the sorted cell sample. Thus, the inventors have determined an optimal interrogation zone with a preferred excitation distance. In one example, the excitation distance is greater than 25 μm, 50 μm, or 100 μm. In another example, the excitation distance is less than 400 μm. In one example, the excitation distance is between 25 μm and 400 μm or between 25 μm and 1000 μm. In another example, taking into account the decrease in fluorescence intensity, the excitation distance is between 50 μm and 400 μm. In another example, the excitation distance is between 50 μm and 250 μm. The optimal excitation distance may depend to some extent on the flow rate, however they may not be significantly affected by the flow rate. Thus, in one example, the flow rate is greater than 5 m / s.

[0311] In other embodiments, the flow rate is 5 m / s to 20 m / s. The interrogation beam and the sorting beam are separated by a distance that is referred to as the "inter-beam distance" IBD in Figure 2 . In some examples, the inter-beam distance is 15 μm to 1000 μm.

[0312] Similarly, it may be desirable to minimize this distance to prevent divergence of the flow in the flow environment and to minimize the loss of orientation that an asymmetric particle moving in the flow may experience. However, the inter-beam distance can depend on the time taken to detect the emission, identify the particle type, process the data to determine how to sort that particular particle, and then send a signal to the sorting beam to generate a beam with appropriate properties to sort the particle. After extensive experimental analysis and modeling, the inventors have determined the parameters for these events and found the optimal inter-beam distance range for the flow rate range.Figure 13 and Table 1 below show the range of inter-beam distance (IBD) for specific flow rates according to the example. In this example, a minimum IBD of 16 μm is achieved at a sample flow rate of 1 ms -1 , where the propagation delay is 1.6×10 -5 s. At the same propagation delay, a flow rate of 20 ms -1 requires a minimum IBD of 320 μm. Thus, in some examples, the IBD is from about 10 μm to about 400 μm.

[0313] In some examples, the IBD is from about 40 μm to about 300 μm, or from about 50 μm to about 200 μm.

[0314] Cell velocity (m / s) IBD (microns) 1 16 2 32 3 48 4 64 5 80 6 96 7 112 8 128 9 144 10 160 11 176 12 192 13 208 14 224 15 240 16 256 17 272 18 288 19 304 20 320

[0315] Table 1 - Distance between beams vs cell flow velocity

[0316] In some examples, the microfluidic flow remains intact and continuous until it has passed through the interrogation and sorting device beams. The microfluidic flow 115 can remain intact until it intercepts one or more collection containers 160. In an alternative example, the microfluidic flow can become discontinuous and break into droplets at some point after the sorting device 140. In a static current cytometer known in the art, the microfluidic flow is intentionally disturbed by the charge applied by an ultrasonic transducer. For example, Cossarizza (2017) (see details of reference 1 pointed out below) describes in Section 1.4 how to apply charge to the flow, and then the droplets retain the charge after breaking at a pre-determined distance from the nozzle orifice / hole. The distance from the orifice / hole to the point where the flow becomes discontinuous is called the "breakup distance". In traditional flow cytometry, an electrostatic plate interacts with the charged droplets to bias the droplet flow direction according to the cell characteristics identified via the interrogation device. However, the vibration applied by the ultrasonic transducer may interfere with the detection via the interrogation device. In addition, if the interrogation device interacts with the fluid flow at the point where the droplets begin to form, the uneven and irregular surface of the flowing stream results in undesirable and unpredictable refraction of the interrogation beam and the emitted illumination pattern. In cases where these irregularities can be predicted, the problem can be improved by using normalization techniques. However, these require computer processing time, which in turn slows down the sorting trigger event, thus limiting the cell throughput and efficiency.

[0317] In addition, in cases where the sorting device does not require the formation of charged droplets, it is not desirable to intentionally disturb the flow. It is also assumed that applying charge to cells may adversely affect their cell surface membrane or cell health. For sperm cells, this assumption results in a reduced fertility of the sorted sperm cells. To avoid these problems of electrostatic cell sorting, some examples do not include a device for inducing droplet formation, such as an ultrasonic transducer.

[0318] In a particular example, the microfluidic flow includes a flow that is continuous until a break distance measured from the orifice. The break distance can be configured according to the sorting method used in the example and can be achieved by appropriately controlling performance metrics of the microfluidic flow, such as flow rate and cross-sectional dimensions. In one example, the present invention provides an electrostatic device downstream of a sorting device. In this example, the electrostatic device is arranged to attract or repel droplets such that they are collected in a container different from the droplets, and these droplets a) are not affected by electrostatic sorting or are attracted in a direction opposite to the desired droplets. This arrangement and processing achieves at least one of the following: a) attracting droplets in which undesired particles are retained, and b) attracting droplets without particles. Both options result in an increase in the concentration of desired particles per unit volume by a) removing the undesired particles and the surrounding liquid from the collected volume of desired particles, or b) removing the liquid from the collected volume of desired particles. As described below with reference to the vaporization device 150, an increase in the concentration of desired particles in the collected volume can be beneficial.

[0319] The break distance can be adjusted or maintained by controlling parameters or performance metrics, such as the flow rate of the microfluidic flow and its cross-sectional dimensions.

[0320] In one example, the flow control device 110 includes one or more features on an inner or outer surface for extending the break distance. The feature can include a hydrodynamic feature that is adapted to smooth the surface of the microfluidic flow as it exits the orifice.

[0321] In another example, the sorting device includes a laser for applying at least one radiation pressure pulse to one or more particles within a fluid flow, wherein the laser is adapted to apply sufficient power to cause: a change in the direction of the particles; and / or the formation of droplets containing the particles. The required power can be determined experimentally.

[0322] In this example, selected particles are irradiated with laser power sufficient to cause droplet formation. The optimal laser power can be determined using experiments. In some examples, two or more laser pulses interact with the flow, one before the particle in the flow and one after the particle in the flow. This causes the fluid flow to split both before and after the particle to initiate droplet formation around the particle. In addition to producing droplets, the power applied to the resulting droplets also has the potential to cause a bias towards different flow paths within the flow environment. This enables the selection and separation of particles from other particles within the microfluidic flow. Compared to other methods such as vibration, using this method to facilitate droplet formation can also increase the particle concentration within the droplets.

[0323] The sheath 170 can partially or completely surround the microfluidic stream 115 and extend substantially parallel to the microfluidic stream. The sheath protects the microfluidic stream from gaseous streams that may disrupt the positioning of the stream and from contaminants (such as dust and dirt particles) that may accumulate on auxiliary equipment. Optical components for providing interrogation and / or sorting beams can be positioned adjacent to the transparent portion of the sheath or can be located in apertures or portions suitably configured in the sheath. In one example, the sheath includes an air curtain that is applied substantially parallel to the microfluidic stream in a direction substantially aligned with the flow of the microfluidic stream. When viewed in a cross-section perpendicular to the longitudinal axis or Z-axis of the stream, the air curtain can be linear (adjacent to the microfluidic stream), semi-circular (partially surrounding the microfluidic stream), or circular (surrounding the microfluidic stream). The air curtain can be applied in the form of an annular ring that includes a plurality of air jets positioned to eject air that shields the microfluidic stream. In an additional or alternative example, the sheath includes a solid structure made of a suitable impermeable material. The sheath itself can be spaced from the surface of the microfluidic stream by a gaseous interface and protects the stream from gaseous streams in the broader flow environment. Alternatively, the sheath can completely surround the microfluidic stream without a gaseous interface between the sheath and the inner wall of the sheath. In the presence of a gaseous interface, the sheath can include apertures or slots to allow some mixing between the gases inside and outside the sheath, for example, to moderate the gaseous flow inside the sheath. Additionally, the sheath can protect the stream 115 from contamination by dust and other particles. Further, the sheath can prevent aerosols from diffusing from the microfluidic stream into the surrounding environment. Such aerosols can accumulate on sensitive optical devices, which reduces their ability to sense accurately. The sheath can also be adapted to cover the optical components that make up at least one of the illuminator 120, the detector 130, and the interrogation optics (130 and 120) and / or the sorting device 140. The sheath 170 can partially or completely extend into the collection container and optionally also includes a sheath bifurcation to accommodate the separation of the stream. The sheath can take the form of an air curtain, a cylindrical or other shaped tube, a series of flat plates, or any other suitable shielding arrangement for the stream. In another example, the sheath includes discrete components that shield the optics of at least one of the illuminator 120, the detector 130, and / or the sorting device 140. Figure 14 An example of a sheath is shown that includes a discrete sheath component 1410 that covers the objective lens 1420. The sheath component 1410 can be coupled to a larger sheath 170 or the larger sheath can be omitted, with one or more sheath components 1410 used to protect the beams from various optical components to the microfluidic stream within a gaseous or liquid environment.

[0324] In one example, the sheath surrounds the microfluidic stream starting from a break distance and ending at or around the entrance of the collection container. In this example, the sheath does not interfere with interrogation and optionally does not interfere with the sorting beam or the detector.

[0325] In another example, a sheath surrounds a microfluidic flow that begins at or around a pore and ends at a break distance or at a point downstream of a sorting laser. In this example, the sheath may include at least one window or aperture such that an interrogation beam, sorting beam, or emission from a particle can be transmitted through the window or aperture.

[0326] In some examples, the distance from the microfluidic flow to the sheath is from 5 mm to 20 mm.

[0327] The sheath may be arranged to completely or partially surround the fluid flow.

[0328] The inventors have found that, in cases where the flow environment is gaseous, the gaseous environment within or around the sheath 170 can be environmentally controlled. This provides advantages including improved cell viability. The environmental control can also be modified so that some evaporation occurs in the exterior (sheath flow) of the microfluidic flow 115. The environmental control can include temperature and humidity control of the gaseous environment, and the introduction of a gaseous flow 175 (such as a warm air flow) at or along the microfluidic flow 115 to further promote evaporation. The illuminator 120, detector 130, and sorting device 140 may be arranged for sealed engagement with the sheath 170 to improve the controllability of the environment within the sheath.

[0329] In one example, the microfluidic flow may flow within a sheath, conduit, or channel, the cross-section of which can be circular, square, rectangular, triangular, elliptical, or another desired shape. The sheath, conduit, or channel may be formed of any one or more of a polymer, glass, ceramic, or other solid substrate, or may be a preformed component, such as a PTFE tube or a glass capillary. The sheath, conduit, or channel may have a depth between about 10 μm and about 2500 μm, may have a width between about 50 μm and about 2000 μm, and may have a length between about 10 μm and about 200 mm. For example, the depth of the sheath, conduit, or channel may be between about 20 μm and about 10 mm, between about 30 μm and about 5000 μm, between about 40 μm and about 1000 μm, between about 50 μm and about 500 μm, between about 60 μm and about 100 μm, and between about 70 μm and about 90 μm. For example, the width of the sheath, conduit, or channel may be between about 50 μm and about 2000 μm, between about 60 μm and about 1500 μm, between about 70 μm and about 1000 μm, between about 80 μm and about 500 μm, and between about 90 μm and about 100 μm. For example, the length of the sheath, conduit, or channel may be between about 10 μm and about 10 mm, between about 100 μm and about 5000 μm, between about 1000 μm and about 2500 μm, and between about 1500 μm and about 2000 μm.

[0330] The vaporization device 150 can be arranged upstream or downstream of the sorting device 140 to direct energy into the microfluidic stream 115, the energy being sufficient to evaporate a portion of the liquid in the stream. The sorting device can additionally or alternatively include a vaporization device. Figure 15 An example of a vaporization device 1550 is shown that emits directed energy to vaporize a portion of the stream. The vaporization device includes a laser having sufficient power to vaporize a portion of the stream. Figure 15 A portion 1553 of the outer layer 1552 of the stream 1515 that is being evaporated is shown, while the inner particle or "sample" stream 1551 is substantially unaffected. The laser can be timed to evaporate the stream only when there are no cells or cells are not desired in the vicinity, to avoid additional energy affecting the cell health, motility, or viability of the desired cells in the sample or particle stream 1551. This can be important in cases where radiant energy has been applied to the cells to cause a change in direction of the cells into different flow paths.

[0331] In another example, the break distance of the microfluidic stream can be controlled such that droplets are formed after the sorting device 140, and only the droplets containing the unwanted cells are subject to the vaporization energy of the vaporization device 150. In this example, the vaporization device receives a signal from the detector 130, and the analysis unit directs the vaporization device to implement timed laser pulses to coincide with the droplets or regions of the microfluidic stream to be vaporized.

[0332] In the case of vaporization or evaporation of a portion of the liquid in the stream, a suitable extractor can be provided in the vaporization region to remove the vapor from the gaseous environment. The extractor can be a negative pressure air extraction fan or a solid-phase moisture-absorbing material that absorbs moisture without causing air movement that could affect collection or sorting.

[0333] The above examples of vaporization and evaporation have the effect of increasing the concentration of the desired cells in the culture medium collected in the collection container 160, and this provides many advantages. As described above, this can be achieved by reducing the proportion of the sheath flow 1552 compared to the particle stream 1551, promoting evaporation along the stream 1515, applying vaporization energy 1550, or a combination of these.

[0334] Concentrated bovine X cells are typically provided in standardized units called "straws", each straw containing a standard number of X cells. Thus, increasing the concentration of X cells in the output fluid while maintaining a constant flow rate enables the production of more standardized straws per hour, for example. When the cell concentration in the output fluid is below the desired level, a re-concentration processing step may be required to re-concentrate the cells. By reducing the concentration as described herein, this can reduce the need for such downstream processing to re-concentrate. One such processing step is centrifugation, which can affect cell health and motility. Processing steps after collection are also time-consuming, which exposes sensitive cells to adverse temperatures for longer periods. Centrifugation and other concentration processes can cause torsional or other mechanical forces that may damage the cells. It has also been noted an "accompanying" effect, where sperm cells in close proximity to other sperm cells have higher survival rates, motility, and fertility. Thus, some examples can provide a solution to the re-concentration problem affecting sperm by increasing the cell concentration in the output fluid and thus avoiding or ameliorating the need for re-concentration.

[0335] In some examples, at least one irradiator (e.g., IR or UV irradiator 120) can be oriented to deliver substantially perpendicular radiation to each cell passing through interrogation region 125. At least one and preferably two or more detectors 130 can also be oriented to absorb emissions from the particles. In one example, two detectors are arranged perpendicular to each other to capture responsive fluorescence emitted from different directions of the irradiated cells. The unique architecture of examples using fluid flow in a gaseous environment can enable the detectors to be positioned perpendicular to each other, while planar chip designs only allow detection and interrogation from a single axis (i.e., at the shortest distance through the planar chip). The detectors can be photomultiplier tubes at any suitable angle to each other and to the direction of flow 115. For example, 90 degrees to each other and to the direction of the flow. The detectors can be photomultiplier tubes or other detectors arranged to collect responsive fluorescence pulses from the stained particles. In one example, the detectors include avalanche photodiodes. In one example, the detectors are driven by a voltage driver that drives the detectors. For example, a control voltage can be applied to the detectors, and an additional tuning voltage can be applied to the detectors to tune the gain of the analog output. An amplifier can optionally be connected to the detector output to amplify and optionally normalize or rectify the output signal. This amplifier is particularly important when emissions from the cells are detected, due to the very fast flow rates and low emission signals that can be detected from the stained and fluorescent cells. The output signal is transmitted to analysis unit 135 as described above.

[0336] In an alternative example using an IR laser without cell staining, a laser such as a quantum cascade laser (QCL) provides a high spectral power density at specific wavelengths in the mid-IR and THz ranges corresponding to molecular bond vibrations. In this case, the detector is tuned to detect light transmitted or scattered at different angles.

[0337] An additional optical illumination method applicable to the previous configurations disclosed herein adds polarization as a sensing modality to IR-based interrogation of particles in a stream. If the molecules probed by mid-IR vibrational spectroscopy are arranged in a particular manner within the particle being measured (e.g., DNA in a helical configuration), the absorption measured at the absorption band of the molecule will depend on the polarization of the mid-IR light. Thus, in one example, the illuminator generates light polarized in left and right circular polarizations and measures the difference. The observed difference, the so-called vibrational circular dichroism (VCD), can provide a particularly sensitive measurement of chiral or helical molecules, and / or provide information about the folding or configuration of specific particles / molecules within the analyzed particle / cell / droplet. If the particles can be successfully distinguished by the detector and the analysis device, the sorting device can sort the particles based on these molecular properties. In the case of using two detectors, this results in two pulsed measurement signals or channels, which are proportional to the intensity or power of the received signal or fluorescence, and correspond to a specific angle between the detector directions.

[0338] In one example, the interrogation beam and the sorting beam propagate in the same direction through a single or common optical objective such as an objective lens or a convex mirror to interact with the particles and the microfluidic stream. The common optical objective is the final optical focusing component closest to the object, which in this case is the particle in the microfluidic stream. The advantages of this feature of the present invention include:

[0339] · Reducing the optical components (e.g., objective lens) around the hole, interrogation, and sorting regions. This can enable better observation of the stream for alignment and observation purposes, and can also facilitate positioning the interrogation beam close to the hole to reduce the excitation distance;

[0340] · Enhancing the ease of aligning the beams since multiple lasers will typically be focused on a single focal point;

[0341] · Minimizing optical aberrations and enhancing the focusing ability when focusing through multiple phases (e.g., through air, liquid, and / or solid window materials).

[0342]

[0343] Shared optical components reduce the complexity of the device and also simplify the setup, where the interrogation beam and the sorting beam must be directed to focus on the desired part of the microfluidic flow. For example, the interrogation beam is focused at the desired excitation distance, and the sorting beam is then focused at the inter-beam distance. This advantage is enhanced at the small distances involved in the operation of the exemplary device. When the beams propagate towards a common optical objective, the beam positioning can be further simplified by angling the beams relative to each other. The directions in which the two beams intersect the objective determine the positions at which their foci are formed in the microfluidic flow. As described in more detail below, the inter-beam distance can then be easily configured by adjusting the angle between the beams. The excitation distance can be configured by adjusting the angle at which the interrogation beam intersects the common objective. Alternatively or additionally, the interrogation beam can intersect the common objective at 90 degrees, such that the positioning of the objective sets the excitation distance. Then, the inter-beam distance can be adjusted by adjusting the angle between the co-propagating interrogation beam and sorting beam before they intersect the common objective.

[0344] The inter-beam distance can be adjusted by modifying the angle of the beam propagating from the final objective lens. Methods for enabling co-propagation of the interrogation beam and the sorting beam through the same objective include using a dichroic mirror that allows a specific wavelength to pass through but reflects other wavelengths. Alternatively, if the sorting beam and the interrogation beam have similar or the same wavelength, the beams can be polarized, and a polarizing mirror can be used to reflect one polarity of the beam while allowing the other polarity of the beam to pass through.

[0345] Figures 22a to 22c Examples of the co-propagating beam arrangement of the present invention are illustrated. In these examples, the interrogation beam 2205 (solid outline) propagates through an optical objective 2210 that focuses (2215) the interrogation beam towards a microfluidic flow 2220 emitted from an orifice 2225 of a flow control device 2230. The co-propagating sorting beam (short dashed line) 2235 co-propagates through the same (common optical) objective and is focused (2215) onto the microfluidic flow 2220 at a downstream location. The excitation distance ED can be observed as the distance between the lower surface of the flow control device and the interaction point of the interrogation beam and the flow. The inter-beam distance IBD can be observed as the distance between the interaction point of the interrogation beam and the flow and the interaction point of the sorting beam and the flow. An optional third illumination beam (long dashed line) 2240 is illustrated, which is widely focused to illuminate the interrogation / sorting region. The illumination beam provides light to enable imaging of the interrogation / sorting region.

[0346] Figure 22aAn example of a sorting operation is illustrated, where radiation pressure causes a change in the direction of a flow or selected particles within a flow in a gaseous fluid environment. Cells are collected in the first or second container 2222. Particles are selected based on characteristics determined after interrogation by a probing beam. Although the particles are shown in a gaseous fluid environment, the sorting operation can also be performed in a liquid fluid environment where the flow is surrounded by a solid microfluidic chip.

[0347] Figure 22b An example of an apparatus is illustrated where a focused beam 2215 passes through a window 2235 and the microfluidic flow is enclosed within a solid microfluidic chip. The semi-focused beam can be adjusted to account for the refractive properties of the window 2235. In examples where a window is used, the sorting process using this configuration may require a further step of adjusting the focus of at least one of the probing beam and the sorting beam to account for any refraction or aberration caused by the window 2235. An illumination beam 2240 also propagates through the window to illuminate the interrogation region and the sorting region. When passing the probing beam through the window, an optical correction component may be required to reduce distortion.

[0348] For example, the objective lens includes a correction ring to prevent distortion. Additionally, a numerical aperture less than 0.4 can help manage the optical aberrations caused by using a window. In some examples, the window is optically flat to avoid beam distortion. Additionally, the window is required to be made of a material resistant to the beams and any damage they may cause.

[0349] Figure 22c An example of the present invention is illustrated where the microfluidic flow propagates into a gaseous fluid environment. In this example, the probing beam and the sorting beam are focused to interact with the flow while the illumination beam 2240 illuminates the region.

[0350] As described above, one aspect of the present invention is to achieve an excitation distance (ED) within a specified range (e.g., the range described above), and an optimal inter-beam distance (IBD) within a specified range (e.g., the range described above). The importance of these two distances can be observed in Figures 22a to 22c the illustrated configurations. The ED is preferably greater than the minimum ED to ensure that the beams do not pinch the edges of the flow focusing device. Similarly, maintaining a relatively low IBD enables the particles in the flow to remain confined and directed. These properties decrease with the distance from the flow focusing device. Maintaining them increases the orientation efficiency and the enrichment of the preferred particles selected after interrogation. The IBD is preferably adjustable to enable effective discrimination of particles based on, for example, emission intensity, flow rate, and fluid viscosity.

[0351] One option for optimizing the IBD is to position the interrogation beam substantially perpendicular to the sorting beam while still pointing along the z-axis of the flow. For example, one of the interrogation beam and the sorting beam will be aligned with the x-axis, while the other will be aligned with the y-axis. In this example, the optical components do not interfere with each other and the beams can be easily aligned independently.

[0352] Alternatively, a co-propagating configuration can be used, as described above. The co-propagating configuration reduces the number of optical components required, but requires a more complex alignment process. Figure 23 An example of a co-propagating beam configuration is shown, in which the interrogation beam 2305 is reflected by a first beam splitter 2310 and defines a first optical path 2315. The optical path optionally passes through a lens 2320 to shape the beam. The interrogation beam is then focused by an objective lens 2210 to interact with the flow 2220. Alternative examples may include using a window as shown Figure 22b in the figure.

[0353] A second sorting beam 2325 propagates towards a second beam splitter 2330 and is reflected to define a second optical path that propagates through the first beam splitter 2310 towards the microfluidic flow 2220. Again, optional lenses can be employed to shape the second beam. It can be observed that the positioning of the second beam at 2325 can be adjusted to achieve the desired IBD. Alternatively, the IBD can be obtained by first aligning the beams focused at the same location in the flow in a concentric manner. Then, at least one of the beams is angled to produce non-parallel beams that result in a distance between the beams. Thus, the sorting beam and the interrogation beam can be angled with respect to each other.

[0354] Optionally, a third illumination beam 2340 can be reflected from a third beam splitter 2345 and propagate through the second beam splitter 2330 and the first beam splitter 2310, and optionally through the lens 2320 to the objective lens 2210, and then illuminate the interrogation and sorting regions, as described above. Alternatively, the third illumination beam 2340 can simply propagate through the second and first beam splitters without the third beam splitter. It can be observed that this unique configuration of beams, beam splitters, and lenses works in concert with the flow focusing device to achieve stable, fast, and accurate cell interrogation, sorting, and optional illumination.

[0355] The beam splitters presented herein include coatings that reflect light at some wavelengths while being substantially transparent to other wavelengths. The beam splitters can be dichroic beam splitters such as dichroic mirrors or harmonic beam splitters. The beam splitters can be short-pass or long-pass beam splitters. Accordingly, lasers with interrogation lasers and sorting lasers of higher wavelengths can be positioned to transmit as well as reflect in the configurations described herein. In some examples, the beam splitter is a polarizing mirror. In one example, the interrogation beam includes an infrared (IR) or mid-IR laser, more preferably a mid-IR quantum cascade laser (QCL). In this example, the beam splitter includes a beam splitter that reflects IR light, such as Thorlabs DMSP805. In another example, the interrogation beam includes a UV laser in the ultraviolet wavelength range. In one example, the UV laser includes a wavelength of about 355 nm. In this case, the first beam splitter reflects UV light, such as 355 nm or in the UV range, such as Thorlabs DMLP550, DMLP567, DMLP605, DMLP638, DMLP650, DMSP550, DMSP567, DMSP605, DMSP638, DMSP650, HBSY12, HBSY22, HBSY11, HBSY21, FELH0550, FELH0600, FESH0550, FESH0600.

[0356] In one example, the wavelengths of the interrogation beam and the sorting beam are different. In another example, the wavelengths of the interrogation laser and the sorting laser overlap. For example, a 355 nm interrogation laser and a 355 nm sorting laser can be used, where each beam is polarized in a different plane from the other beam. In this example, the beam splitter includes a polarizing mirror, such as Thorlabs PBS12-355-HP, PBS25-355-HP. In one example, the beam splitter reflects UV light and transmits green light. For example, the beam splitter can reflect at about 355 nm and transmit at about 532 nm. In another example, the beam splitter reflects green light and transmits UV light.

[0357] In one example, the sorting beam includes about 355 nm, 405 nm, 515 nm, 532 nm, 800 nm, 1030 nm, or 1064 nm. The second beam splitter 2330 can include reflection at about 532 nm and transmission at about 1064 nm and / or 660 nm. This enables the illumination beam to pass through for imaging purposes.

[0358] In one example, the system includes an illumination beam at a different wavelength from the sorting beam and the interrogation beam to ensure that it does not interfere with the detector or sorting. For example, the illumination can be an LED emitting at approximately 565 nm, 590 nm, 595 nm, 617 nm, 625 nm, 660 nm, 680 nm, 700 nm, 730 nm, 780 nm, 810 nm. The system can include one or more imaging cameras adjacent to the detector.

[0359] In one example, the illumination beam is directed along an axis that is substantially aligned with the camera optical path entrained over the interrogation region and the sorting region. This enables clear and high-quality imaging of the cells / particles while they are being interrogated and optionally sorted. Preferably, the camera is aligned to provide guidance for system alignment and can provide feedback information regarding the beam size, shape, power, and intensity distribution. In one example, the illumination beam includes a wavelength (color) that does not overlap with the interrogation beam and the sorting beam so that filtering can be performed during or prior to signal processing.

[0360] In one example, separation of the interrogation beam and the sorting beam to achieve IBD is accomplished by at least one of the following:

[0361] a. The first beam splitter and / or the second beam splitter are angled relative to each other. This results in the angles of the beams not being parallel after the beam splitter that effects the beam separation.

[0362] b. The first beam splitter and / or the second beam splitter are parallel and at least one of the beams is angled relative to the other beam, i.e., they are not parallel.

[0363] c. The objective lens is movable and the first beam splitter is tiltable.

[0364] d. The beams are positioned so that they are offset onto the beam splitter. In this case, the first beam splitter and the second beam splitter can be substantially parallel.

[0365] In another example, at least one of the interrogation beam and the sorting beam passes through beam expansion optics before reaching the beam splitter. Beam expansion (or control of the beam diameter) can be important for optimal compatibility with the optical elements used (e.g., spreading the power spatially to mitigate thermal damage). The beam expansion optics can include a pinhole. The pinhole can be placed at the internal focus and used as a spatial filter to enhance the resolution of the beam profile, for example using a Keplerian beam expander. The inventors have found that a higher-resolution beam profile results in a more accurate beam profile at the microfluidic flow and thus more accurate / efficient / precise sorting.

[0366] In some examples, it is preferred to include a lens 2320 to shape the beam into an elongated shape for enhanced interrogation and / or sorting. In one example, the lens includes a cylindrical lens to flatten and elongate the interrogation beam such that the interrogation beam provides a wider focus. This modification of the beam provides a wider (and thinner in the z-axis) focus such that the energy of the laser is more uniform across the width of the flow and each cell experiences less variation in light intensity due to its positioning variations. Another key advantage of the thinner focus is that cells can be better resolved spatially and temporally along the z-axis during interrogation and detection. The cylindrical lens stretches the beam in the x-axis to ensure a uniform distribution of the laser energy (flux) across a portion of the microfluidic flow. In one example, the laser exhibits a Gaussian power distribution along its length. In some examples, at least one of the interrogation beam and the sorting beam is focused into a line. This can be an elliptical Gaussian at the focal plane. The inventors have found that a height (z-axis) 1 / e 2 between about 1 μm and 10 μm provides an effective focus for interrogation or sorting. In some examples, the beam width 1 / e 2 (perpendicular to the x-axis of the flow and beam propagation) can be between about 1 to 5 times the width of the microfluidic flow. Thus, the beam width can be 150 μm to 750 μm. In one example, the beam width and height of the interrogation beam and the sorting beam differ by less than 20%.

[0367] In some examples, both the interrogation beam and the sorting beam propagate through the same cylindrical lens. The advantage of this configuration is that it allows the optical components to be positioned closely together and the beam profiles to be concentric or overlapping. Additionally, the lens causes both the interrogation beam and the sorting beam to be shaped.

[0368] In one example, the cross-section of the microfluidic channel through which the fluid flows after being emitted from the orifice can be circular, square, rectangular, triangular, elliptical, or another desired shape. The flow control device includes a delivery microchannel and an aggregation chamber and / or a confinement chamber. At least one of the flow control device and the microfluidic channel can be formed of any one or more of a polymer, glass, ceramic, or other solid substrate, or can be a preformed component, such as a PTFE tube or a glass capillary. At least one of the flow control device and the microfluidics can have an internal channel depth between about 10 μm and about 2500 μm, can have a width between about 50 μm and about 2000 μm, and can have a length between about 10 μm and about 20 mm. For example, the depth of the microfluidic channel can be between about 20 μm and about 10 mm, between about 30 μm and about 5000 μm, between about 40 μm and about 1000 μm, between about 50 μm and about 500 μm, between about 60 μm and about 100 μm, and between about 70 μm and about 90 μm. For example, the width of the microfluidic channel can be between about 50 μm and about 2000 μm, between about 60 μm and about 1500 μm, between about 70 μm and about 1000 μm, between about 80 μm and about 500 μm, and between about 90 μm and about 100 μm. For example, the length of the microfluidic channel 402 can be between about 10 μm and about 10 mm, between about 100 μm and about 5000 μm, between about 1000 μm and about 2500 μm, and between about 1500 μm and about 2000 μm.

[0369] In some examples, a variety of materials and methods can be used to form any of the above components of the present disclosure. In some cases, the various materials selected are suitable for the various methods. For example, the microfluidic aggregation device or channel, sheath, window, objective lens, support bracket, or various components of the present disclosure can be formed of a solid material, where the channel can be formed via micromachining, film deposition processes (such as spin coating and chemical vapor deposition), laser fabrication, lithography techniques, etching methods including wet chemical or plasma processes, etc. In one example, at least a portion of the microfluidic channel or the flow control device is formed of silicon by etching features in a silicon chip. Techniques for precisely and efficiently fabricating the various fluid systems and devices of the present disclosure from silicon are known. In another example, the various components of the systems and devices of the present disclosure can be formed of a polymer, for example, an elastomeric polymer, such as polydimethylsiloxane (PDMS), polytetrafluoroethylene (PTFE), etc. In another example, the channels of the present disclosure can be formed of a polymer, glass, ceramic, or other solid substrate, or can be a preformed component, such as a PTFE tube or a glass capillary.

[0370] The different components can be made of different materials. For example, at least one of the flow control device and the microfluidic channel can be made of an opaque material (such as silicon), and the window 2235 can be made of a transparent or at least partially transparent material (such as glass or a transparent polymer) for observing and / or controlling the interrogation and sorting processes.

[0371] Optionally, the bottom wall, top wall, or side wall can be formed of an optically transparent material to enable efficient transmission of electromagnetic radiation. The components can be coated so as to expose the desired chemical functionality to the fluid contacting the inner channel walls. For example, the components can be manufactured to have inner channel walls coated with another material. The materials for the various components used to manufacture the systems and devices of the present disclosure (e.g., the materials for coating the inner walls of the fluid channels) can desirably be selected from those materials that do not adversely affect the fluid flowing through the fluid system or are not affected by the fluid flowing through the fluid system (e.g., materials that are chemically inert in the presence of the fluids used within the device). In one embodiment, the various components of the present disclosure are made of polymers and / or flexible and / or elastomeric materials and can conveniently be formed from a curable fluid, thereby facilitating manufacture via molding (e.g., replica molding, injection molding, cast molding, etc.). The curable fluid can generally be any fluid that can be induced to cure or spontaneously cure into a solid capable of containing and / or transporting the fluids intended for use in or with the described microfluidic systems. In one embodiment, the curable fluid includes a polymer liquid or a liquid polymer precursor (i.e., a “prepolymer”). Suitable polymer liquids can include, for example, thermoplastic polymers, thermosetting polymers, or mixtures of such polymers heated above their melting points. As another example, suitable polymer liquids can include a solution of one or more polymers in a suitable solvent, which forms a solid polymer material when the solvent is removed, for example, by evaporation. Such polymer materials that can be cured from a molten state or by solvent evaporation are well known to those of ordinary skill in the art. A non-limiting list of examples of such polymers includes polymers of the general classes of silicone polymers, epoxy polymers, and acrylate polymers. Silicone polymers, including PDMS, have several beneficial properties that simplify the manufacture of the microfluidic structures of the present disclosure. For example, such materials are inexpensive, readily available, and can be cured from a prepolymerized liquid via heat curing. For example, PDMS can generally be cured by exposing the prepolymerized liquid to a temperature of about, for example, about 65° C. to about 75° C. for an exposure time of, for example, about one hour. Additionally, silicone polymers (such as PDMS) can be elastomeric and can thus be used to form very small features with relatively high aspect ratios, which is necessary in certain embodiments of the present disclosure. In this regard, flexible (e.g., elastomeric) molds or masters may be advantageous. In additional examples, the components of the present invention can be formed from recycled polymers or biodegradable polymers such as polylactic acid (PLA) and polyhydroxyalkanoates (PHA).

[0372] The measurement of fluorescence from cells can be affected by many factors, including the orientation and confinement of cells within the interrogation region 125, the level of retained staining of cells at the interrogation region 125, and biological factors such as whether the cells are dead or abnormal. For sperm cells, the measurement of fluorescence also depends on the identity of the sperm sex chromosome (X or Y). Due to the presence of the X or Y chromosome, the difference in the measured signal is only about 3%. This combination of factors makes accurate and efficient sorting of sperm cells challenging. Signals from the detector can each correspond to a rapid "trigger" applied to each cell to cause a detected signal. By integrating a single responsive fluorescence emission pulse associated with a single cell over a predetermined period, a pulse integral signal can be derived for each cell. These pulse integral signals for each channel can be generated at the detector device 130 or at the analysis unit 135.

[0373] In one example, a measurement from a channel corresponding to the fluorescence intensity of a cell represents a measurement data point. In some examples, two or more measurements from two or more channels are combined to represent a measurement data point. This can include, for example, the pulse integration of measurements taken from substantially perpendicular directions. Figure 2 A plot of measurement data points for channel 1 (e.g., 0 degrees) and channel 2 (e.g., 90 degrees) is shown. In additional examples, the detector can be positioned to detect emissions at other angles with respect to each other while being substantially perpendicular to the axis of the flow. Each data point represents the fluorescence pulse integration level measured in at least one perpendicular direction and, in some examples, represents the fluorescence pulse integration level measured in two or more perpendicular directions.

[0374] Figure 1 The analysis unit 135 analyzes these signals or measurement data points to determine whether a cell should be classified as having a first characteristic A (e.g., part of an X or Y population) (e.g., P1 or P2), and if so, controls the sorting device 140 to sort the cell. In some examples, sorting can be achieved by changing the orientation of the cell by applying radiation pressure to collect the cell or direct it to waste. This can be accomplished by using a nudge laser to apply radiation pressure to nudge Y cells into different parts of the flow and then separating the two flow paths, for example, using a microfluidic channel. Increasing the proportion of cells correctly classified as having a preferred characteristic (e.g., sperm cells carrying X) increases the efficiency of the sorting device. Alternatively, other sorting methods can be used, for example, an ablation laser can apply a directed energy pulse to the cell. This allows all other cells having a desired characteristic B (e.g., X chromosome) or that are not classifiable to remain undisturbed by the sorting device 140. In an alternative example, Y-type sperm cells can be sorted by moving the cells to different flow paths.

[0375] In some examples, the sorting device 140 includes an ablation laser that applies laser energy pulses ("sorting beam") to the stream 115 as unwanted cells pass by. This sorting method overcomes or at least ameliorates some of the drawbacks of more conventional sorting techniques such as charged droplets and charged deflection plates. These conventional methods require a stream charging wire to intersect the microfluidic stream, typically upstream of the orifice and interrogation beam. The intersection of the wire with the stream can cause disruptions and turbulence in the stream, which can reduce confinement and orientation, resulting in impaired sorting efficiency. In one example, the present invention does not include a stream charging wire and does not include a droplet forming device. Various properties of the ablation laser can be optimized, including: pulse rate; wavelength; power; beam shaping and / or patterning. The energy applied to the cell to render the cell immotile is described as the ablation threshold and is the minimum amount of energy per unit area required to cause permanent material modification, damage, or removal. For example, the ablation threshold can correspond to ablating the cell, resulting in rupture of the cell surface membrane or otherwise damaging the cell sufficiently to rapidly induce permanent immotility.

[0376] Alternatively, a sorting beam of lower energy can be used to ablate the cells, which does not rapidly render the cells permanently immotile but is comparable to the cells becoming immotile after freezing and thawing. This ablation method is referred to as "pretreating" the unwanted cells. It requires the cells to be subjected to electromagnetic radiation that is sufficient to transfer energy to the selected cells, which is below a predetermined ablation threshold corresponding to rupturing the cell membrane and above a predetermined pretreatment threshold corresponding to the cells not surviving after freezing and thawing. An advantage of this method is that debris from the unwanted cells is not released into the stream, which can have a negative impact on the remaining desired cells. Another advantage of a method that does not rupture the cell membrane is that any downstream genetic analysis of the cells can be more effectively achieved because there is less free DNA from ruptured cells in the culture medium.

[0377] Figure 2 Illustrated is a portion of another system 200 for classifying and sorting cells, which includes a flow control device 210 that flows a microfluidic stream 215 from an orifice 213 to a collector 260. System 200 also includes an interrogation beam generator 220 (such as an IR or UV illuminator), one or more detectors 230, and a sorting laser 240.

[0378] System 200 also optionally includes a droplet detector 265 that detects whether the microfluidic stream 215 breaks up into droplets 217 above a threshold height (breakoff distance). This can be used as a control input for operating system 200, for example, increasing the flow rate of the particle stream and / or sheath stream if droplets are detected above the breakoff distance.

[0379] System 200 also optionally includes a sheath 270 that extends at least partially between the flow control device 210 and the collector 260 and, in this example, extends to the sorting laser 240. In this regard, the sheath may completely surround the stream 215, or there may be one or more baffles with an air gap therebetween.

[0380] The flow control device 210 includes an aggregation chamber 211 where the particles and the sheath flow converge such that the sheath flow moves coaxially around the internal particle stream. This aggregation has the effect of orienting the asymmetric cells in the particle stream and confining the cells within a narrow range of lateral dimensions. The aggregation chamber is fluidly coupled to and tapers towards a microfluidic channel 212 that terminates in a pore 213 in the flow control device 210. The dimensions of the channel 212 can be determined experimentally to establish a stable laminar flow at the pore 213. In a particular example, the channel 212 includes a length of at least 10 microns to 10 mm from the outlet of the flow aggregation chamber to the pore outlet, where the pore outlet is defined as a plane perpendicular to the longitudinal axis or z-axis of the flow aligned with the terminus of the flow aggregation device. In one example, the channel includes a length of 50 microns to 1 mm, where this length allows for the re-establishment of laminar flow after the orientation and confinement of the particles in the hydrodynamic orientation and confinement aggregation chamber. The channel enables the microfluidic flow to flow stably when ejected into a gaseous environment.

[0381] The pore 213 may have a cross-section that extends more in one axis or dimension (major axis) than in a perpendicular axis or dimension (minor axis). In a rectangular-shaped pore, this results in a non-uniform aspect ratio, i.e., the ratio of the length in one direction to the length in another direction is greater than or less than 1, which may be referred to as an unequal aspect ratio. In a particular example, the aspect ratio lengths include at least 10 microns to 1 mm, such as an aspect ratio of: 1:100 to 2:3; or 1:50 to 1:10.

[0382] The microfluidic stream 215 is ejected from the pore with a cross-sectional shape corresponding to the cross-sectional shape of the pore. When the stream is ejected into free space rather than into a solid conduit (such as a microfluidic channel), surface tension acts on the liquid to make the cross-sectional shape symmetric and tend towards circular. This effect can occur at a distance 216 after which the stream assumes a stable circular cross-sectional shape. This varying cross-sectional shape causes internal hydrodynamic forces within the stream that contract along the longer axis, thereby resulting in further orientation of the cells that are not yet aligned along the shorter axis. This is visualized in Figure 6 which is described in more detail below. Thus, the asymmetric dimensions of the pore 213 along the two cross-sectional axes further improve the orientation of the asymmetric cells. A variety of shapes can be used, such as a rectangular shape or an oval shape.

[0383] The aperture 213 can alternatively or additionally be adjustable such that the shape, size, orientation, or dimensions of the aperture cross-section can be controlled. In one example, the present invention provides a system including an adjustable aperture, where at least one of size, shape, or aspect ratio is adapted to be adjusted. This can be achieved, for example, by stretching a flexible material having a slit forming the aperture. The aperture can be adjusted to control various properties of the microfluidic flow 215, such as the flow rate of one or both of the particle flow and the sheath flow, the relative proportion of the particle flow and the sheath flow, the height of droplet formation, the orientation of the particles, or the confinement of the particles.

[0384] The aperture 213 can be adjusted to a cleaning mode, in which the aperture presents a maximum size, and during this cleaning mode, a high flow rate of sheath flow (with or without particle flow) is applied so that any particles or other debris stuck around the aperture or other parts of the flow control device 210 are removed and washed away.

[0385] The flow control device 210 further includes a pressure sensor 267 associated with the collection chamber, the sheath flow, or the particle flow. Pressure measurements can be used to control various properties of the flow, such as the flow rate, the concentration of desired cells, the height of droplet formation.

[0386] Figure 3 and Figure 4 respectively illustrate a longitudinal cross-sectional view and a transverse cross-sectional view of a flow control device 300 according to an example. Figure 4 The transverse cross-section of Figure 3 The longitudinal cross-section of passes through the section line AA. The flow control device 300 includes a delivery tube 330 assembled within a housing 305. Specifically, the delivery tube 330 is firmly and accurately received within a cavity 310 of the housing 305, which is defined by the inner surface of the housing. The delivery tube 330 includes a lumen 340 for transporting a particle flow 345, which is a moving liquid, such as an aqueous solution containing particles such as sperm cells. The lumen 340 leads to a delivery tube inlet 332 at the input end of the delivery tube 330 and to a delivery tube outlet 333 at the distal end of the delivery tube.

[0387] The delivery tube 330 also includes a ridge, fin, or protrusion 335 that extends longitudinally along the delivery tube. The ridge 335 engages the inner surface of the cavity 310 of the housing 305 to secure the delivery tube within the housing. In an alternative example, the housing 305 includes a ridge, fin, or protrusion that extends longitudinally along the housing and also engages the outer surface of the delivery tube. The size of the ridge 335 can be designed to ensure a friction fit with the featureless wall of the cavity 310, or the walls of the cavity can include corresponding grooves in which these ridges are located. A variety of other mechanical fastening mechanisms can alternatively be used. By extending longitudinally, the ridge 335 improves the lateral positioning of the distal end of the delivery tube 330 such that the delivery tube outlet 333 is firmly and accurately positioned within the housing 305.

[0388] A variety of alternative engagement structures are possible. Although the ridges 335 extend longitudinally, they can also be angled relative to the longitudinal axis to form a helical shape along the exterior of the delivery tube. Additionally, although the ridges have been shown as continuous, they can be discontinuous, having portions that engage the cavity wall at different longitudinal positions. In another alternative arrangement, longitudinally extending ridges can extend from the cavity to engage the delivery tube. In this alternative, the delivery tube 330 may or may not have ridges 335 that extend to the cavity wall. In yet another alternative, the outer circumference of the delivery tube 330 can be sized to directly mate with the inner wall of the cavity to ensure a friction fit. The outer surface of the delivery tube and / or the inner surface of the cavity 310 can include depressions to form channels between the housing 305 and the delivery tube 330.

[0389] In Figure 3 and Figure 4 examples, one or more sheath flow channels 360 are formed between the delivery tube 330 and the housing 305 to convey a sheath flow 365, such as an aqueous solution. The sheath flow channels 360 can extend from a sheath flow inlet 362 at the input end of the delivery tube and include channels formed between the ridges 335. The sheath flow channels extend along the exterior of the delivery tube 330 to an aggregation chamber 370 that is defined by a volume formed within the housing 305 at the end of the delivery tube 330, and the particle stream 345 is discharged from the delivery tube outlet 333 into this aggregation chamber. The aggregation chamber 370 is also fluidly coupled to a delivery microchannel 375 having a pore 313, and the combined particle and sheath flow exits through this pore for downstream processing and is also referred to herein as the microfluidic flow 115.

[0390] A central particle stream 345 is surrounded by one or more coaxial flowing sheath streams 365. The shape and size of the aggregation chamber 370, the geometries and dimensions of the sheath flow channel 360 and the lumen 340, and the flow rates of the particle stream 345 and the sheath stream 365 all contribute to controlling the combined fluid flow from the particle delivery outlet 375. Example use cases include controlling the orientation and confinement of particles within the combined particle and fluid stream 360. In some examples, the particle stream and the sheath stream may be concentrically arranged. In other examples, the central axis of the particle stream may be offset relative to the central axis of the sheath stream. This may be useful in examples where the downstream interrogation and / or sorting beam has a focal offset from the microfluidic stream 115 described previously; as described with respect to Figure 1 、 Figure 12 a、 Figure 12 b、 Figure 13 a and Figure 13 b.

[0391] As described above, longitudinally extending ridges or other engagement structures ensure accurate and stable lateral positioning of the delivery tube outlet 333 within the aggregation chamber 370. In more conventional arrangements, a delivery needle is introduced into a sheath fluid with a tapered volume, however the distal end of the needle is subject to fluid flow impact and lateral movement, resulting in the resulting particle stream moving within the surrounding sheath fluid stream or even partially mixing with the sheath fluid stream, leading to poor orientation and confinement of the particle fluid stream. This can make downstream processing difficult, inaccurate, and inefficient.

[0392] In some examples, accurate longitudinal positioning of the delivery tube outlet 333 within the aggregation chamber 370 can also contribute to optimizing the control and stabilization of the orientation and / or confinement of particles or other flow properties of the microfluidic stream 360 delivered from the delivery microchannel or orifice 375. In the examples of Figure 3 and Figure 4 , this is achieved by sizing the ridges 335 of the delivery tube 330 to be complementary to the dimensions of the cavity 310 of the housing 305 to prevent the delivery tube 330 from being inserted into the cavity beyond a predetermined longitudinal position.

[0393] The cavity 310 can be divided into several parts, including a first part 310-S1 having a longitudinal cross-sectional shape (such as a rectangle), which is arranged to engage with corresponding first parts of the delivery tube 330-S1 at multiple longitudinal positions. The first part of the cavity can have a substantially uniform transverse cross-section along the longitudinal direction, for example, the transverse cross-section is a circular shape with a fixed diameter. In other examples, the cross-sectional shape can be asymmetric to promote some asymmetry in the particle flow within the sheath flow. For example, the cross-sectional shape can be elliptical, hemispherical, triangular, or can be a combination of a smaller rectangle and a larger rectangle. The first part S1 of the cavity 310 is for receiving the ridge 335 of the delivery tube 330. The second part 310-S2 of the cavity tapers such that it has a reduced size when extending towards the distal end of the delivery tube. The end of the ridge 335 having a larger size prevents the delivery tube from extending beyond this point, thereby ensuring an accurate and stable longitudinal positioning of the delivery tube outlet 333 within the collection chamber 370. The end of the ridge can be shaped as complementary to the internal shape of the cavity 310 as shown, thereby further improving this positioning. In other arrangements, a groove in the wall of the cavity 310 can be used to receive the ridge 335, and the length of the groove is controlled to control the longitudinal positioning of the delivery tube outlet 333 within the collection chamber 370. In contrast, in a more conventional arrangement, a delivery needle can be placed into a sheath fluid with a tapered volume, however, if the distal end of the needle is not correctly positioned, the sheath flow may interact turbulently with the particle flow, resulting in unwanted mixing, chaotic misalignment of the particle flow, and poor particle orientation and confinement.

[0394] The tapered second part 310-S2 of the cavity 310 can include a taper angle α with respect to the longitudinal axis, and the tapered second part 330-S2 of the delivery tube 330 can include a taper angle β with respect to the longitudinal axis. The taper angles can be adjusted to achieve control over the acceleration of the sheath flow in a portion of the sheath flow channel. The third part 310-S3 of the cavity can include a uniform size extending along a longitudinal length. Similarly, the third part 330-S3 of the delivery tube 330 can include a uniform but smaller size extending along a similar longitudinal length. The portion of the sheath channel 360 formed between these two parts 310-S3, 330-S3 does not accelerate the sheath flow 365 and allows it to stabilize to ensure laminar flow and reduce turbulence.

[0395] The fourth part 330-S4 of the delivery tube includes a distal end containing the delivery tube outlet 333. This end can be shaped to enhance the orientation and / or confinement of the particulate, as described in more detail below. This end region can be complementary to a further tapered fourth part 310-S4 of the cavity 310. An aggregation chamber 370 is formed in a fifth part 310-S5 of the cavity 310, where the delivery tube outlet 333 is positioned to discharge a particulate stream 345 into a sheath stream 365 entering the aggregation chamber 370. The aggregation chamber and other components of the flow control device 300 are configured to cause a combined laminar flow of the particulate and the sheath stream flowing out of the particulate delivery outlet 375, where the particulate is primarily oriented along one axis and primarily confined within a plane containing that axis.

[0396] Additional portions of the delivery tube and / or the sheath flow channel 360 may be included, or some of the described portions may be removed from some examples, such as removing the third part 330-S3 from the delivery tube as needed, to impart target characteristics to the particulate stream, such as but not limited to confinement. Alternatively, a geometry different from the geometry of the illustrated sheath flow channel may be employed.

[0397] In some examples, the geometry of these portions may vary along the delivery tube and / or the cavity to promote some asymmetry of the particulate stream within the sheath flow of the final microfluidic flow.

[0398] The sheath flow 365 through the sheath flow channel 360 can be symmetric or asymmetric. For example, a larger volume in the upper half of the sheath flow channel 360 can cause the particulate stream 345 to shift downward. The sheath flow can also be made to rotate around the delivery tube to generate a vortex, which can contribute to particulate stream confinement. The different cross-sectional volumes of the sheath flow channel 360 along its length enable fine control of the sheath flow, including acceleration and stabilization of the flow. The volume of the sheath flow channel 360 also controls the flow rate of the sheath flow through the channel. The ridges 335 and the channels formed between them can also be used to stabilize the sheath flow, as the sheath flow can be introduced from outside the flow control device as a turbulent flow.

[0399] Figure 4 A cross-section along section line AA is shown, Figure 3 wherein the input region of the housing 305 and the delivery tube 330 can be seen. This shows four evenly spaced ridges 335 extending from the delivery tube 330, but any number of ridges can alternatively be used. Various other portions of the delivery tube and the housing are shown with Figure 3The same reference numerals are used to illustrate those parts. One of the ridges 335L in the ridge portion is longer than the other ridges and corresponds to a groove 315 in the outer wall of the cavity 310 of the housing. This arrangement ensures that the delivery tube can only be received into the housing in a single orientation generally illustrated by R. In an example where there are multiple grooves in the cavity wall 310, each groove is for receiving a single ridge 335, and the groove 315 for the longer ridge 335L is deeper so that the longer ridge 335L will still only fit within that one groove to ensure a pre-determined rotational alignment of the delivery tube 330 within the housing 305. In an alternative arrangement where grooves are used for all the ridges, one of these grooves can be wider than the others to receive a wider but not necessarily longer ridge. In another alternative, a pin and corresponding hole arrangement can be used to correctly align the delivery tube within the housing. For example, the pin can extend through the housing into a ridge of the delivery tube, or the delivery tube or the ridge can include a pin that extends through a hole in the housing. In another arrangement, magnets in one of the delivery tube or the housing can be used with another magnet (or metal feature) within the corresponding housing or delivery tube. Optionally or additionally, various other mechanical rotational alignment features can be used.

[0400] The outer visible surfaces of the housing and the delivery tube can be marked to assist the user in aligning the delivery tube when inserting it into the housing, thereby ensuring rotational alignment.

[0401] In some examples, the lateral positioning of the sheath flow channel 360 and / or the delivery tube outlet 333 can be asymmetric to generate a particle flow that is offset within the sheath flow surrounding the microfluidic flow exiting from the hole 313.

[0402] Some examples can utilize different configurations of the flow control device 300 for providing the microfluidic flow.

[0403] Figure 5 A cross-sectional view of a hole 513 according to one example in the flow control device 510 is illustrated, with a microfluidic flow of combined particles and sheath flow exiting from the hole. The hole is rectangular and has a length Dx on one (long) axis X that is longer than the length Dy on the perpendicular (short) axis Y. This provides a hole with an unequal aspect ratio. Also refer to Figure 6, when the stream 615 exits the aperture 613, the shape of the stream can be seen, where the stream has the same cross-sectional shape as the aperture, which is rectangular in this case, as illustrated by the dashed outline on the right. An asymmetric cell 606 having a flat circular shape is also shown within the stream 615. Once the stream 615 exits the aperture 613, surface tension acts on the surface of the stream such that its cross-section is contrary to a circular shape. This shape change causes hydrodynamic forces 618 to be generated within the stream along the longest dimension Dx as that dimension shrinks. These forces 618 are used to orient the cell 606 along the shortest dimension Dy. These surface tension effects can act in concert with the effects of the aggregation chambers 211, 370 to improve cell alignment, which in turn improves cell interrogation and thus cell sorting.

[0404] Figure 6 Illustrated is how surface tension acts on a microfluidic stream 615 exiting an aperture 613 having an asymmetric or non-circular cross-sectional area such that the microfluidic stream assumes a circular cross-sectional shape with a uniform distribution of surface tension. This action causes hydrodynamic forces 618 within the microfluidic stream to orient the asymmetric particles 606 in a preferred direction to interact with interrogation and / or sorting beams. The change in cross-sectional shape is illustrated at 619, and also the changed orientation of the particles 606 having such a changed shape.

[0405] Figure 7 Illustrated is a cross-sectional view of an aperture 713 in an inlet arrangement 710 according to another example, from which a microfluidic stream with a combined particle and sheath flow exits. The aperture 713 has an elliptical shape having a length Dx on one axis X that is longer than the length Dy on the perpendicular axis Y. This provides an aperture with unequal aspect ratios. Similar to Figure 5 the example of, the longer extension in one direction (X) compared to the extension in the perpendicular direction (Y) causes surface tension effects to change the cross-sectional shape of the stream from the cross-sectional shape of the aperture 713 to a circular shape as the stream enters the flow environment below the aperture. This in turn generates hydrodynamic forces that assist in orienting the cells along the desired axis.

[0406] Although rectangular and elliptical shapes have been described, other shapes having a longer dimension on one axis compared to the perpendicular axis can alternatively be used. Examples include: parallelograms; trapezoids; polygons. Single-axis asymmetric shapes such as semi-circles or triangles can also be used, or the shape can be completely asymmetric along two perpendicular axes.

[0407] The aperture 713 emits the microfluidic flow into the flow environment and the confinement chamber (if used) for downstream processing. In some examples, at least one of the delivery microchannel 375 leading to the aperture or the aperture itself has a width Dx (X-axis) of 10 μm to 200 μm. Preferably, the width Dx is between 50 μm and 150 μm. In some examples, the width is equal to the depth Dy (Y-axis).

[0408] In Figure 18 In one example shown and applicable to any flow control device or method described herein, the delivery microchannel 1801 adjacent to the aperture includes a longitudinally expanding taper from a smaller delivery microchannel cross-sectional area upstream to a larger delivery microchannel cross-sectional area downstream (e.g., at the aperture itself), where the cross-sectional area is defined perpendicular to the flow axis. The longitudinally expanding taper can be restricted to a short length of the microchannel adjacent to the aperture, while the remainder of the microchannel has a constant cross-sectional area. For example, the short length can be between 1% and 10% of the total length of the microchannel. In an example, the longitudinally expanding taper can be a transition from a flared delivery microchannel to the aperture. It has been found that such a transition from a flared delivery microchannel to the aperture produces a more stable microfluidic flow 1802 within the downstream flow environment. The sample flow 1810 is surrounded by the sheath flow 1805, and the use of a flared aperture design provides enhanced confinement, interrogation, and downstream sorting.

[0409] In some examples, the length (Z-axis) of the delivery microchannel 1801 from the exit of the downstream confinement chamber to the aperture 313 is at least 10 microns to 10 mm. In this case, the aperture is defined as the point aligned with the terminus of the flow focusing device in a plane perpendicular to the flow z-axis. In one example, the channel length 375 is in the range of 50 microns to 1 mm. This length can be beneficial for the re-establishment of a fully formed laminar flow after the upstream orientation and subsequent orientation and confinement of particles in the confinement chamber.

[0410] In Figure 19 In one example shown, the width at a downstream point in the delivery microchannel is reduced compared to the width at an upstream point in the microchannel. For example, the width W2 compared to the upstream width W1 at the entry point 1905 of the delivery microchannel 1910. This reduction in the width shown from W1 to W2 has the beneficial effect of further increasing the confinement of particles. The reduction in width can produce a taper along a portion of the length of the delivery microchannel as Figure 19 shown or along its entirety. In one example, W2 is between 50% and 95% of W1. In a particular example, the width is reduced from 100 μm to 150 μm to 50 μm to 99 μm. In a specific example, the width is reduced from approximately 125 μm to approximately 50 μm.

[0411] Figure 19The longitudinally decreasing taper of the microchannel shown can be combined with Figure 18 the longitudinally expanding taper shown.

[0412] The cross-sectional shape of the delivery microchannel 375, its inlet and outlet or orifice 313 can be the same or different, and can include: circular, oval, triangular, square or rectangular with various aspect ratios. In one example, the delivery microchannel 375, its inlet and orifice 313 include the same rectangular cross-section with an aspect ratio greater than 1:1.

[0413] In another example, the orifice cross-sectional shape includes a square or rectangle. Without wishing to be bound by theory, a cross-sectional shape with right-angled corners is understood to provide benefits in the following aspects: providing a substantially flat surface for the interrogation beam to enter the microfluidic flow and for any light emission to leave the flow. In one example, the flow is emitted into a rectangular microfluidic channel, and in this case, the shapes of the outlet orifice and the channel are substantially aligned. This ensures a smooth transition without turbulence. In another example, the microfluidic flow is emitted into a gaseous free-space fluid flow environment. In this case, through the surface tension reshaping effect, the shape of the flow will immediately be induced to a circular cross-section. If the interrogation occurs early enough after the flow ejection, the substantially flat surface after ejection will still be presented to the interrogation beam, and the advantages of the orifice shape can still be utilized to improve the interrogation and detection of cell emission. Thus, in one example, the excitation distance is less than 250 μm. In another example, the excitation distance is less than 200 μm. The flow rate also determines such a distance beyond which the flow reforms to a substantially circular cross-section. Thus, in one example, the flow rate is greater than 5 m / s. In other embodiments, the flow rate is from 5 m / s to 20 m / s. The inventors have found that if a flow rate below 5 m / s is used in a gaseous fluid environment, the flow breaks and oscillations occur in the flow, which adversely affects the interrogation and sorting efficiency.

[0414] Figure 8Illustrated is a cross-sectional view of adjustable holes 813R, 813F in a flow control device 810, from which a microfluidic flow with a combined particle and sheath flow exits. The holes may have a circular or slightly elliptical cross-sectional shape in a rest state 813R and an elliptical shape with a greater proportion of length on one axis X than on the perpendicular axis Y in a stressed state 813F. This can be achieved using a flexible plastic film that is stretched along the X-axis to achieve a hole shape with a more unequal aspect ratio. In one example, size-tunable membrane nanopores or micropores can be used - see, for example, the following literature: Roberts, G.S., Kozak, D., Anderson, W., Broom, M.F., Vogel, R., and Trau, M. (2010), "Tunable Nano / Micropores for Particle Detection and Discrimination: Scanning Ion Occlusion Spectroscopy", Small, Vol. 6 (No. 23), pp. 2653 - 2658,

[0415] Doi: 10.1002 / smll.201001129.

[0416] In different arrangements, the cross-sectional shape may remain substantially the same between the rest state and the stressed state, where the hole only changes in size or cross-sectional area. Different combinations of cross-sectional shapes can be employed, such as: circular, elliptical, rectangular, triangular, parallelogram, and any other shape.

[0417] The adjustable holes can be used to accommodate different batches of cells, which can be in different aqueous solutions or have other properties that may affect factors such as the orientation efficiency, the speed of interrogating and / or sorting cells, and the flow properties that may affect the desired cell concentration and other characteristics. The hole size and / or shape can be controlled in coordination with other controllable and / or measurable properties, such as flow rate and break-off distance, in order to achieve the desired operating state of the sorting and separation device.

[0418] Figure 9 Illustrated is a side view of an adjustable hole arrangement including O-rings 913R, 913F, which form a circular or elliptical hole in a rest state 913R. For perspective, an aggregation chamber and channels, as well as the flow below the hole 913R, are also shown. A force can be applied upward on the O-ring to deform the hole 913F into a stressed state. The stressed state hole 913R may maintain the same cross-sectional shape but have a smaller diameter.

[0419] Examples can include an illuminator, a detector, and a sorting device, each of which includes a plurality of optical components. In cases where it is necessary to separate components from a microfluidic flow, such as in the case of using channels or conduits, or in the case of using a sheath to cover the components, the optical architecture can include one or more optically transparent windows to allow light radiation to pass through, so as to focus the radiant energy on the particles, which can achieve at least one of the following: torque and / or pressure on the particles; vaporization of some of the microfluidic flow, for example to achieve sorting and / or concentration and / or droplet formation of the particles; interrogation of the particles, for example by promoting fluorescence emission. Potential materials include: Si, Ge, ZnSe, certain polymers. If needed, Si, ZnSe (or the like), polymers can be compatible with visible light, NIR or SWIR (500 nm to 1600 nm) optical interrogation or manipulation. ZnSe or the like can be used to allow visible light observation, interrogation and possible manipulation (directed torque, pressure to change direction or laser-based cell damage or ablation). Another feature can be an anti-reflection coating applied to both sides of each window, such as an AR coating designed for external air and internal water. Another feature can be a tilted or wedge-shaped window to further reduce the effect of reflection. Another feature can be a channel or fluid flow width that exceeds the spot size of the laser (e.g., QCL), so that a small offset of the channel or fluid flow positioning relative to the beam does not generate false signals.

[0420] In a microfluidic flow ejected into a gaseous environment, the emission from the particles may be affected by the refractive index difference between the liquid flow and the gaseous environment. Therefore, in order to optimally detect the emission, the one or more detectors can be positioned to account for this refractive effect of the emission. In one example, the one or more detectors are positioned to collect maximum emission from the particles. In one example, two detectors are used, and the angle between the detectors is greater than 90° and less than 120°. In another example, two detectors are used, and the angle between the detectors is less than 90° and less than 120°.

[0421] Detector positioning may also be affected by the instability of the microfluidic flow before droplet formation. This phenomenon is in Figure 16ais illustrated, where a stable or laminar portion 1656 of the microfluidic flow forms an unstable region 1657 before the fluid separates into droplets 1658. In the unstable region 1657, the radius of the microfluidic flow fluctuates, causing the angle of incidence of an incident light beam (such as the interrogation beam 1620 as illustrated) to change. This can cause the focal point to change, and in fact, the hydrodynamic forces within this region may cause turbulence and change the paths of the entrained particles. This in turn may lead to inaccurate particle interrogation and sorting, or in the case of a sorting light beam, inaccurately targeting unwanted particles. Therefore, the positioning of the incident light beam may need to be located well above this unstable region 1657 to avoid its effects, as the point at which these effects begin may vary.

[0422] In some examples, the length of the delivery microchannel 375 (the longitudinal axis of the flow or the z-axis) is at least 10 microns to 10 mm from the outlet of the aggregation chamber 370 to the aperture 313, where the aperture is defined as the point aligned with the terminus of the flow aggregation device in a plane perpendicular to the z-axis of the flow. In one example, the delivery microchannel includes a length of 50 microns to 1 mm, where this length allows for the re-establishment of laminar flow after the particles have been oriented and confined in the upstream orientation and confinement chamber. The inventors have found that a minimum length of 10 microns is required to ensure that the flowing fluid has the opportunity to stabilize and assume a laminar profile before flowing out of the aperture.

[0423] In Figure 16b In another example as illustrated, droplet formation can be facilitated before the formation of the unstable region by applying a droplet formation or vaporization light beam 1622 at the stable portion 1656 of the microfluidic flow. Greater control of droplet formation can make the positions of the detector and the light beam forming device more reliable, and can also shorten the distance of system operation. For example, in the case of using a vaporization sorting light beam, this can be achieved by being closer to the interrogation beam in the following way: using the droplet formation beam 1622 to facilitate the early formation of droplets.

[0424] In another example illustrated in FIG. 17, the detection device 1700 includes one or more illuminators 1720 and a detector 1730, the one or more illuminators being arranged to direct a light beam into the microfluidic flow 1715, the detector being arranged to detect the response emission from the particles 1706 impacted by the light beam. One or more interrogation light beams can be used with a plurality of detectors arranged in an arc to collect the response emissions from the particles in the microfluidic flow. In other examples, the plurality of detectors can be arranged linearly as Figure 17B illustrated. In the example, the light beam 1722 can be an infrared light beam or a UV light beam, and the excitation light 1727 can be fluorescence. The light beam 1722 can be controlled to provide an elliptical intensity pattern on the microfluidic flow 1715, although other intensity patterns, such as circular, can be generated.

[0425] Although ideally, the particles 1706 may be intended to be well confined within the narrow cross-sectional portion of the microfluidic flow, in practice, the particles may spread over a larger cross-sectional portion of the microfluidic flow, as illustrated by the particles 1706 at three representative positions. Particles at different lateral positions within the flow may result in different excitation light 1727 towards the detector 1730. For example, the fluorescence 1727 may be transmitted in different directions and / or propagated different distances through the microfluidic flow. Different directions may also result in different diffraction angles through the interface between the microfluidic flow 1715 and the flow environment between it and the detector.

[0426] In an example, the detector 1730 may include a plurality of detection units 1732, such as photodiodes in a photoelectric array, and the plurality of detection units are arranged within an angular range starting from the point where the interrogation beam 1722 intersects the particle 1706. This angular range may extend in more than one plane. For example, the photoelectric array may be arranged to capture the excitation light from the particles at different positions 1706 within the interrogation beam 1722.

[0427] The output 1733 from each detection unit 1732 is sent to an analyzer 1735, which is arranged to integrate the outputs from the detection units into a signal that can be used to classify the particles. Different weights may be applied to some outputs. For example, the outputs at the edges of the detector 1730 may correspond to emissions that are more severely degraded than others, and in this case, these outputs may be amplified to normalize the amplitude of the output 1733. Due to different propagation paths, there may also be different delays and phases between different emissions 1727, and the analyzer 1735 may be configured to compensate for these emissions or otherwise normalize the signal. In one example, the circular cross-section of the microfluidic flow causes refraction of the fluorescence emission during the transition of the medium from the first refractive index to the second refractive index. In this example, the independent detection of the emission signals at different positions on the array (which may be linear, arcuate, or other shapes) enables the analyzer to independently scale at least one of the independently detected emissions to produce a normalized signal that takes into account the refraction effect. This results in enhanced signal processing characteristics and higher accuracy in determining cell characteristics and downstream sorting. The detector may be used with a collection objective lens.

[0428] In some examples, although the possible refractive properties of the flow or the different positions of the particles within the beam 1722, the analyzer 1735 may use the differences in the propagation paths and / or refractive properties to improve the classification of the particles. The correspondence between the output 1733 and the particle classification may be determined experimentally or using, for example, machine learning.

[0429] Any and all references to publications or other documents, including but not limited to patents, patent applications, articles, web pages, books, etc. presented anywhere in this application, are hereby incorporated by reference in their entirety.

[0430] As described elsewhere, the disclosed examples are described only for illustrative purposes and not restrictively. Other examples are possible and are covered by this disclosure, which will be apparent from the teachings contained herein. Accordingly, the breadth and scope of this disclosure should not be limited by any of the above examples, but should be defined only in accordance with the claims supported by this disclosure and their equivalents. Additionally, the examples of this subject disclosure may include methods, systems, and apparatuses / devices, and may also include any and all elements from any other disclosed methods, systems, and devices, including any and all elements corresponding to event determination systems, devices, and methods in combination. In other words, elements from one or another of the disclosed examples may be interchangeable with elements from other disclosed examples. Further, one or more features / elements of the disclosed examples may be removed and still result in patentable subject matter (and thus, result in more examples of this subject disclosure). Moreover, some examples correspond to systems, devices, and methods that are specifically lacking one and / or another element, structure, and / or step (if applicable) compared to the teachings of the prior art, and thus represent patentable subject matter and may be distinguished therefrom (i.e., claims directed to such examples may include one or more negative limitations to point out the lack of one or more features in the prior art teachings).

[0431] It should be understood that although the different features have been described separately for ease and simplicity of explanation, these features may be combined in different ways in different examples. For example, features of the flow control device, such as Figure 18 , Figure 19 , Figure 20a , Figure 3 , Figure 4 , Figure 5 , Figure 7 , Figure 8 , Figure 9 as shown. Similarly, different aspects of the downstream processing may be combined, such as Figure 1 , Figure 2 , Figure 11 a to Figure 12 b, Figure 14 , Figure 17a, Figure 17b, Figure 20a , Figure 20b , Figures 22a to 22c , Figure 23 , Figures 24a to 24c as shown.

[0432] The various inventive concepts disclosed herein may be embodied as one or more methods, as described above. The acts performed as part of the method may be ordered in any suitable way. Accordingly, examples may be constructed in which the acts are performed in an order different from the recited order, which may include performing some acts simultaneously, even though shown as sequential acts in the illustrative embodiments.

[0433] References

[0434] 1. Cossarizza A, Chang HD, Radbruch A, et al., "Guidelines for the use of flow cytometry and cell sorting in immunological studies", Eur J Immunol, 2017; 47(10): 1584 - 1797, doi:

[0435] 10.1002 / eji.201646632.

Claims

1. A method for processing particles in a particle stream, the method comprising: delivering a microfluidic stream from a microfluidic orifice into a flow environment, the microfluidic stream comprising a particle stream containing a plurality of particles; directing interrogation electromagnetic radiation to the particles in the microfluidic stream and monitoring the response emission from the irradiated particles; subsequently directing sorting electromagnetic radiation to at least some of the particles in the microfluidic stream so as to sort the particles into at least two populations based on the monitored response emission of the particles; the microfluidic stream comprising a continuous phase flow of a liquid.

2. The method according to claim 1, wherein the flow environment comprises one or more of the following: a microchannel, which optionally comprises a substantially transparent material; a substrate exposed to a fluid environment; a liquid fluid environment; a gaseous fluid environment.

3. The method according to claim 2, wherein the flow environment comprises a gaseous fluid environment and is at least partially surrounded by a gaseous sheath that is substantially parallel to the microfluidic stream and moves relative to the gaseous environment.

4. The method according to any one of the preceding claims, wherein the sorting comprises one or more of the following operations: directing subsequent sorting electromagnetic radiation to the particles in one of the populations in the microfluidic stream to apply radiation pressure on the particles; directing the subsequent sorting electromagnetic radiation to the particles in one of the populations in the microfluidic stream to ablate the particles; directing the subsequent sorting electromagnetic radiation to particles that are biological cells, wherein the subsequent sorting electromagnetic radiation is configured to transfer energy to selected cells, the energy being lower than a predetermined ablation threshold equivalent to rupturing the cell membrane and higher than a predetermined pretreatment threshold equivalent to the cells not surviving after a freezing and thawing process.

5. The method according to any one of the preceding claims, wherein the interrogation electromagnetic radiation and the sorting electromagnetic radiation are directed through a common optical objective.

6. The method according to claim 5, wherein the interrogation electromagnetic radiation and the sorting electromagnetic radiation are directed through the common optical objective at an angle relative to each other.

7. The method according to claim 6, wherein the angle between the interrogation electromagnetic radiation and the sorting electromagnetic radiation directed through the common optical objective is adjusted so as to define an inter-beam distance between the focus of the interrogation electromagnetic radiation within the microfluidic stream and the focus of the sorting electromagnetic radiation within the microfluidic stream.

8. The method according to any one of claims 5 to 7, wherein the microfluidic stream is delivered from a flow control device having the microfluidic orifice, the flow control device being shaped to define a region above the microfluidic orifice through which the interrogation electromagnetic radiation and / or the sorting electromagnetic radiation are directed.

9. The method according to claim 8, wherein the objective optical component is at least partially positioned within the region.

10. The method according to any one of the preceding claims, wherein the probing electromagnetic radiation is propagated as a probing light beam that intersects the microfluidic flow at an excitation distance from the microfluidic orifice, the excitation distance being between 25 μm and 1000 μm.

11. The method according to claim 10, wherein the excitation distance is less than 400 μm and greater than one of: 25 μm, 50 μm, 100 μm.

12. The method according to any one of the preceding claims, wherein the probing electromagnetic radiation and the subsequent sorting electromagnetic radiation are propagated as a respective probing light beam and a respective sorting light beam that intersect the microfluidic flow and separate a beam separation distance, the beam separation distance including one or more of: at least 10 μm; Between 10 μm and 400 μm.

13. The method according to any one of the preceding claims, wherein at least one of the probing electromagnetic radiation and the sorting electromagnetic radiation is controlled to be propagated as a light beam that is equal to or wider than the microfluidic flow when intersecting the microfluidic flow.

14. The method according to any one of the preceding claims, wherein monitoring the response emission from the irradiated particles includes using the output from a plurality of sensors arranged around the microfluidic flow, the plurality of sensors being arranged to capture response emissions from different directions.

15. The method according to claim 14, wherein the output from the sensors is normalized to compensate for differences in one or more of the following properties of the response emission captured from the respective sensors: amplitude; phase; propagation delay; refractive effect when entering the flowing environment from the microfluidic flow.

16. The method according to any one of the preceding claims, the method comprising: Adjust the cross-section of the microfluidic orifice.

17. The method according to any one of the preceding claims, wherein the flow velocity of the microfluidic flow is from 5 m / s to 20 m / s.

18. A method of processing particles in a particle stream, the method comprising: Conveying a microfluidic flow from a microfluidic orifice into a flowing environment, the microfluidic flow including a particle stream containing a plurality of particles; Directing probing electromagnetic radiation through a common optical objective to the particles in the microfluidic flow and monitoring the response emission from the irradiated particles; Subsequently directing sorting electromagnetic radiation through the common optical objective lens to at least some of the particles in the microfluidic flow so as to sort the particles into at least two populations based on the monitored response emission of the particles.

19. The method according to claim 18, wherein the probing electromagnetic radiation and the sorting electromagnetic radiation are directed through the common optical objective at an angle relative to each other.

20. The method according to claim 19, wherein the angle between the probing electromagnetic radiation and the sorting electromagnetic radiation directed through the common optical objective is adjusted so as to define a beam separation distance between the focus of the probing electromagnetic radiation within the microfluidic flow and the focus of the sorting electromagnetic radiation within the microfluidic flow.

21. The method according to any one of claims 18 to 20, wherein the microfluidic flow is delivered from a flow control device having the microfluidic orifice, the flow control device being shaped to define a region above the microfluidic orifice, and the interrogation electromagnetic radiation and / or the sorting electromagnetic radiation is guided through the region.

22. The method according to claim 21, wherein the objective optical component is at least partially positioned within the region.

23. The method according to any one of claims 18 to 22, wherein the interrogation electromagnetic radiation propagates as an interrogation beam, the interrogation beam intersecting the microfluidic flow at an excitation distance from the microfluidic orifice, the excitation distance being between 25 μm and 1000 μm.

24. The method according to claim 23, wherein the excitation distance is less than 400 μm and greater than one of the following: 25 μm, 50 μm, 100 μm.

25. The method according to any one of claims 18 to 24, wherein the interrogation electromagnetic radiation and the subsequent sorting electromagnetic radiation propagate as a respective interrogation beam and a respective sorting beam, the respective interrogation beam and the respective sorting beam intersecting the microfluidic flow and separating the inter-beam distance, the inter-beam distance including one or more of the following: at least 10 μm; between 10 μm and 400 μm.

26. The method according to any one of claims 18 to 25, wherein the sorting electromagnetic radiation is controlled to propagate as a sorting beam, the sorting beam being wider than the microfluidic flow when intersecting the microfluidic flow.

27. The method according to any one of claims 18 to 26, wherein monitoring the response emission from the irradiated particles includes using the outputs from a plurality of sensors arranged around the microfluidic flow, the plurality of sensors using a photoelectric array with detectors arranged to capture response emissions from different directions.

28. The method according to claim 27, wherein the outputs from the sensors are normalized to compensate for differences in one or more of the following properties of the response emission captured from the respective sensors: amplitude; phase; propagation delay; refractive effect when entering the flow environment from the microfluidic flow.

29. The method according to any one of claims 18 to 28, the method including adjusting the cross-section of the microfluidic orifice.

30. The method according to any one of claims 18 to 29, wherein the flow rate of the microfluidic flow is from 5 m / s to 20 m / s.

31. The method according to any one of claims 18 to 30, wherein the flow environment includes one or more of the following: a microchannel, the microchannel optionally including a substantially transparent material; a substrate exposed to a fluid environment; a liquid fluid environment; a gaseous fluid environment.

32. The method according to claim 31, wherein the flow environment includes a gaseous fluid environment and is at least partially surrounded by a gaseous sheath, the gaseous sheath being substantially parallel to the microfluidic flow and moving relative to the gaseous environment.

33. The method according to any one of claims 18 to 31, wherein the sorting comprises one or more of the following operations: Directing the subsequent sorting electromagnetic radiation to particles in one of the populations in the microfluidic flow to apply radiation pressure on the particles; Directing the subsequent sorting electromagnetic radiation to particles in one of the populations in the microfluidic flow to ablate the particles; Directing the subsequent sorting electromagnetic radiation to particles that are biological cells, wherein the subsequent sorting electromagnetic radiation is configured to transfer energy to selected cells, the energy being lower than a predetermined ablation threshold equivalent to rupturing the cell membrane and higher than a predetermined pretreatment threshold equivalent to the cells not surviving after a freezing and thawing process.

34. A method of processing particles in a particle stream, the method comprising: Delivering a microfluidic flow from a microfluidic orifice to a flowing environment, the microfluidic flow comprising a particle stream containing a plurality of particles; Directing a probing beam to the particles in the microfluidic flow and monitoring the response emission from the irradiated particles; Subsequently sorting the particles into at least two populations based on the monitored response emission of the particles; wherein the excitation distance between the orifice and the probing beam is less than 1000 um.

35. The method according to claim 34, wherein the excitation distance is less than 400 um.

36. The method according to claim 34 or 35, wherein the excitation distance is greater than one of the following: 25 um, 50 um, 100 um.

37. The method according to any one of claims 34 to 36, wherein the flow rate of the microfluidic flow is from 5 m / s to 20 m / s.

38. The method according to any one of claims 34 to 37, wherein sorting the particles comprises directing sorting electromagnetic radiation to at least some of the particles in the microfluidic flow to sort the particles into the at least two populations.

39. The method according to claim 38, wherein the sorting comprises one or more of the following operations: Directing the subsequent sorting electromagnetic radiation to particles in one of the populations in the microfluidic flow to apply radiation pressure on the particles; Directing the subsequent sorting electromagnetic radiation to particles in one of the populations in the microfluidic flow to ablate the particles; Directing the subsequent sorting electromagnetic radiation to particles that are biological cells, wherein the subsequent sorting electromagnetic radiation is configured to transfer energy to selected cells, the energy being lower than a predetermined ablation threshold equivalent to rupturing the cell membrane and higher than a predetermined pretreatment threshold equivalent to the cells not surviving after a freezing and thawing process.

40. The method according to claim 38 or 39, wherein the probing electromagnetic radiation and the sorting electromagnetic radiation are directed through a common optical objective.

41. The method according to claim 40, wherein the interrogation electromagnetic radiation and the sorting electromagnetic radiation are guided through the common optical objective at an angle relative to each other.

42. The method according to claim 41, wherein the angle between the interrogation electromagnetic radiation and the sorting electromagnetic radiation guided through the common optical objective is adjusted to define the inter-beam distance between the focus of the interrogation electromagnetic radiation within the microfluidic flow and the focus of the sorting electromagnetic radiation within the microfluidic flow.

43. The method according to any one of claims 38 to 42, wherein the microfluidic flow is delivered from a flow control device having the microfluidic orifice, the flow control device being shaped to define a region above the microfluidic orifice through which the interrogation electromagnetic radiation and / or the sorting electromagnetic radiation are guided.

44. The method according to claim 43, wherein the objective optical component is at least partially positioned within the region.

45. The method according to any one of claims 38 to 44, wherein the interrogation electromagnetic radiation and the subsequent sorting electromagnetic radiation propagate as a respective interrogation beam and a respective sorting beam, the respective interrogation beam and the respective sorting beam intersecting the microfluidic flow and separated by an inter-beam distance, the inter-beam distance including one or more of the following: at least 10 μm; between 10 μm and 400 μm.

46. The method according to any one of claims 38 to 44, wherein the interrogation electromagnetic radiation and / or the sorting electromagnetic radiation are controlled to propagate as a sorting beam that is wider than the microfluidic flow when intersecting the microfluidic flow.

47. The method according to any one of claims 34 to 46, wherein the microfluidic flow includes a continuous phase flow of a liquid.

48. The method according to any one of claims 34 to 47, wherein the flow environment includes one or more of the following: a microchannel, the microchannel optionally including a substantially transparent material; a substrate exposed to a fluid environment; a liquid fluid environment; a gaseous fluid environment.

49. The method according to claim 48, wherein the flow environment includes a gaseous fluid environment and is at least partially surrounded by a gaseous sheath that is substantially parallel to the microfluidic flow and moves relative to the gaseous environment.

50. The method according to any one of claims 34 to 49, wherein monitoring the response emission from the irradiated particles includes using the output from a plurality of sensors arranged around the microfluidic flow, the plurality of sensors using a photoelectric array having detectors arranged to capture response emissions from different directions.

51. The method according to claim 50, wherein the output from the sensors is normalized to compensate for differences in one or more of the following properties of the response emission captured from the respective sensors: amplitude; phase; propagation delay; refractive effect when entering the flow environment from the microfluidic flow.

52. The method according to any one of claims 34 to 51, the method comprising adjusting the cross-section of the microfluidic pore.

53. An apparatus for processing particles in a particle stream, the apparatus comprising: means for conveying a microfluidic stream from a microfluidic pore into a flow environment, the microfluidic stream comprising a particle stream containing a plurality of particles; means for directing interrogating electromagnetic radiation to the particles in the microfluidic stream and monitoring the response emission from the irradiated particles; means for directing sorting electromagnetic radiation to at least some of the particles in the microfluidic stream so as to sort the particles into at least two populations based on the monitored response emission of the particles; wherein the means for conveying the microfluidic stream is configured to maintain the microfluidic stream as a continuous phase stream of liquid when receiving the electromagnetic radiation.

54. The apparatus according to claim 53, wherein the flow environment comprises one or more of the following: a microchannel, which optionally comprises a substantially transparent material; a substrate exposed to a fluid environment; a liquid fluid environment; a gaseous fluid environment.

55. The apparatus according to claim 54, wherein the flow environment comprises a gaseous fluid environment and is at least partially surrounded by a gaseous sheath that is substantially parallel to the microfluidic stream and moves relative to the gaseous environment.

56. The apparatus according to any one of claims 53 to 55, wherein the means for directing the sorting electromagnetic radiation is configured to: Direct the subsequent sorting electromagnetic radiation to the particles in one of the populations in the microfluidic flow to apply radiation pressure on the particles; and / or direct the subsequent sorting electromagnetic radiation to the particles in one of the populations in the microfluidic stream to ablate the particles; and / or direct the subsequent sorting electromagnetic radiation to particles that are biological cells, wherein the subsequent sorting electromagnetic radiation is configured to transfer energy to the selected cells, the energy being lower than a predetermined ablation threshold equivalent to rupturing the cell membrane and higher than a predetermined pretreatment threshold equivalent to the cells not surviving after freezing and thawing.

57. The apparatus according to any one of claims 53 to 56, the apparatus comprising a common optical objective through which the interrogating electromagnetic radiation and the sorting electromagnetic radiation are directed.

58. The apparatus according to claim 57, the apparatus being configured to direct the interrogating electromagnetic radiation and the sorting electromagnetic radiation through the common optical objective at an angle relative to each other.

59. The apparatus according to claim 58, wherein the angle between the interrogating electromagnetic radiation and the sorting electromagnetic radiation directed through the common optical objective can be adjusted so as to define the beam separation between the focus of the interrogating electromagnetic radiation in the microfluidic stream and the focus of the sorting electromagnetic radiation in the microfluidic stream.

60. The device according to any one of claims 57 to 59, wherein the member for conveying the microfluidic stream comprises a flow control device having the microfluidic aperture through which the microfluidic stream is conveyed, the flow control device being shaped to define a region above the microfluidic aperture through which the interrogation electromagnetic radiation and / or the sorting electromagnetic radiation is guided.

61. The device according to claim 60, wherein the objective optical component is at least partially positioned within the region.

62. The device according to any one of claims 53 to 61, wherein the interrogation electromagnetic radiation is configured to propagate as an interrogation beam that intersects the microfluidic stream at an excitation distance from the microfluidic aperture, the excitation distance being between 25 μm and 1000 μm.

63. The device according to claim 62, wherein the excitation distance is less than 400 μm and greater than one of: 25 μm, 50 μm, 100 μm.

64. The device according to any one of claims 53 to 63, wherein the interrogation electromagnetic radiation and the subsequent sorting electromagnetic radiation are configured to propagate as respective interrogation beams and respective sorting beams that intersect the microfluidic stream and are separated by a beam separation distance that comprises one or more of: at least 10 μm; between 10 μm and 400 μm.

65. The device according to any one of claims 53 to 64, wherein at least one of the interrogation electromagnetic radiation and the sorting electromagnetic radiation is controlled to propagate as a beam that is equal to or wider than the microfluidic stream when intersecting the microfluidic stream.

66. The device according to any one of claims 53 to 65, the device comprising a plurality of sensors arranged around the microfluidic stream and configured to capture response emissions from different directions.

67. The device according to claim 66, the device being configured to normalize the output from the sensors to compensate for differences in one or more of the following properties of the response emissions captured from the respective sensors: amplitude; phase; propagation delay; refractive effects when entering the flow environment from the microfluidic stream.

68. The device according to any one of claims 53 to 67, wherein the cross-section of the microfluidic aperture is adjustable.

69. The device according to any one of claims 53 to 68, the device being configured to provide a flow rate of the microfluidic stream of 5 m / s to 20 m / s.

70. A device for processing particles in a particle stream, the device comprising: a member for: conveying a microfluidic stream from a microfluidic aperture into a flow environment, the microfluidic stream comprising a particle stream containing a plurality of particles; a common objective; a member for: guiding interrogation electromagnetic radiation through the common optical objective to the particles in the microfluidic stream and monitoring response emissions from the irradiated particles; Subsequently, the sorted electromagnetic radiation is guided through the common optical objective lens to at least some of the particles in the microfluidic flow so as to sort the particles into at least two populations according to the monitored response emission of the particles.

71. The apparatus according to claim 70, the apparatus being configured to guide the interrogation electromagnetic radiation and the sorted electromagnetic radiation through the common optical objective at an angle relative to each other.

72. The apparatus according to claim 71, wherein the angle between the interrogation electromagnetic radiation and the sorted electromagnetic radiation guided through the common optical objective can be adjusted so as to define the inter-beam distance between the focus of the interrogation electromagnetic radiation within the microfluidic flow and the focus of the sorted electromagnetic radiation within the microfluidic flow.

73. The apparatus according to any one of claims 70 to 72, wherein the member for conveying the microfluidic flow is a flow control device having the microfluidic orifice, the flow control device being shaped to define a region above the microfluidic orifice through which the interrogation electromagnetic radiation and / or the sorted electromagnetic radiation is guided.

74. The apparatus according to claim 73, wherein the objective optical component is at least partially positioned within the region.

75. The apparatus according to any one of claims 70 to 74, wherein the interrogation electromagnetic radiation is configured to propagate as an interrogation beam that intersects the microfluidic flow at an excitation distance from the microfluidic orifice, the excitation distance being between 25 μm and 1000 μm.

76. The apparatus according to claim 75, wherein the excitation distance is less than 400 μm and greater than one of: 25 μm, 50 μm, 100 μm.

77. The apparatus according to any one of claims 70 to 76, wherein the interrogation electromagnetic radiation and the subsequent sorted electromagnetic radiation are configured to propagate as a respective interrogation beam and a respective sorted beam, the respective interrogation beam and the respective sorted beam intersecting the microfluidic flow and separated by an inter-beam distance that includes one or more of: at least 10 μm; between 10 μm and 400 μm.

78. The apparatus according to any one of claims 70 to 77, wherein the sorted electromagnetic radiation is controlled to propagate as a sorted beam that is wider than the microfluidic flow when intersecting the microfluidic flow.

79. The apparatus according to any one of claims 70 to 78, the apparatus including a photoelectric array having detectors arranged to capture response emissions from different directions.

80. The apparatus according to claim 79, the apparatus being configured to normalize the output from the sensors to compensate for differences in one or more of the following properties of the response emissions captured from the respective sensors: amplitude; phase; propagation delay; refractive effect when entering the flow environment from the microfluidic flow.

81. The device according to any one of claims 70 to 80, wherein the cross-section of the microfluidic pore is adjustable.

82. The device according to any one of claims 70 to 81, the device being configured to provide a flow rate of the microfluidic flow of 5 m / s to 20 m / s.

83. The method according to any one of claims 70 to 82, wherein the flow environment comprises one or more of the following: a microchannel, which optionally comprises a substantially transparent material; a substrate exposed to a fluid environment; a liquid fluid environment; a gaseous fluid environment.

84. The device according to claim 83, wherein the flow environment comprises a gaseous fluid environment and is at least partially surrounded by a gaseous sheath, the gaseous sheath being substantially parallel to the microfluidic flow and moving relative to the gaseous environment.

85. The device according to any one of claims 70 to 84, wherein the member for guiding the probing electromagnetic radiation is configured to: Direct the subsequent sorting electromagnetic radiation to the particles in one of the populations in the microfluidic flow to apply radiation pressure on the particles; and / or direct the subsequent sorting electromagnetic radiation to a particle in one of the populations in the microfluidic flow to ablate the particle; and / or direct the subsequent sorting electromagnetic radiation to a particle that is a biological cell, wherein the subsequent sorting electromagnetic radiation is configured to transfer energy to a selected cell, the energy being lower than a predetermined ablation threshold equivalent to rupturing the cell membrane and higher than a predetermined pretreatment threshold equivalent to the cell not surviving after a freezing and thawing process.

86. A device for processing particles in a particle stream, the device comprising: a member for: conveying a microfluidic flow from a microfluidic pore into a flow environment, the microfluidic flow comprising a particle stream containing a plurality of particles; a member for: directing a probing beam to the particles in the microfluidic flow and monitoring the response emission from the irradiated particles; a member for: sorting the particles into at least two populations based on the monitored response emission of the particles; wherein the excitation distance between the pore and the probing beam is configured to be less than 1000 um.

87. The device according to claim 86, wherein the excitation distance is configured to be less than 400 um.

88. The device according to claim 86 or 87, wherein the excitation distance is configured to be greater than one of: 25 um, 50 um, 100 um.

89. The device according to any one of claims 86 to 88, wherein the flow rate of the microfluidic flow is configured to be 5 m / s to 20 m / s.

90. The device according to any one of claims 86 to 89, wherein the member for sorting the particles comprises a member for: directing sorting electromagnetic radiation to at least some of the particles in the microfluidic flow so as to sort the particles into the at least two populations.

91. The device according to claim 90, wherein the member for guiding the sorting electromagnetic radiation is configured to: Direct the subsequent sorting electromagnetic radiation to the particles in one of the populations in the microfluidic flow to apply radiation pressure on the particles; and / or directing the subsequent sorting electromagnetic radiation to particles in a population in the microfluidic stream to ablate the particles; and / or directing the subsequent sorting electromagnetic radiation to particles that are biological cells, wherein the subsequent sorting electromagnetic radiation is configured to transfer energy to selected cells, the energy being below a predetermined ablation threshold equivalent to rupturing the cell membrane and above a predetermined pretreatment threshold equivalent to the cells not surviving after freezing and thawing.

92. The apparatus according to claim 90 or 91, the apparatus being configured to direct the interrogation electromagnetic radiation and the sorting electromagnetic radiation through a common optical objective.

93. The method according to claim 92, the apparatus being configured to direct the interrogation electromagnetic radiation and the sorting electromagnetic radiation through the common optical objective at an angle relative to each other.

94. The apparatus according to claim 93, wherein the angle between the interrogation electromagnetic radiation and the sorting electromagnetic radiation directed through the common optical objective can be adjusted to define the inter-beam distance between the focus of the interrogation electromagnetic radiation in the microfluidic stream and the focus of the sorting electromagnetic radiation in the microfluidic stream.

95. The apparatus according to any one of claims 86 to 94, wherein the member for conveying the microfluidic stream is a flow control device having the microfluidic orifice, the flow control device being shaped to define a region above the microfluidic orifice through which the interrogation electromagnetic radiation and / or the sorting electromagnetic radiation is directed.

96. The apparatus according to claim 95, wherein the objective optical component is at least partially located within the region.

97. The apparatus according to any one of claims 90 to 96, wherein the interrogation electromagnetic radiation and the subsequent sorting electromagnetic radiation are configured to propagate as a respective interrogation beam and a respective sorting beam, the respective interrogation beam and the respective sorting beam intersecting the microfluidic stream and separated by an inter-beam distance that includes one or more of the following: at least 10 μm; between 10 μm and 400 μm.

98. The apparatus according to any one of claims 90 to 97, wherein the sorting electromagnetic radiation is controlled to propagate as a sorting beam that is wider than the microfluidic stream when intersecting the microfluidic stream.

99. The apparatus according to any one of claims 86 to 98, wherein the member for conveying the microfluidic stream is configured to maintain the microfluidic stream as a continuous phase flow of liquid.

100. The apparatus according to any one of claims 86 to 99, wherein the flow environment includes one or more of the following: a microchannel, the microchannel optionally including a substantially transparent material; a substrate exposed to a fluid environment; a liquid fluid environment; a gaseous fluid environment.

101. The apparatus according to claim 100, wherein the flow environment comprises a gaseous fluid environment and is at least partially surrounded by a gaseous sheath that is substantially parallel to the microfluidic flow and moves relative to the gaseous environment.

102. The apparatus according to any one of claims 86 to 101, the apparatus comprising a photoelectric array having detectors arranged to capture response emissions from different directions.

103. The apparatus according to claim 102, the apparatus being configured to normalize the output from the sensors to compensate for differences in one or more of the following properties of the response emissions captured from the respective sensors: amplitude; phase; propagation delay; refractive effects when entering the flow environment from the microfluidic flow.

104. The apparatus according to any one of claims 86 to 103, wherein the cross-section of the microfluidic pore is adjustable.

Images