Flow control devices, components and methods thereof

Abstract

There is provided a flow control device comprising: a transfer tube having an internal cavity, the transfer tube extending within a housing along a longitudinal axis to a focusing chamber; one or more channels defined between the outer surface of the transfer tube and the inner surface of the housing, the channels extending within the housing toward the lumen outlet of the transfer tube and at least partially aligned with the longitudinal axis of the transfer tube; and a focusing chamber fluidly coupled to the aperture in the flow control device.

Description

1. Technical Field

[0001] The present disclosure relates to the manipulation of particles in fluid streams for downstream interrogation, orientation, displacement and / or sorting or other processes. In particular, but not exclusively, the present disclosure relates to the manipulation of biological cells, such as sperm cells, in laminar liquid flows. 2. Background Technology

[0002] Microfluidic laminar flow has been used to control the flow of particles, such as biological cells, to enable processing, analysis, or sorting of these particles. Orientation of asymmetric particles, such as sperm cells, can be used for optimal processing. For example, a laser beam can be focused on passing cells, which emit light of various wavelengths that can be detected and interpreted by interrogation equipment. Therefore, it may be desirable to orient these cells to optimize the performance of a processing, analysis, or sorting system.

[0003] Such an arrangement could be used to identify which cell types are contained in a particle stream, for example, whether they contain an X or Y chromosome. This could then be used to sort the cells into different containers. In the dairy industry, this could be used to ensure that brood cows are inseminated with X-chromosome sperm cells, thereby producing more valuable female calves than male calves.

[0004] Improving the accuracy with which particles can be identified and the speed with which this can be achieved while avoiding damage to the particles continues to be of great interest.

[0005] In this specification, where reference has been made to patent specifications, other external documents or other sources of information, this has generally been for the purpose of providing a context for discussing the features of the invention. Unless specifically stated otherwise, reference to such external documents is not to be construed as an admission that such documents or such sources of information are prior art, or form part of the common general knowledge in the art, in any jurisdiction. 3. Summary of the Invention

[0006] In one aspect, a flow control device is provided, comprising: a transfer tube having an inner lumen, the transfer tube extending along a longitudinal axis within a housing to a focusing chamber; one or more channels defined between an outer surface of the transfer tube and an inner surface of the housing, the channels extending within the housing toward an inner lumen outlet of the transfer tube and at least partially aligned with the longitudinal axis of the transfer tube; and the focusing chamber fluidically coupled to a hole in the flow control device.

[0007] In one example, the one or more channels extend substantially parallel to or at an angle relative to the longitudinal axis of the transfer tube. The one or more channels may be defined by corresponding protrusions extending along the transfer tube and / or the housing. At least some of these protrusions may extend from the transfer tube to contact the inner surface of the housing, or extend from the inner surface of the housing to contact the transfer tube. The protrusions may be arranged to contact the inner surface of the housing or the transfer tube at multiple longitudinal and lateral locations to position the transfer tube outlet at a predetermined location within the focusing chamber.

[0008] In one example, the one or more channels a. comprise a substantially semicircular or triangular cross-section; and / or b. extend from the sheath fluid inlet to the focusing chamber; and / or c. are configured to provide a single flow path or multiple flow paths for the sheath fluid between the sheath fluid inlet and the focusing chamber over at least a portion of the longitudinal extension of the transfer tube within the housing; and / or d. have a larger cross-sectional area at a downstream location than at an upstream location of the one or more channels.

[0009] The one or more channels may be spaced circumferentially around the entire outer cross-section of the transfer tube and merge into a focusing chamber defined at least in part by the tapered transfer tube and the inverse tapered housing wall.

[0010] The one or more channels may be defined by two or more ridges that define a cross-sectional shape of the channel, and wherein the ridges are sized to provide a friction fit with the housing such that there is substantially zero volume between the ridges and the inner surface of the housing.

[0011] In one example, the flow control device includes a coupling structure configured to secure the transfer tube and the housing together during use to form at least one of the one or more channels between the housing and the transfer tube and to position the transfer tube outlet at a predetermined longitudinal, lateral, and / or rotational position within the focusing chamber.

[0012] The housing may have a first portion having a longitudinal cross-sectional shape arranged to engage the first portion of the transfer tube at a plurality of longitudinal locations to position the transfer tube outlet at predetermined lateral locations within the focusing chamber.

[0013] The engagement structure may include longitudinally extending ridges within the cavity of the housing, the ridges configured to position the transfer tube outlet according to a predetermined lateral relationship to the housing when the transfer tube outlet is received in the housing.

[0014] In one example, the engagement structure includes a rotational alignment feature arranged to secure the transfer tube outlet at a predetermined rotational angle relative to the housing.

[0015] In one example, the interior lateral cross-sectional shape of the housing in which the transfer tube is positioned is different from the exterior lateral cross-sectional shape of the transfer tube at corresponding longitudinal locations, and wherein the one or more channels are defined by the difference in lateral cross-sectional shapes at different longitudinal locations.

[0016] In one example, an inner surface of the housing defines a cavity for receiving a transfer tube, the cavity terminating in a focusing chamber, wherein the cavity has a geometry that differs from the geometry of the focusing chamber at the terminal end; the geometry comprising one or more of the following: size, cross-sectional shape, and longitudinal taper angle. The end of the transfer tube can be positioned within the focusing chamber at a predetermined distance from the terminal end.

[0017] In one example, the flow control device includes a second focusing chamber fluidly coupled between a first focusing chamber and the aperture, wherein the first focusing chamber has a geometry different from the geometry of the second focusing chamber. The geometries can differ in one or more of: size, cross-sectional shape, longitudinal taper angle. The cross-sectional shape of the first focusing chamber can have a lower aspect ratio than the cross-sectional shape of the second focusing chamber. The cross-sectional shape of the first focusing chamber can be circular, and the cross-sectional shape of the second focusing chamber can be substantially rectangular. The second focusing chamber can be fluidly coupled to the aperture via a transfer microchannel having a smaller dimension along a lateral axis than the second focusing chamber.

[0018] In one example, the flow control device includes a confinement chamber fluidly coupled to the second focusing chamber, the confinement chamber tapering longitudinally from the second focusing chamber along a lateral axis.

[0019] In one aspect, a flow control device is provided, comprising: a transfer tube having an inner lumen extending along a longitudinal axis within a housing to a first focusing chamber; a housing comprising a second focusing chamber fluidly coupled between the first focusing chamber and an aperture in the flow control device; wherein a geometry of the first focusing chamber is different from a geometry of the second focusing chamber.

[0020] In one example, the geometries differ in one or more of the following: size, cross-sectional shape, longitudinal taper angle.

[0021] In one example, the cross-sectional shape of the first focusing chamber can have a lower aspect ratio than the cross-sectional shape of the second focusing chamber.The cross-sectional shape of the first focusing chamber can be circular, and the cross-sectional shape of the second focusing chamber can be rectangular.

[0022] In one example, the second focusing chamber is fluidly coupled to the well via a transfer microchannel having a smaller dimension in a lateral axis than the second focusing chamber.

[0023] In one example, the flow control device includes a confinement chamber fluidly coupled to the second focusing chamber, the confinement chamber tapering longitudinally from the second focusing chamber along a lateral axis.

[0024] In one example, the cross-sectional shape of the second focusing chamber includes two opposing straight lines and one or more of: a curve between the two straight lines; a V-shaped line or a chined line between the two straight lines.

[0025] In another aspect, there is provided a particle processing system comprising a flow control device and a particle interrogation device and / or a sorting device as defined above.

[0026] In another aspect, a method for controlling fluid flow associated with transporting particles is provided. The method includes: transporting a particle stream of a liquid containing particles in a lumen of a transfer tube extending along a longitudinal axis within a housing to a focusing chamber; transporting a sheath stream of liquid in one or more channels between the transfer tube and the housing toward the focusing chamber, wherein the channels extend toward an outlet of the lumen of the transfer tube within the housing and are at least partially aligned with the longitudinal axis of the transfer tube; and generating a microfluidic stream using the focusing chamber and emitting the microfluidic stream from an aperture, the microfluidic stream comprising a laminar flow of liquid from the sheath stream surrounding the liquid from the particle stream.

[0027] In another aspect, a method for controlling fluid flow associated with transporting particles is provided. The method includes: transporting a particle flow of a liquid containing particles in a lumen of a transport tube to a first focusing chamber; transporting a sheath flow of the liquid to the first focusing chamber; and generating a microfluidic stream using the first focusing chamber and a second focusing chamber fluidically coupled to the first focusing chamber, wherein the first focusing chamber has a different geometry than the second focusing chamber.

[0028] In another aspect, a method for controlling fluid flow associated with transporting particles is provided. The method includes: transporting a particle stream of a liquid containing particles in a lumen of a transfer tube extending along a longitudinal axis within a housing to a first focusing chamber; transporting a sheath stream of liquid in one or more channels between the transfer tube and the housing toward the focusing chamber, wherein the channels extend toward an outlet of the lumen of the transfer tube within the housing and are at least partially aligned with the longitudinal axis of the transfer tube; generating a microfluidic stream using the first focusing chamber and a second focusing chamber fluidically coupled to the first focusing chamber, wherein the first focusing chamber has a different geometry than the second focusing chamber; and emitting a microfluidic stream from an aperture, the microfluidic stream comprising a laminar flow of liquid from the sheath stream surrounding the liquid from the particle stream.

[0029] In one example, one or more of these methods uses a flow control device as defined above.

[0030] In one example, a flow control device for controlling fluid flow associated with transporting particles is provided. The flow control device includes: a transfer tube having an inner lumen, the transfer tube being configured to transport a particle flow of a liquid containing particles from a transfer tube inlet to a transfer tube outlet, the transfer tube extending within a housing along a longitudinal axis to a focusing chamber; one or more channels being configured to transport a sheath flow of the liquid toward the focusing chamber, the one or more channels being defined between an outer surface of the transfer tube and an inner surface of the housing, the channels extending within the housing in a direction substantially aligned with the longitudinal axis of the transfer tube; and a focusing chamber configured to combine the particle flow and the sheath flow to generate a microfluidic stream, which is emitted from an aperture in the flow control device, the microfluidic stream comprising a laminar flow of the liquid from the sheath flow surrounding the liquid from the particle flow.

[0031] In one example, the housing includes a cavity for receiving the transfer tube, the cavity terminating in a focusing chamber, wherein the geometry of the cavity is different from the geometry of the focusing chamber at the terminal end; these geometries include one or more of the following: size, cross-sectional shape, longitudinal cone angle.

[0032] In one example, the end of the transfer tube is positioned within the lumen at a predetermined distance from the terminal end.

[0033] In one example, the end of the transfer tube is positioned within the focusing chamber at a predetermined distance from the terminal end.

[0034] In one example, the one or more channels extend in a direction parallel to the extension of the transfer tube within the housing, or parallel to the flow direction of the particle stream.

[0035] In one example, the one or more channels extend along the screw axis, wherein the longitudinal axis of the transfer tube within the housing or the flow direction of the particle stream is the axis of rotation of the screw axis.

[0036] In one example, the flow control device includes a plurality of channels for conveying a sheath flow of liquid between the transfer tube and the housing toward the focusing chamber, wherein a downstream outlet of each of the channels is regularly spaced around the transfer tube relative to a flow path of the particle stream.

[0037] In one example, the one or more channels are defined by corresponding protrusions extending along the transfer tube and / or the housing.

[0038] In one example, the protrusion extends from the transfer tube toward the inner surface of the housing or from the inner surface of the housing toward the transfer tube in a direction substantially perpendicular to the direction in which the channel defined by the protrusion extends.

[0039] In one example, the protrusion extends from the transfer tube to engage the inner surface of the housing, or extends from the inner surface of the housing to engage the transfer tube.

[0040] In one example, the interior lateral cross-sectional shape of the housing in which the transfer tube is positioned is different from the exterior lateral cross-sectional shape of the transfer tube at corresponding longitudinal locations, and wherein the one or more channels are defined by the difference in lateral cross-sectional shapes at different longitudinal locations.

[0041] In one example, the one or more channels: a. comprise a substantially semicircular or triangular cross-section; and / or b. extend from the sheath fluid inlet to the focusing chamber; and / or c. are configured to provide a single flow path or multiple flow paths for the sheath fluid between the sheath fluid inlet and the focusing chamber over at least a portion of the longitudinal extension of the transfer tube within the housing; and / or d. have a larger cross-sectional area at a downstream location than at an upstream location of the one or more channels.

[0042] In one example, the one or more channels are defined by two or more ridges that define a cross-sectional shape of the channel, and wherein the ridges are sized to smoothly engage the housing such that there is substantially zero volume between the ridges and the inner surface of the housing.

[0043] In one example, the one or more channels are angled relative to a longitudinal axis of the transfer tube.

[0044] In one example, the one or more channels are arranged at regular intervals around the entire outer cross-sectional circumference of the transfer tube and merge into a focusing chamber defined at least in part by the tapered transfer tube and the inverted tapered housing wall.

[0045] In one example, a longitudinal cross-section of the exterior of the transfer tube adjacent to the transfer tube outlet includes one or more of the following: conical; hemispherical; cylindrical; inclined on one lateral axis; inclined at two angles on one lateral axis; partially inclined on a second perpendicular lateral axis; a notch on the lateral axis.

[0046] In one example, an interior shape of the longitudinal cross-section of the housing corresponding to a longitudinal cross-section of the exterior of the transfer tube includes a constant or tapered lateral cross-sectional shape and / or size.

[0047] In one example, the transfer tube and the housing define a plurality of longitudinal lumen segments therebetween for conveying the sheath fluid, the longitudinal lumen segments having different cross-sectional shapes and / or sizes from one another.

[0048] In one example, the inner shape of the housing of any of the longitudinal segments or the outer shape of the transfer tube comprises one or more of: a longitudinal taper; a circular or elliptical or rectangular cross-sectional shape.

[0049] In one example, the interior shape of the housing has a first portion having a longitudinal cross-sectional shape arranged to engage the first portion of the transfer tube at a plurality of longitudinal locations to position the transfer tube outlet at a predetermined lateral location within the focusing chamber.

[0050] In one example, a first portion of the interior shape of the housing has a rectangular longitudinal cross-sectional shape and a first portion of the transfer tube has a rectangular longitudinal cross-sectional shape, and wherein the interior shape of the housing has a second cross-sectional shape with a reduced lateral width arranged to position the transfer tube outlet at a predetermined longitudinal position within the focusing chamber.

[0051] In one example, the transfer tube has a transfer tube inlet portion, a transfer tube tapered portion, and a transfer tube distal portion, wherein the transfer tube inlet portion has a transfer tube inlet cross-sectional size, the transfer tube distal portion has a transfer tube distal cross-sectional size that is smaller than the transfer tube inlet cross-sectional size, and wherein the interior shape of the housing has a cavity inlet portion, a cavity tapered portion, and a cavity distal portion, wherein the cavity inlet portion has a cavity inlet cross-sectional size, the cavity distal portion has a cavity distal portion size.

[0052] In one example, the delivery tube distal cross-sectional dimension and the lumen distal cross-sectional dimension are both uniform in the longitudinal direction.

[0053] In one example, the cross-sectional shape of any one of the delivery tube inlet portion, the delivery tube tapered portion, and the delivery tube distal portion is: circular, non-circular, elliptical, or rectangular.

[0054] In one example, the cross-sectional shape of any one of the cavity entrance portion, the cavity tapered portion, and the cavity distal portion is: circular, non-circular, elliptical, or rectangular.

[0055] In one example, the cross-sectional shape of any one of the delivery tube inlet portion, the delivery tube tapered portion, and the delivery tube distal portion is different from the cross-sectional shape of the corresponding lumen inlet portion, lumen tapered portion, and lumen distal portion.

[0056] In one example, the transmission tube taper angle of the transmission tube taper portion is the same as the cavity taper angle of the cavity taper portion.

[0057] In one example, a transfer tube tapering angle of the transfer tube tapering portion is smaller than a cavity tapering angle of the cavity tapering portion.

[0058] In one example, the transfer tube has a conical shape at the transfer tube outlet.

[0059] In one example, the transfer tube is tilted on an axis at the transfer tube outlet.

[0060] In one example, the transfer tube is partially inclined on the second vertical axis at the transfer tube outlet.

[0061] In one example, the transfer tube has a notch on the notch axis at the transfer tube outlet.

[0062] In one example, the lumen includes one or more of: a non-circular cross-sectional shape along at least a portion of its length; multiple portions having different cross-sectional dimensions.

[0063] In one example, the lumen includes multiple portions having different cross-sectional dimensions.

[0064] In one example, the focusing chamber includes a substantially constant lateral cross-sectional shape along the longitudinal axis.The lateral cross-sectional shape can have an aspect ratio of substantially 1:1.

[0065] In one example, the downstream second focusing chamber includes a longitudinally extending protrusion.

[0066] In one example, the height of the longitudinally extending protrusion tapers along its length.

[0067] In one example, the longitudinally extending protrusions form a V-shaped inner surface of the focusing chamber.

[0068] In one example, the V-shaped inner surface forms at least one side of the lateral rectangular cross-sectional shape of the focusing chamber.

[0069] In one example, the second V-shaped inner surface forms a second side of the lateral rectangular cross-sectional shape of the focusing chamber.

[0070] In one example, the focusing chamber tapers longitudinally such that the height of the focusing chamber varies in at least one axis perpendicular to the longitudinal axis.

[0071] In one example, the focusing chamber tapers longitudinally such that the focusing chamber varies in height and width in two axes perpendicular to the longitudinal axis.

[0072] In one example, the flow control device includes a second focusing chamber fluidly coupled between a first focusing chamber and an aperture, wherein the first focusing chamber has a geometry different from the geometry of the second focusing chamber; the geometries comprising one or more of: size, cross-sectional shape, longitudinal taper angle, longitudinally extending protrusion.

[0073] In one example, the second focusing chamber includes a lateral cross-sectional shape having a higher aspect ratio than the lateral cross-sectional shape of the first focusing chamber.

[0074] In one example, the second focusing chamber includes a longitudinally extending protrusion.

[0075] In one example, the height of the longitudinally extending protrusion tapers along its length.

[0076] In one example, the longitudinally extending protrusions form a V-shaped inner surface of the second focusing chamber.

[0077] In one example, the V-shaped inner surface forms one side of the lateral cross-sectional shape of the second focusing chamber.

[0078] In one example, the second V-shaped inner surface forms a second side of the lateral rectangular cross-sectional shape of the second focusing chamber.

[0079] In one example, the second focusing chamber extends longitudinally with a substantially constant cross-sectional dimension in the first lateral axis.

[0080] In one example, the second focusing chamber extends longitudinally with a substantially constant cross-sectional dimension in a second lateral axis that is perpendicular to the first lateral axis.

[0081] In one example, the focusing chamber tapers longitudinally such that the height of the focusing chamber varies in at least one axis perpendicular to the longitudinal axis.

[0082] In one example, the focusing chamber tapers longitudinally such that the focusing chamber varies in height and width in two axes perpendicular to the longitudinal axis.

[0083] In one example, the second focusing chamber is fluidically coupled to the well via a transfer microchannel.

[0084] In one example, the transport microchannel has a cross-sectional shape having a higher aspect ratio than the first focusing chamber and a smaller dimension along one lateral axis than the second focusing chamber.

[0085] In one example, the transport microchannel has one of the following cross-sectional shapes: circular, elliptical, triangular, square, rectangular.

[0086] In one example, the transport microchannel increases in cross-sectional area toward the aperture.

[0087] In one example, the transport microchannel is formed in an inverted conical shape toward the aperture.

[0088] In one example, the flow control device includes a confinement chamber fluidly coupled to the second focusing chamber, the confinement chamber tapering longitudinally from the second focusing chamber in at least one lateral axis.

[0089] In one example, the lateral axis comprises an axis of the second focusing chamber having the same dimension as its longest lateral axis.

[0090] In one example, the flow control device includes a coupling structure configured to secure the transfer tube and the housing together during use to form at least one of the one or more channels between the housing and the transfer tube and to position the transfer tube outlet at a predetermined longitudinal, lateral, and / or rotational position within the focusing chamber.

[0091] In one example, the housing has a first portion having a longitudinal cross-sectional shape arranged to engage the first portion of the transfer tube at a plurality of longitudinal locations to position the transfer tube outlet at predetermined lateral locations within the focusing chamber.

[0092] In one example, the engagement structure includes longitudinally extending ridges within the cavity of the housing, the ridges configured to position the transfer tube outlet according to a predetermined lateral relationship to the housing when the transfer tube outlet is received in the housing.

[0093] In one example, the ridges are formed with the housing and / or the transfer tube.

[0094] In one example, the engagement structure includes a rotational alignment feature arranged to secure the transfer tube outlet at a predetermined rotational angle relative to the housing.

[0095] In one example, the rotational alignment feature is a protrusion from one of the transfer tube or the housing and a corresponding recess in one of the housing or the transfer tube.

[0096] In one example, a flow control device includes a transfer tube having an inner lumen for conveying a particle stream of a liquid containing particles from a transfer tube inlet to a transfer tube outlet; the transfer tube extends longitudinally within a housing to a focusing chamber adjacent to the transfer tube outlet; the flow control device is configured to convey a sheath stream of liquid toward the focusing chamber; a focusing chamber configured to join the particle stream and the sheath stream so as to emit a microfluidic stream from a hole in the flow control device, the microfluidic stream comprising a laminar flow of liquid from the sheath stream surrounding liquid from the particle stream; wherein the focusing chamber comprises a lateral cross-sectional shape that is substantially constant along the longitudinal axis and has an aspect ratio of substantially 1:1.

[0097] In one example, the transfer tube and the housing together define one or more longitudinally extending channels for conveying the sheath fluid.

[0098] In one example, the one or more longitudinally extending channels are defined by corresponding longitudinally extending protrusions extending laterally from the transfer tube portion and / or the housing portion.

[0099] In one example, the internal lateral cross-sectional shape of the housing portion in which the transfer tube portion is positioned is different from the external lateral cross-sectional shape of the transfer tube portion at a corresponding longitudinal position, and wherein the one or more longitudinally extending channels are defined by the difference in lateral cross-sectional shapes at different longitudinal positions.

[0100] In one example, the longitudinally extending channel has a semicircular or triangular cross-section.

[0101] In one example, the longitudinally extending channel includes at least one of: a. a semicircular or triangular cross-section; b. extending from the sheath fluid inlet to the focusing chamber; c. a configuration within the housing that provides a unique flow path for the sheath fluid between the sheath fluid inlet and the focusing chamber over at least a portion of the longitudinal extension of the transfer tube.

[0102] In one example, the longitudinally extending channel is angled relative to the longitudinal axis of the transfer tube.

[0103] In one example, a longitudinal cross-section of the exterior of the transfer tube adjacent to the transfer tube outlet includes one or more of the following: conical; hemispherical; cylindrical; inclined on one lateral axis; inclined at two angles on one lateral axis; partially inclined on a second perpendicular lateral axis; a notch on the lateral axis.

[0104] In one example, an interior shape of the longitudinal cross-section of the housing corresponding to a longitudinal cross-section of the exterior of the transfer tube includes a constant or tapered lateral cross-sectional shape and / or size.

[0105] In one example, the transfer tube and the housing define a plurality of longitudinal lumen segments therebetween for conveying the sheath fluid, the longitudinal lumen segments having different cross-sectional shapes and / or sizes from one another.

[0106] In one example, the inner shape of the housing of any of the longitudinal segments or the outer shape of the transfer tube comprises one or more of: a longitudinal taper; a circular or elliptical or rectangular cross-sectional shape.

[0107] In one example, the lumen includes one or more of: a non-circular shape along at least a portion of its length; multiple portions having different cross-sectional dimensions.

[0108] In one example, the focusing chamber tapers longitudinally.

[0109] In one example, the flow control device includes a second focusing chamber fluidly coupled between the first focusing chamber and the aperture, the second focusing chamber including a lateral cross-sectional shape having a higher aspect ratio than the first focusing chamber.

[0110] In one example, the second focusing chamber extends longitudinally with a substantially constant cross-sectional dimension in the first lateral axis.

[0111] In one example, the second focusing chamber extends longitudinally with a substantially constant cross-sectional dimension in a second lateral axis that is perpendicular to the first lateral axis.

[0112] In one example, the second focusing chamber is fluidly coupled to the well by a transfer microchannel having a cross-sectional shape having a higher aspect ratio than the first focusing chamber and a smaller dimension along one lateral axis than the second focusing chamber.

[0113] In one example, the flow control device includes a confinement chamber fluidly coupled to the second focusing chamber, the confinement chamber tapering longitudinally from the second focusing chamber along a lateral axis.

[0114] In one example, a particle processing system comprises a flow control device as defined above, an interrogation device and / or a sorting device arranged to direct electromagnetic radiation at particles in a microfluidic stream emitted by the flow control device. The flow control device is arranged to orient the asymmetric particles so that the electromagnetic radiation is directed at predetermined facets of the asymmetric particles.

[0115] In one example, the transfer tube and housing form together as a single unified unit of material or are formed separately as respective units of material.

[0116] In one example, the flow control device includes a coupling structure configured to secure the transfer tube and the housing together during use to form at least one longitudinally extending channel between the housing and the transfer tube and to position the transfer tube outlet at a predetermined longitudinal, lateral, and / or rotational position within the focusing chamber.

[0117] In one example, the cavity of the housing has a first portion having a longitudinal cross-sectional shape arranged to engage the first portion of the transfer tube at a plurality of longitudinal locations to position the transfer tube outlet at a predetermined lateral location within the focusing chamber.

[0118] In one example, the engagement structure includes longitudinally extending ridges within the cavity of the housing, the ridges configured to position the transfer tube outlet according to a predetermined lateral relationship to the housing when the transfer tube outlet is received in the housing.

[0119] In one example, the engagement structure includes a rotational alignment feature arranged to secure the transfer tube outlet at a predetermined rotational angle relative to the housing.

[0120] In one example, a method of processing a particle stream emitted from a transfer tube includes receiving a first fluid stream comprising one or more particles and passing the stream through at least one of: a flow control device as defined above or herein; a particle processing system as defined above or herein; a housing as defined above or herein; and a transfer tube as defined above or herein.

[0121] In one example, the processing method includes one or more of: inspecting the particle; displacing the particle into a different flow path within the processing channel; orienting the particle.

[0122] In one example, the method includes exposing the particles to one or more radiation sources.

[0123] In one example, the method includes directing electromagnetic radiation at the particle to expose the particle to the electromagnetic radiation to displace the particle by a predetermined amount and / or orient the particle to a predetermined orientation and / or ablate the particle.

[0124] In one example, the method includes inspecting particles and further includes the steps of determining one or more characteristics of the particles within the stream to produce a particle signature; and selecting a subpopulation of particles based on the particle signature.

[0125] In one example, the particles comprise cells or sperm cells.

[0126] In one example, the step of determining one or more characteristics includes detecting fluorescent light emitted from the particles.

[0127] In one example, the step of selecting a subpopulation of particles includes sorting the particles using electromagnetic sorting.

[0128] In one example, electromagnetic sorting methods include pulsed or continuous energy flow to displace charged or uncharged particles.

[0129] In one example, the sorting method includes a radiation source configured to direct radiation onto the particles to achieve at least one of a force and a torque on each particle so as to cause each particle to be at least one of displaced and oriented relative to an axis defined by the direction of fluid flow along the microfluidic channel.

[0130] In one example, a flow control device includes a housing having a particle flow outlet fluidly connected to a cavity for receiving a transfer tube, the transfer tube having an inner cavity for conveying a source fluid containing particles from a transfer tube inlet to a transfer tube outlet; a coupling structure arranged to secure the transfer tube in the cavity to form a passage between the housing and the transfer tube; and a focusing chamber fluidly connected to the particle flow outlet, the passage for conveying a sheath fluid to the focusing chamber; the coupling structure being arranged to position the transfer tube outlet at a predetermined longitudinal, lateral, or rotational position within the focusing chamber.

[0131] In one example, the cavity of the housing has a first portion having a longitudinal cross-sectional shape arranged to engage the first portion of the transfer tube at a plurality of longitudinal locations to position the transfer tube outlet at a predetermined lateral location within the focusing chamber.

[0132] In one example, the first portion of the cavity has a rectangular longitudinal cross-sectional shape and the first portion of the transfer tube has a rectangular longitudinal cross-sectional shape, and wherein the cavity has a second cross-sectional shape with a reduced transverse width arranged to position the transfer tube outlet at a predetermined longitudinal position within the focusing chamber.

[0133] In one example, the engagement structure includes longitudinally extending ridges on at least one of the transfer tubes or within the cavity of the housing, the ridges being configured to position the transfer tube outlet according to a predetermined lateral relationship to the housing when the transfer tube outlet is received in the housing.

[0134] In one example, the ridges are configured to form channels between the housing and the transfer tube for conveying sheath fluid to the focusing chamber.

[0135] In one example, the channel has a semicircular or triangular cross-section.

[0136] In one example, the ridges are angled relative to the longitudinal axis of the transfer tube.

[0137] In one example, the engagement structure includes a rotational alignment feature arranged to secure the transfer tube outlet at a predetermined rotational angle relative to the housing.

[0138] In one example, the rotational alignment feature is a protrusion from one of the transfer tube or the housing and a corresponding recess in one of the housing or the transfer tube.

[0139] In one example, the transfer tube has a conical shape at the transfer tube outlet.

[0140] In one example, the transfer tube is tilted on an axis at the transfer tube outlet.

[0141] In one example, the transfer tube is partially inclined on the second vertical axis at the transfer tube outlet.

[0142] In one example, the transfer tube has a notch at the transfer tube outlet on the notch axis.

[0143] In one example, the lumen has a non-circular shape along at least a portion of its length.

[0144] In one example, the lumen includes multiple portions having different cross-sectional dimensions.

[0145] In one example, the transfer tube has a transfer tube inlet portion, a transfer tube tapered portion, and a transfer tube distal portion, wherein the transfer tube inlet portion has a transfer tube inlet cross-sectional size, the transfer tube distal portion has a transfer tube distal cross-sectional size that is smaller than the transfer tube inlet cross-sectional size, and wherein the cavity of the housing has a cavity inlet portion, a cavity tapered portion, and a cavity distal portion, wherein the cavity inlet portion has a cavity inlet cross-sectional size, the cavity distal portion has a cavity distal portion size.

[0146] In one example, the delivery tube distal cross-sectional dimension and the lumen distal cross-sectional dimension are both uniform in the longitudinal direction.

[0147] In one example, the transmission tube taper angle of the transmission tube taper portion is the same as the cavity taper angle of the cavity taper portion.

[0148] In one example, a transfer tube tapering angle of the transfer tube tapering portion is smaller than a cavity tapering angle of the cavity tapering portion.

[0149] In one example, the cross-sectional shape of any one of the delivery tube inlet portion, the delivery tube tapered portion, and the delivery tube distal portion is: circular, non-circular, elliptical, or rectangular.

[0150] In one example, the cross-sectional shape of any one of the cavity entrance portion, the cavity tapered portion, and the cavity distal portion is: circular, non-circular, elliptical, or rectangular.

[0151] In one example, the cross-sectional shape of any one of the delivery tube inlet portion, the delivery tube tapered portion, and the delivery tube distal portion is different from the cross-sectional shape of the corresponding lumen inlet portion, lumen tapered portion, and lumen distal portion.

[0152] In one example, a flow control device as defined above or herein, a merging flow microchannel connected to a particle flow outlet, and a downstream module are arranged to direct electromagnetic radiation at particles transported by the merging flow microchannel.

[0153] In one example, the flow control device is arranged to orient the asymmetric particle such that the electromagnetic radiation is directed at a predetermined facet of the asymmetric particle.

[0154] In one example, the transfer tube and the direction of the electromagnetic radiation are rotationally aligned about a longitudinal axis of the particle flow.

[0155] In one example, the downstream module includes one or more of the following: a particle inspection module; a particle displacement module arranged to displace particles into different flow paths within the merging flow microchannel; and a particle orientation module arranged to expose particles to one or more radiation sources to orient the particles.

[0156] In one example, a housing for a flow control device includes: a cavity in fluid communication with a particle flow outlet, the cavity being in fluid communication with and configured to receive a transfer tube for conveying a source fluid containing particles to the transfer tube outlet; a coupling structure arranged to, in use, secure the transfer tube in the cavity to form a passage between the housing and the transfer tube and to form a focusing chamber in fluid communication with the particle flow outlet, the passage for conveying a sheath fluid to the focusing chamber; the coupling structure being arranged to position the transfer tube outlet at a predetermined longitudinal, lateral, or rotational position within the focusing chamber.

[0157] In one example, the cavity has a first portion having a longitudinal cross-sectional shape arranged to engage the first portion of the transfer tube at a plurality of longitudinal locations to position the transfer tube outlet at a predetermined lateral location within the focusing chamber.

[0158] In one example, the first portion of the cavity has a rectangular longitudinal cross-sectional shape configured to engage with a first portion of a transfer tube having a rectangular longitudinal cross-sectional shape, and wherein the cavity has a second cross-sectional shape with a reduced transverse width arranged to position the transfer tube outlet at a predetermined longitudinal position within the focusing chamber.

[0159] In one example, the engagement structure includes longitudinally extending ridges within the cavity of the housing, the ridges configured to position the transfer tube outlet according to a predetermined lateral relationship to the housing when the transfer tube outlet is received in the housing.

[0160] In one example, the ridges are configured to form channels between the housing and the transfer tube for conveying sheath fluid to the focusing chamber.

[0161] In one example, the engagement structure includes a rotational alignment feature arranged to secure the transfer tube outlet at a predetermined rotational angle relative to the housing.

[0162] In one example, a transfer tube for a flow control device for controlling fluid flow associated with transporting particles is provided. The transfer tube includes: an inner lumen for transporting a source fluid containing particles from a transfer tube inlet to a transfer tube outlet; a coupling structure arranged to secure the transfer tube within the lumen of a housing, forming a passage between the housing and the transfer tube, and a focusing chamber in fluid communication with the particle flow outlet, the passage being for transporting a sheath fluid to the focusing chamber; the coupling structure being arranged to position the transfer tube outlet at a predetermined longitudinal, lateral, or rotational position within the focusing chamber.

[0163] In one example, the transfer tube includes a first portion having a longitudinal cross-sectional shape arranged to engage a first portion of the cavity of the housing at a plurality of longitudinal locations to position the transfer tube outlet at predetermined lateral locations within the focusing chamber.

[0164] In one example, a first portion of the transfer tube has a rectangular longitudinal cross-sectional shape configured to engage a second portion of the cavity having a reduced transverse width to position the transfer tube outlet at a predetermined longitudinal position within the focusing chamber.

[0165] In one example, the engagement structure includes longitudinally extending ridges on the transfer tube, the ridges configured to position the transfer tube outlet according to a predetermined lateral relationship to the housing when the transfer tube outlet is received in the housing.

[0166] In one example, the ridges are configured to form channels between the housing and the transfer tube for conveying sheath fluid to the focusing chamber.

[0167] In one example, a transfer tube for use in a flow control device for controlling fluid flow associated with transporting particles is provided. The transfer tube includes an inner lumen for transporting a source fluid containing particles from a transfer tube inlet to a transfer tube outlet; the transfer tube is tilted about one axis at the transfer tube outlet and partially tilted about a second, perpendicular axis and / or has a notch in the notch axis.

[0168] In one example, the transfer tube is simultaneously partially inclined on a second vertical axis and has a notch on a notch axis at the transfer tube outlet, wherein the notch axis is identical to the second vertical axis.

[0169] In one example, a flow control device for controlling fluid flow associated with transporting particles is provided. The flow control device includes a housing having a particle flow outlet in fluid communication with a lumen for receiving a transfer tube, the transfer tube having an inner lumen for transporting a source fluid containing particles from a transfer tube inlet to a transfer tube outlet; wherein the transfer tube has a transfer tube inlet portion, a transfer tube tapered portion, and a transfer tube distal portion, the transfer tube inlet portion having a transfer tube inlet cross-sectional dimension, the transfer tube distal portion having a transfer tube distal cross-sectional dimension that is smaller than the transfer tube inlet cross-sectional dimension, and wherein the lumen of the housing has a lumen inlet portion, a lumen tapered portion, and a lumen distal portion, the lumen inlet portion having a lumen inlet cross-sectional dimension, and the lumen distal portion having a lumen distal portion dimension.

[0170] In one example, the delivery tube distal cross-sectional dimension and the lumen distal cross-sectional dimension are both uniform in the longitudinal direction.

[0171] In one example, the transmission tube taper angle of the transmission tube taper portion is the same as the cavity taper angle of the cavity taper portion.

[0172] In one example, a transfer tube tapering angle of the transfer tube tapering portion is smaller than a cavity tapering angle of the cavity tapering portion.

[0173] In one example, the cross-sectional shape of any one of the delivery tube inlet portion, the delivery tube tapered portion, and the delivery tube distal portion is: circular, non-circular, elliptical, or rectangular.

[0174] In one example, the cross-sectional shape of any one of the cavity entrance portion, the cavity tapered portion, and the cavity distal portion is: circular, non-circular, elliptical, or rectangular.

[0175] In one example, the cross-sectional shape of any one of the delivery tube inlet portion, the delivery tube tapered portion, and the delivery tube distal portion is different from the cross-sectional shape of the corresponding lumen inlet portion, lumen tapered portion, and lumen distal portion.

[0176] In one example, the lumen has a non-circular shape along at least a portion of its length.

[0177] In one example, the lumen includes multiple portions having different cross-sectional dimensions.

[0178] Aspects of the invention may also be broadly said to include the parts, elements and features referred to or indicated in the specification of this application, either individually or collectively, in any or all combinations of two or more of said parts, elements or features, and where specific integers are referred to herein that have known equivalents in the art to which the invention relates, such known equivalents are deemed to be incorporated herein as if individually set forth. 4. Description of the Figures

[0179] Examples will now be described, by way of example only, with reference to the accompanying drawings, in which:

[0180] Figure 1 shows a perspective view of a flow control device according to one example;

[0181] Figure 2 Shown with Figure 1 side and end views of a system of flow control devices;

[0182] Figure 3 shows a longitudinal cross-section of a flow control device according to one example;

[0183] Figure 4A Shown by Figure 3 A cross-sectional view of a flow control device taken along section line AA;

[0184] Figures 4B to 4I Shown by Figure 3 An alternative cross section of the flow control device along the section line AA;

[0185] Figure 5A and Figure 5B Shown Figure 3 A detailed longitudinal cross-section of the control chamber area of ​​the flow control device;

[0186] Figure 6A shows a side view and an end view of a transfer tube according to one example;

[0187] Figure 6B shows a longitudinal cross-section of a flow control device according to one example;

[0188] Figure 6BA and Figure 6BB Shown according to an example Figure 6B A side cross-sectional view of a flow control device;

[0189] Figure 6C shows a longitudinal cross-section of a flow control device according to one example;

[0190] Figure 6D and Figure 6E shows a longitudinal cross-section of a flow control device according to one example;

[0191] Figure 6F Shown for Figure 6B Examples of orientation efficiency and resolution measurements at different flow rates;

[0192] Figure 7A 、 Figure 7B and Figure 7C shows a side view of a transfer tube according to other examples;

[0193] Figure 8 An end view and longitudinal sections through different axes and a perspective view of a conical end of a transfer tube according to one example are shown;

[0194] Figure 9 shows an end view and longitudinal cross-section through different axes and a perspective view of the end of a transfer tube tilted on one axis according to one example;

[0195] Figure 10 shows end views and longitudinal sections through different ends of a transfer tube tilted on two axes according to one example;

[0196] Figure 11 shows an end view and longitudinal cross-sections through different axes and a perspective view of a distal end of a transfer tube according to one example, the distal end of the transfer tube being inclined on one axis, partially inclined on a second perpendicular axis and including a notch passing through the second axis;

[0197] Figure 12 shows an end view of a transfer tube including a notch through one axis and a longitudinal cross-section and perspective view through a different axis according to one example;

[0198] Figure 12A shows a longitudinal cross-section through an axis of a plurality of other tapered ends of a transfer tube according to some examples;

[0199] Figure 13 shows cross-sectional views of flow control devices having different combinations of housing cavity and transfer tube geometries according to some examples;

[0200] Figure 14 illustrates experimental results of particle flow confinement at a particle velocity of 200 mm / s according to one example;

[0201] Figure 15 illustrates experimental results of particle flow confinement at a particle velocity of 100 mm / s according to one example;

[0202] Figure 16 illustrates experimental results of particle flow confinement at a particle velocity of 50 mm / s according to one example;

[0203] Figure 17 illustrates experimental results of particle flow confinement at a particle velocity of 500 mm / s according to one example;

[0204] Figure 18 An experimental result of particle flow positions under different particle velocity conditions according to an example is illustrated;

[0205] Figure 19illustrates experimental results of particle flow restriction results using different transfer tube ends according to one example;

[0206] Figure 20 An experimental result of the particle flow position on the short axis under different particle velocity conditions according to one example is illustrated;

[0207] Figure 21 Experimental results of particle flow positions of an elliptical transfer tube outlet cross section under different particle velocity conditions according to one example are illustrated.

[0208] Figure 22 A method of controlling a flow associated with particles according to one example is illustrated.

[0209] Figure 23A and Figure 23B Summary plots of experimental orientation efficiency (y-axis) versus PODT tip position on the z-axis (bottom x-axis, [mm]) and maximum cell velocity (top x-axis, [m / s]), respectively, are shown.

[0210] Figure 24 It illustrates how velocity components induce drag and can lead to preferred orientation of aspherical particles in the focusing device geometry described herein.

[0211] Figure 25A shows a simulated v x / v y (y-axis) versus maximum cell velocity (top x-axis, [m / s]) and PODT tip position on the z-axis (bottom x-axis, [m]).

[0212] Figure 25B shows a simulated v xx / v yy (y-axis) versus maximum cell velocity (top x-axis, [m / s]) and PODT tip position on the z-axis (bottom x-axis, [m]).

[0213] Figure 26A 、 Figure 26B and Figure 26C The effects of misaligned or tilted tips are illustrated;

[0214] Figure 27a and Figure 27b illustrates an example longitudinal position of the transfer tube in the second focusing chamber;

[0215] Figure 28a -e illustrates an example configuration of a second focusing chamber; and

[0216] Figure 29 A cross section of a hole according to one example is illustrated. 5. Specific implementation methods

[0217] As used herein, the term "comprising" means "consisting at least in part of." When interpreting each statement in this specification that includes the term "comprising," features other than the one or those features beginning with the term may also be present. Related terms such as "including" are to be interpreted in the same manner.

[0218] As used herein, the term "about" means a reasonable amount of deviation of the modified term such that the end result is not significantly changed. For example, when applied to a value, the term should be interpreted as including a deviation of + / - 5% of the value.

[0219] All definitions, as defined and used herein, should be understood to control over dictionary definitions, definitions in documents incorporated by reference, and / or ordinary meanings of the defined terms.

[0220] Unless explicitly indicated to the contrary, the indefinite articles "a" and "an" as used herein in the specification and claims should be understood to mean "at least one."

[0221] The terms "can" and "may" are used interchangeably in this disclosure and indicate that a referenced element, component, structure, function, functionality, objective, advantage, operation, step, process, apparatus, system, equipment, result, or clarification has the capability of being used, included, or produced, or otherwise represents the proposition indicated in the statement in which the term is used (or referred to) for the specific example.

[0222] As used herein in the specification and claims, the phrase "and / or" should be understood to mean "either or both" of the elements so combined, i.e., elements that are present in combination in some cases and separately in other cases. Multiple elements listed with "and / or" should be interpreted in the same manner, i.e., "one or more" of the elements so combined. In addition to the elements specifically identified by the "and / or" clause, other elements may optionally be present, whether related or unrelated to those specifically identified. Thus, as a non-limiting example, when used in conjunction with open language such as "comprises," "comprising," a reference to "A and / or B" may refer to just A (optionally including elements other than B) in one example; to just B (optionally including elements other than A) in another example; to both A and B (optionally including other elements) in yet another example; and so on.

[0223] As used herein in the specification and claims, the phrase "at least one" with respect to a list of one or more elements should be understood to mean at least one element selected from any one or more elements in the list of elements, but does not necessarily include at least one of each element specifically listed within the list of elements, and does not exclude any combination of elements in the list of elements. This definition also allows for the optional presence of elements other than the elements specifically identified within the list of elements to which the phrase "at least one" refers, whether related or unrelated to those specifically identified elements. 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") may refer to at least one (optionally including more than one) A in one example, without B (and optionally including elements other than B); to at least one (optionally including more than one) B in another example, without A (and optionally including elements other than A); to at least one (optionally including more than one) A and at least one (optionally including more than one) B (and optionally including other elements); and so on.

[0224] Reference to a numerical range disclosed herein (e.g., 1 to 10) is also intended to include reference 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), as well as any range of rational numbers within that range (e.g., 2 to 8, 1.5 to 5.5, and 3.1 to 4.7), and thus, all subranges of all ranges explicitly disclosed herein are hereby explicitly disclosed. These are merely examples of what is specifically intended, and all possible combinations of numerical values ​​between the lowest and highest values ​​recited are considered to be expressly stated in this application in a similar manner.

[0225] Whenever a range (e.g., a size range, a time range, or a composition range) is given in the specification, all intermediate ranges and subranges, as well as all individual values ​​included in the given range, are intended to be included in the present disclosure. In the present disclosure and claims, "and / or" means additionally or alternatively. In addition, any use of a term in the singular also encompasses the plural form.

[0226] Specific details are set forth below, such as specific examples or examples for explanation and not limitation purposes. Those skilled in the art will appreciate that, in addition to these specific details, other examples may also be adopted. In some cases, detailed descriptions of well-known methods, nodes, interfaces, circuits and devices are omitted to avoid unnecessary details from blurring the description. Those skilled in the art will appreciate that the functions described may be implemented in one or more nodes using hardware circuits (e.g., analog and / or discrete logic gates, ASICs, PLAs, etc., interconnected to perform specialized functions) and / or using software programs and data in conjunction with one or more digital microprocessors or general-purpose computers. Nodes communicating using air interfaces also have suitable radio communication circuits. In addition, where appropriate, the technology may additionally be considered to be fully embodied in any form of computer-readable memory, such as solid-state memory, magnetic disks or optical disks comprising a set of suitable computer instructions that will cause a processor to perform the technology described herein.

[0227] Hardware implementations may include or encompass, 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 application specific integrated circuits (ASICs) and / or field programmable gate arrays (FPGAs), and, where appropriate, state machines capable of performing these functions. Memory may be used to store temporary variables, maintain and transfer data between processes, non-volatile configuration settings, standard message formats, etc. Any suitable form of volatile memory and non-volatile storage may be employed, including random access memory (RAM) implemented as a metal oxide semiconductor (MOS) or integrated circuit (IC), as well as storage implemented as a hard drive and flash memory.

[0228] Some or all of the described devices or functionalities may be instantiated in a cloud environment such as Docker, Kubenetes, or Spark. The cloud functionality may be instantiated at the network edge, device edge, on-premises, or in a remote server coupled via a network such as 4G or 5G. Alternatively, the functionality may be implemented in dedicated hardware.

[0229] As used herein, the term "restriction" refers to the restriction of the cross-sectional shape and size of the particle stream in the sample stream. For example, the diameter of a circular cross-section of the stream can be restricted, or the dimensions of the major and minor axes of an elliptical cross-section stream can be restricted, which can result in a single narrow trajectory in which the deviation of any polar axis of the particle from a defined central longitudinal axis of the stream is minimized. This constriction of the flow also has the effect of reducing the average distance of individual particles from the nominal central flow vector along the axis of the flow. It is generally desirable to increase the restriction to result in accurate focusing of the interrogation beam and the sorting beam used for downstream interrogation and sorting. If the particles are not accurately confined, the measurement may be less accurate, which can negatively impact the yield of selected cells and the speed with which cells can be accurately detected.

[0230] The term "orientation" of asymmetric particles (including cells) refers to the predominant angle of the faces of a representative sample of the particles relative to an axis substantially perpendicular to the axis of flow of the particles. In the absence of any features to impart an orienting torque on the particles, it is expected that the orientations of the faces will be randomly distributed and face any angle of about 360°. A sample of cells to which an orienting torque has been imparted by an orienting feature will have a non-random angular orientation that preferentially directs the faces of the particles at particular angles such that a predominant angle can be determined or observed.

[0231] The "orientation efficiency" of a method or apparatus for orienting asymmetric particles corresponds to the percentage or proportion of particles in a sample of particles that are oriented at a predominant angle within predetermined tolerances.

[0232] "Cells" and "X cells" are referred to herein as examples of specific types of particles that may be desired to be retained within a microfluidic sorting arrangement. Where the term "cell" is used herein, it may be replaced with the term "particle," and there is no requirement that the cell / particle be a living cell. One skilled in the art will readily appreciate that reference to an X cell is intended to refer to any other cell or particle having characteristics suitable for interrogation and sorting according to the present invention. Specifically, an X cell may be replaced herein with any type of particle or cell, including substantially symmetrical and asymmetrical cells, neurons, red blood cells, labeled cells, viruses, or microbial populations, as would be known to one skilled in the art.

[0233] As used herein, the term "microfluidic stream" refers to a liquid stream having at least one geometrically constrained dimension at which surface forces dominate volume forces. In one example, this can include a liquid stream having a sub-millimeter diameter or other cross-sectional dimension. In one example, a microfluidic stream can be a continuous phase flow of a liquid, such as an unbroken stream of one or more aqueous solutions. This can be a laminar flow having a particle flow comprising particles and a sheath flow surrounding the particle flow. A microfluidic stream can alternatively or additionally comprise a dispersed flow of droplets. A microfluidic stream can be associated with one or more performance metrics, such as flow rate, cross-sectional diameter and / or size, distance to droplet formation.

[0234] As used herein, the term "flow environment" refers to an environment through which a microfluidic stream can flow. One example includes a microchannel that may include a material, such as glass, forming an elongated lumen or path through which the microfluidic stream flows. The path may be completely surrounded by material between each end of the path; or the path may have at least one boundary that exposes the microfluidic stream to a fluid environment, wherein the material forming the substrate is in contact with other boundaries of the microfluidic stream. 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 stream may not be in contact with a material substrate, but rather 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.

[0235] Figure 1 1 is a perspective view of a flow control device according to an example. The flow control device 100 includes a transfer tube 130 mounted within a housing 105 during use. The figure shows the two components 105, 130 coupled together for use and separated. The two components 105, 130 can be individually replaceable with each other and can be consumable parts of a larger system. Alternatively, the components can be formed as part of a single integral structure. The housing 105 includes a cavity 110, and the transfer tube 130 is located in the cavity and can be coupled to the processing microchannel 180 at a hole from the flow control device during use. The transfer tube 130 includes an inner cavity 145 extending from an inlet end as shown to a distal end within the cavity 110.

[0236] The housing 105 and the transfer tube 130 can be made of a variety of materials, including: plastics, polymers, metals, glass, ceramics, and composite materials. In one example, the flow control device includes both a transfer microchannel and a focusing and / or confinement chamber. At least one of the flow control device and the microfluidic channel can be formed from any one or more of a polymer, glass, ceramic, or other solid substrate, or can be a preformed component, such as PTFE tubing or a glass capillary. In some examples, a variety of materials and methods can be used to form any of the above-mentioned components of the present disclosure. For example, the flow focusing device, tubes, channels, and chambers of the present disclosure can be formed from solid materials, wherein the channels can be formed via micromachining, film deposition processes (such as spin coating and chemical vapor deposition), laser manufacturing, photolithography, etching methods including wet chemical or plasma processes, and the like. In one example, at least a portion of the microfluidic channel or flow control device is formed from silicon by etching a feature structure in a silicon chip. Technologies for accurately and efficiently manufacturing the various fluid systems and devices of the present disclosure from silicon are known. In another example, the various components of the system and equipment of the present disclosure can be formed by polymers, for example, elastomeric polymers such as polydimethylsiloxane (PDMS), polytetrafluoroethylene (PTFE) and the like. In another example, the channel of the present disclosure can be formed by polymers, glass, ceramics or other solid substrates, or can be preformed components, such as PTFE tubing or glass capillaries. 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) or by a transparent or at least partially transparent material (such as glass or a transparent polymer) for observing and / or controlling the flow process. The components can be coated so that the desired chemical function is exposed to the fluid contacting the inner channel wall. For example, the components can be manufactured to have an inner channel wall coated with another material. The materials for manufacturing the various components of the system and equipment of the present disclosure (for example, the material for coating the inner wall of the fluid channel) can be desirably selected from those materials that will not adversely affect the fluid flowing through the fluid system or will not be affected by the fluid flowing through the fluid system (for example, a chemically inert material in the presence of the fluid to be used in the device). In one embodiment, the various components of the present disclosure are made of polymer and / or flexible and / or elastomeric materials, and can be easily formed by hardenable fluids, thereby being convenient to manufacture via molding (for example, replication molding, injection molding, casting molding, etc.). Hardenable fluids can basically be any fluid that can be induced to solidify or spontaneously solidify into a solid that can accommodate and / or convey the fluid envisioned for use in or with the described microfluidic system. In one embodiment, hardenable fluids include polymer liquids or liquid polymer precursors (i.e., " prepolymers "). Suitable polymer liquids can include, for example, thermoplastic polymers, thermosetting polymers, or mixtures of these polymers that are heated to a point higher than their melting point.As another example, a suitable polymer liquid may include a solution of one or more polymers in a suitable solvent that forms a solid polymer material when the solvent is removed, for example, by evaporation. Such polymer materials that can be solidified from, for example, 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 from the general class 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 cheap, readily available, and can be cured from a pre-polymerized liquid via heat curing. For example, PDMS can typically be cured by exposing the pre-polymerized 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. In addition, silicone polymers such as PDMS can be elastomeric and can therefore be used to form very small feature structures with relatively high aspect ratios, which is necessary in certain embodiments of the present disclosure. In this regard, a flexible (e.g., elastomeric) mold or master may be advantageous. In further examples, components of the present invention may be formed from recycled polymers or biodegradable polymers such as polylactic acid (PLA) and polyhydroxyalkanoates (PHA).

[0237] In some examples, materials are pre- or post-treated to increase smoothness. Surprisingly, this has been found to significantly improve some of the material's flow characteristics, such as reducing turbulence or drag. For example, stainless steel can be electropolished to improve smoothness, and 3D-printed plastics can be sanded, surface-melted, and / or coated with a smooth surface material.

[0238] The transfer tube 130 includes a plurality of ridges or protrusions that extend longitudinally and, in use, engage the inner wall of the lumen. In practice, this secures the transfer tube within the housing, such that the lumen 145 is securely and accurately positioned within the housing at the distal end of the transfer tube. In some examples, this feature also influences fluid flow characteristics.

[0239] In one example, accurate positioning enables the particle flow from the transmission tube to be carefully controlled within the microfluidic material stream, which is emitted into the processing microchannel 180 for downstream processing. For example, the flow of particles in the microfluidic material stream can benefit from the improved characteristics selected from the group consisting of: enhanced positional stability, reduced mechanical stress on the particles, reduced agglomeration or more uniform distribution of particles, increased or more accurate restrictions and / or more effective particle orientation. The microfluidic material stream includes a laminar combination of a particle flow from the inside of the transmission tube and a sheath flow from around the outside of the transmission tube, wherein the sheath flow surrounds the particle flow in the microfluidic material stream. The stable laminar flow (wherein the particles are expected to be arranged) generated by the flow control device enables more effective downstream processing, as described in more detail below. The microfluidic material stream can be emitted into the processing microchannel 180 as shown in the figure, or emitted into another flow environment such as a gas or liquid. The microfluidic material stream is emitted from the hole of the transmission microchannel in the flow control device. The other end of the transport microchannel is coupled to one or more focusing chambers within the flow control device, which facilitate desired alignment of particles within the stable laminar flow of the microfluidic stream. As described herein, the focusing chambers may include upstream focusing chambers, downstream focusing chambers, or downstream confinement chambers. In some examples, downstream processing may include detecting particle (e.g., sperm) characteristics, particle orientation, particle displacement, and / or sorting the particles based on the characteristics.

[0240] An advantage of some examples is the controlled and stable confinement of particles within a narrow stream emitted from a transfer tube. In certain examples, this confined stream is passed into a process microchannel 180, and the uninterrupted fluid stream undergoes a sorting step to sort the particles based on certain detected particle characteristics. The controlled confinement achieved can improve sorting efficiency.

[0241] In certain examples, the process microchannel 180 may have a width in the range of, for example, 10 microns to 500 microns or 100 microns to 400 microns, and a depth in the range of 5 microns to 250 microns. In some examples, the width and depth are the same, and the cross-section of the process microchannel can be of any shape. Particularly preferred shapes for the cross-sectional shape are circular or square. The dimensions of the process microchannel support laminar flow with minimal turbulence. In some examples, the process microchannel has a planar form, wherein the length and width in the plane are greater than the depth transverse to the plane. In an alternative example, the depth may be greater than the length and / or width of the microchannel. In an alternative example, the process microchannel may include one or more capillaries and may be characterized by curved sections and angles.

[0242] In some alternative examples, the microfluidic stream from the flow control device 100 is ejected into free space or another flow environment rather than into the processing microchannel 180. Particles within the microfluidic stream can then be interrogated and sorted without the processing microchannel 180. This can provide several advantages, including faster flow rates and reduced diffraction of the interrogation and sorting beams. This, in turn, can lead to greater accuracy in focusing these beams and a reduction in their power, thereby reducing their impact on desired particles.

[0243] In use, the sheath flow of the liquid passes through the channels formed between the ridges 135 and the inner surface of the cavity 110. Alternatively or additionally, these ridges can be formed on the inner surface of the cavity to create channels when engaged with the transfer tube. These channels stabilize the sheath flow, for example, by reducing turbulence that may be present in the introduced fluid and / or by homogenizing the flow. Specifically, turbulence is introduced by having one or more sheath flow channels that enter the flow control device orthogonally with respect to the longitudinal or Z-axis of the flow of the particle flow or sample stream / fluid.

[0244] These channels provide 360-degree confinement and focusing of the particle or sample stream, eliminating the need for multiple sheath streams crossing in different directions. This simplifies the chip architecture for flow control devices.

[0245] A particle flow of a liquid containing particles (preferably, asymmetric particles) passes through lumen 145. Flow control device 100 is configured so that a combined particle flow and sheath flow having the desired characteristics described above appear as a microfluidic stream in the processing microchannel, wherein the coaxially arranged central particle flow and surrounding sheath flow stabilize into a single laminar flow. The particle flow and sheath flow can be independently formed to control a variety of particle flow characteristics and / or combined flow characteristics.

[0246] In one example, asymmetric or non-spherical particles are oriented substantially in a common axis or plane within the microfluidic stream, and / or the particles are aggregated or confined so that the particles flow more evenly. This enables the particles to be more easily studied and / or manipulated downstream, for example, in a processing microchannel or free space flow environment. Some examples also allow the orientation and confinement characteristics of the particles to be maintained over a wide range of flow rates. Other particle and / or flow characteristics can be adjusted additionally or alternatively using a flow control device.

[0247] As will be described in more detail below, the example provides for more accurate lateral and longitudinal positioning of the transfer tube 130 within the housing 105, resulting in more effective focusing of particles within the microfluidic stream. This also results in enhanced positional stability of the particles within the microfluidic stream, which improves downstream interrogation and sorting performance.

[0248] The example also reduces particle orientation changes or shear forces and flow turbulence, thereby reducing mechanical stress on the particles. This in turn can improve the motility of biological cells such as sperm cells.

[0249] The examples also distribute particles more evenly in the microfluidic stream, resulting in less agglomeration and, therefore, better downstream interrogation and sorting performance.

[0250] The example provides improved and more accurate confinement by reducing the displacement of particles from the axis of the microfluidic stream. This also leads to better downstream interrogation and sorting performance.

[0251] Examples provide improved orientation efficiency, increasing the proportion of particles that are optimally oriented for downstream interrogation and sorting processes.

[0252] Figure 2 A system 200 is illustrated according to one example and includes a flow control device 205, 230 having an aperture 235 coupled to a process microchannel 280. A side view of the system 200 is shown in the XY plane at the upper left. The flow control device 205 controls separate sheath and particle flows, which are combined to generate a microfluidic stream having desired characteristics. As described in more detail below, the microfluidic stream is emitted into the process microchannel 280. In one example, the process microchannel terminates prior to downstream interrogation or sorting. In another example, the microfluidic stream is emitted from the aperture 380 of the flow control device 300 into a different flow environment, which may include a free space volume with a static or moving gas atmosphere. In these examples, the process microchannel terminates at the aperture 380 of a transport microchannel 375 within the flow control device.

[0253] Once in the processing microchannel 280 or emitted from the apertures 235 , 380 into another flow environment, the microfluidic streams are stabilized into a merged single laminar flow 260 . Figure 2 The lower left detail of illustrates the flow of particles 277 within particle flow path 275 within sheath flow path 265 in the XY plane. An end view of system 200 is shown in FIG. Figure 2 , where the lower right detail illustrates the flow of particles 277 within particle flow path 275 within sheath flow path 265 of merged flow 260 in the YZ plane.

[0254] In one example, the outer surface of the processing microchannel 280 is transparent along at least a portion of its length and can be constructed from, for example, plastic, glass, or other suitable substrates. A particle interrogation device 285 is positioned to direct a beam of electromagnetic radiation 285L toward a particle 277 passing within the particle flow path 275. Asymmetric particles can be oriented to present a desired face to the incident beam 285L. The lower left image shows a particle in profile and has a narrow elliptical shape, which in some examples may result in a suboptimal surface shape for the beam to be projected onto due to its reduced presentation area. Alternative orientations for ellipsoidal particles and angles of the incident beam may be preferred. The beam 285L can be laser light of a predetermined wavelength and intensity, and it interacts with the particle to generate a plurality of secondary fluorescence wavelengths 285S that can be detected by a detector 290. The intensity and / or spread of the detected fluorescence wavelengths can be used to infer the type of particle being interrogated. For example, a particle can be identified as a sperm cell with an X or Y chromosome.

[0255] In one example, the interrogation beam 285L is directed to interact with particles within the flow environment such that the interrogation beam intersects the microfluidic stream 260 directly without passing through the process microchannel 280. The process microchannel 280 can be omitted, with the microfluidic stream 260 emerging from the aperture 380. Alternatively, a shortened process microchannel 280 can be employed that is coupled to the aperture 380 of the transport microchannel 375 but terminates before the interrogation beam 285L. The interrogation beam 285L can be located near the aperture 235, 375 or the shortened process microchannel terminus, which enables the interrogation event to occur near the focusing chamber where the asymmetric particles are oriented. This ensures maximum orientation and reduces potential turbulence.

[0256] Downstream of the particle interrogation module 285 is a sorting device 295 that directs a second beam of electromagnetic radiation 295L ("sorting beam") toward particles based on the particle type, as identified by the particle interrogation module 285 and the detector 290. The second beam 295L can be a laser having a predetermined wavelength and intensity, and is directed toward one type of cell but not another. For example, the second beam 295L can be directed toward sperm cells with a Y chromosome but not toward sperm cells with an X chromosome. Alternatively, the second beam 295L can be directed toward sperm cells with an X chromosome but not toward sperm cells with a Y chromosome. The second beam 295L can displace the particles it impinges upon along the optical axis of the incident beam. This is configured to cause these particles to be displaced out of the flow path 275 corresponding to the original particle stream and into one or more different flow paths having positions that can correspond to the original sheath stream and / or into one or more different particle trajectories. The two or more types of particles can then be separated by separating the two or more particle flow paths. Different selected particle streams can be collected in different containers to produce discrete cell populations.

[0257] According to one example, operational details of the interrogation or first beam 285L, the displacement or second beam 295L, their associated equipment, and downstream separation of particles are described in published PCT patent document WO 2020 / 013903, which is incorporated herein by reference. However, other particle interrogation and / or particle selection / displacement techniques may alternatively be used with the flow control devices and examples 205, 230 described herein.

[0258] To optimize the operation of downstream particle interrogation and / or particle sorting equipment, the particles 277 may be controlled to orient themselves at a preferred angle relative to the beams 285L, 295L. The particles may also or alternatively be aligned laterally so that they intersect the beams.

[0259] The particles 277 are also controlled to be primarily within the same plane or distance from the interrogation module 285 or the displacement / selection module 295. To achieve this, in some examples, the transfer tube 230 is accurately positioned within the housing 205 of the flow control device. The accurate longitudinal, lateral, and / or rotational position of the transfer tube relative to the housing, and the accurate rotational position of the housing relative to the direction of the light beam, can improve the accuracy and efficiency of these downstream processes. In one example, the transfer tube and / or the housing include an engagement structure that is arranged to improve the longitudinal, lateral, and / or rotational positioning of the transfer tube within the housing, and by extension, the longitudinal, lateral, and / or rotational positioning of the transfer tube with the rest of the system 200.

[0260] In an alternative example, the particle flow control device can be used in a system that does not use an extended microchannel, but can be used with an alternative cell sorting method that uses any method of displacing or selecting particles in space (whether in a fluid, an aerosol droplet, or in a gas phase). Such displacement / selection methods include, but are not limited to, forces obtained from electric fields, electrostatics, electromagnetics, acoustic pulses, or pneumatic pulses. The flow control device can also be used with a selection / sorting method that destroys or destroys unwanted cell populations rather than physical displacement. This is referred to as "ablation" in this article. For example, such selection / sorting methods may include a radiation source (RS) that is configured to direct radiation onto particles to achieve at least one of a force and a torque on each particle so as to cause ablation or at least one of displacement and orientation of each particle relative to an axis defined by the direction of the fluid flow. Ablation can be considered a process in which energy is transferred to particles sufficient to permanently inactivate the particles. In the context of biological cells, this may include rendering the cells inviable for their normal function or purpose. For example, sperm can be ablated to rapidly induce permanent immotility, or they can be ablated to "prime" them so that they cannot survive downstream processes such as freezing and thawing. In the former, ablation may involve rupture of the cell surface membrane, which destroys the integrity of the cell. During the "priming" process, the cell surface membrane remains essentially intact, even though motility may be reduced or stopped.

[0261] Some example methods and apparatus can be used to achieve orientation of non-spherical particles, such as sperm cells. In one example, the angle of the non-spherical particles is controlled to achieve a desired angle relative to one or more radiation beams that can be used for downstream processing of the particles. In one specific example, the particles are oriented to optimize downstream processes that interact with the radiation beam, where the absorption and emission of radiation in asymmetric particles, such as sperm cells, can be highly orientation-dependent. Thus, it may be desirable to orient a preferred facet of the asymmetric particle, which facet has the largest or smallest surface area or some other characteristic, toward the incident radiation beam.

[0262] In an alternative example, downstream processing can be implemented using an orientation module or stage employed downstream of the flow control devices 230, 250 to orient the particles. For example, the orientation module can be positioned to direct electromagnetic radiation toward the particles in the merged stream 260. This can expose the particles to radiation pressure, thereby causing asymmetric particles to adopt a preferred orientation. The operational details of the orientation stage are described in published PCT patent document WO2014 / 017929, which is incorporated herein by reference. However, other particle orientation techniques may be used alternatively or additionally with the example flow control devices 205, 230.

[0263] In some examples, various combinations of one or more downstream processes can be performed by installing corresponding modules or stages downstream of the flow control devices 205, 230, such as those described above. These downstream processes can include particle orientation and / or particle interrogation and / or particle displacement / selection, and can be performed individually or in any combination.

[0264] Figure 3 4 and 5 illustrate a longitudinal cross-sectional view and a transverse cross-sectional view of a flow control device according to an example, respectively. Figure 3 Flow control device 300 includes a transfer tube 330 mounted within housing 305. Specifically, transfer tube 330 is securely and accurately received within cavity 310 of housing 305. Transfer tube 330 includes an inner lumen 340 for conveying a particle stream 345, which is a moving liquid, such as an aqueous solution containing particles, such as sperm cells. Lumen 340 opens to a transfer tube inlet 332 at the input end of transfer tube 330 and opens to a transfer tube outlet 333 at the distal end of the transfer tube.

[0265] The transfer tube 330 also includes a ridge, fin, or protrusion 335 extending longitudinally along the inner surface of the housing or the transfer tube. The ridge 335 engages the inner surface of the cavity 310 of the housing 305 to secure the transfer tube within the housing. In an alternative example, the housing 305 includes a ridge, fin, or protrusion extending longitudinally along the housing and also engaging the outer surface of the transfer tube. The ridge 335 can be sized to ensure a friction fit with the featureless wall of the cavity 310, or the wall of the cavity can include a corresponding groove in which the ridge is located. Various other mechanical fixing mechanisms can be used alternatively. By extending longitudinally, the ridge 335 improves the lateral positioning of the distal end of the transfer tube 330, so that the transfer tube outlet 333 is securely and accurately located within the housing 305. In addition, the channel formed causes the sheath flow to be stabilized and aligned with the Z-axis of the flow before intersecting the sample fluid containing the particles.

[0266] Various alternative engagement structures are possible. Although the ridges 335 extend longitudinally, they may also be angled relative to the longitudinal axis to form a spiral shape along the exterior of the transfer tube. Furthermore, although the ridges have been shown as continuous, they may be discontinuous, with the components engaging the cavity wall at different longitudinal locations. In another alternative arrangement, longitudinally extending ridges may extend from the cavity to engage the transfer tube. In this alternative, the transfer tube 330 may or may not have ridges 335 extending to the cavity wall. In yet another alternative, the outer circumference of the transfer tube 330 may be sized to directly mate with the inner wall of the cavity to ensure a friction fit. The outer surface of the transfer tube and / or the inner surface of the cavity 310 may include recesses to form a channel between the housing 305 and the transfer tube 330.

[0267] In some examples, the ridges on the housing and / or transfer tube are arranged to provide channels through which the sheath fluid flows. In some examples, the sheath flow is uniform. Uniform sheath flow means that the flow distance through each channel is substantially the same. This ensures that when the ridges terminate and the sheath fluid from one channel merges with the sheath fluid from a different channel, minimal turbulence is caused. In this example, the sheath fluid from each channel flows at the same speed and along the same distance as the sheath fluid from the other channels. This uniform sheath flow is achieved by arranging the channels in a cross-section with at least one line of symmetry.

[0268] This configuration improves 360° confinement and focusing of the fluid flow and eliminates the need for multiple sheath crossings, which can result in turbulence and sheath flows in different directions if they interact with each other or with the sample flow.

[0269] Figures 4B to 4I Non-limiting examples of channels having at least one line of symmetry are shown, where shaded areas comprise solid material and open areas comprise flow channels.

[0270] In the above example, the channel may have a fan-shaped shape, wherein the channel is formed at the transmission pipe or the housing. The fan-shaped shape means that one or more edges of the channel have a fan-shaped shape. Figure 4F -H and Figure 6A In some examples, the scalloped portion forms a channel between one or more vertices of the shape of the transfer tube when viewed in cross-section. In other examples, one or more vertices of the shape of the transfer tube are rounded or otherwise shaped to smoothly engage the housing. Figure 4H and Figure 6A A smooth engagement of a rounded ridge or vertex 635 is shown. Smooth engagement means that there is essentially zero volume between the smooth ridge / vertex and the housing wall. At least one vertex, and preferably all vertices, preferably engage the housing for a portion of at least 1% of the housing's inner circumference. In other examples, the smoother vertices engage the housing for a portion of at least 5%, at least 10%, at least 20%, or at least 50%. Generally speaking, the higher the engagement portion, the more secure the transfer tube is within the housing. Conversely, the higher the engagement ratio, the smaller the volume of sheath fluid passing through the channel.

[0271] In other examples, the housing includes a channel formed in the inner surface to enable the sheath fluid to flow through and through the transfer tube. In this example, the transfer tube can have a circular cross-section or can have a channel as described above. Figure 4I A housing with a channel and a circular cross-section transfer tube is shown. In other examples, both the housing and the transfer tube may have channels, such as opposing channels where vertices meet to form an elliptical or circular channel, or staggered channels formed in the housing and transfer tube.

[0272] The inventors have found that when the sheath flow is in the focusing chamber or before the focusing region (e.g. Figure 3 The scalloped profile minimizes hydrodynamic turbulence when the sheath fluid merges with the sample fluid (particle fluid) in the focusing chamber and / or confinement chamber. The scalloped profile also helps maintain hydrodynamic smoothness and non-turbulence when the sheath fluid merges with the sample fluid (particle fluid) in the focusing chamber and / or confinement chamber. The scalloped profile also enables easier manufacturing and facilitates cleaning of the transfer tube and housing without having to remove and use special tools to clean the corners.

[0273] In the above examples, the number of channels can vary. However, in some examples, at least 3 channels, preferably 4, 5, 6, 7, 8, 9, 10, 11 or 12 channels provide enhanced throughput and a secure position of the transfer tube in the housing.

[0274] The engagement structure may extend a portion of the length of the transfer tube. For example, Figure 7C Examples are indicated where the engagement structure extends only a portion of the length of the transfer tube.

[0275] In an alternative example, the flow through the channel can be asymmetric, for example with more flow on one side of the transfer tube 330 than on the other side. This can be used to displace the particle flow within the sheath flow to one side of the central axis. Having a particle stream that is not concentric with the sheath flow may be useful in some downstream interrogation and / or sorting processes. In some examples, the channel can be angled relative to the longitudinal direction so that the flow within the channel is circular around the transfer tube, thereby forming a spiral or partially spiral flow. This may result in a vortex sheath flow surrounding the particle flow, which may provide some additional restriction.

[0276] exist Figure 3 and Figure 4A In the example of FIG. 3 , one or more sheath flow channels 360 are formed between the transfer tube 330 and the housing 305 to carry 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 transfer tube and include channels formed between the ridges 335. The sheath flow channels extend along the exterior of the transfer tube 330 to a focusing chamber 370 defined by a volume formed within the housing 305 at the end of the transfer tube 330, and the particle flow 345 is discharged from the transfer tube outlet 333 into the focusing chamber. The focusing chamber 370 is also fluidically coupled to a transfer microchannel 375, from which the microfluidic stream is output via an orifice 380 to a process microchannel or flow environment for downstream processing.

[0277] The sheath flow inlet 362 is configured to receive the sheath fluid of pumping from the sheath reservoir. In some examples, the inlet can be fed with the sheath fluid at a substantially 90 ° angle to the flow direction Z axis. The inventors have found that receiving the sheath flow at this angle reduces the bubbles in the system and enables the sheath channel to be fully filled before the fluid flows down the channel. In an alternative example, the sheath inlet is positioned to inject sheath fluid in the downstream direction at an angle between 90 ° and 0 ° (as shown in the figure) relative to the Z-axis direction of the sample flow. In one example, the sheath fluid is received in the sheath chamber upstream of the sheath flow channel via the sheath inlet. The sheath chamber receives the sheath fluid and allows the flow to be stabilized before the fluid moves down the channel. Preferably, the cleaning outlet is positioned to be communicated with the sheath chamber fluid to receive air and excess sheath fluid and extract it from the system. Preferably, the cleaning outlet is positioned upstream of the sheath inlet.

[0278] The central particle stream 345 is surrounded by one or more coaxially arranged sheath streams 365. The shape and size of the focusing chamber 370, the geometry and dimensions of the sheath channel 360 and lumen 340, and the flow rates of the particle stream 345 and sheath stream 365 all contribute to controlling the combined fluid flow from the transport microchannel 375. Example use cases include controlling the orientation and confinement of particles within the microfluidic stream 385.

[0279] In one example, the present invention provides a flow control device for controlling a fluid flow associated with transporting particles, the flow control device comprising:

[0280] a transfer tube, the transfer tube comprising an inner cavity for conveying a liquid flow, the transfer tube extending longitudinally within the housing, the housing and the transfer tube together defining a focusing chamber adjacent to the transfer tube outlet; and

[0281] b. two, three, four or more longitudinally extending channels for conveying a sheath flow of liquid between the transfer tube and the housing toward the focusing chamber,

[0282] wherein the downstream outlet of each channel is regularly spaced about the transfer tube at a single longitudinal position relative to the flow path.

[0283] The longitudinal axis depicted in this example is substantially aligned with the direction of sample fluid flow through the transfer tube. The focusing chamber is the area where the sheath fluid flow meets the sample fluid flow. As described herein, the regular spacing of the channel outlets around the transfer tube outlet results in non-turbulent flow that simultaneously compresses and focuses the sample fluid flow from multiple directions. This reduces turbulence and enhances confinement. This multi-directional flow focusing arrangement also avoids the need for multiple sheath flow intersections along the chip.

[0284] The transport microchannel 375 fluidically couples the focusing chamber 370 to the exterior surface of the flow control device 300. The transport microchannel 375 is a conduit having an inlet from the focusing chamber 370 and an orifice 380, which may be in the form of an external orifice or may connect to the process microchannel 280. The cross-sectional shape of the transport microchannel 375, its inlet and outlet or orifice 380, may be the same or different and may include: circular, elliptical, triangular, square, or rectangular with various aspect ratios. In one example, the transport microchannel 375, its inlet, and orifice 380 comprise identical rectangular cross-sections with an aspect ratio greater than 1:1.

[0285] As described above, the longitudinally extending ridges or other engaging structures ensure accurate and stable lateral positioning of the transfer tube outlet 333 within the focusing chamber 370. In a more conventional arrangement, the transfer needle is introduced into a conical volume of sheath fluid, however, the distal end of the needle is impacted by the fluid flow and moves laterally, causing the resulting particle flow to move within or even partially mix with the surrounding sheath fluid flow, resulting in a poorly oriented and poorly confined sample fluid (particle flow) flow. This can make downstream processing difficult, inaccurate, and inefficient.

[0286] In some examples, accurate longitudinal positioning of the transfer tube outlet 333 within the focusing chamber 370 helps optimize the control and stability of the orientation and / or confinement or other flow characteristics of particles passing through the transfer microchannel 375 and the microfluidic stream 385 being transferred from the aperture 380. Figure 3 4 , this is accomplished by sizing the ridge 335 of the transfer tube 330 to complement the dimensions of the cavity 310 of the housing 305 to prevent the transfer tube 330 from being inserted into the cavity beyond a predetermined longitudinal position.

[0287] The inventors have found that the distance between the end of the transfer tube and the opening of the hole (in Figure 6B Example 2 demonstrates that the PODT and shell configurations described herein provide consistently high orientation efficiency over a range of PODT tip distances and flow rates.

[0288] The lumen 310 can be divided into several longitudinal segments or portions, including a first portion 310-S1 having a longitudinal cross-sectional shape (such as a rectangle), which is arranged to engage with a corresponding first portion of the transfer tube 330-S1 at multiple longitudinal locations. The first portion of the lumen can have a substantially uniform transverse cross-section along the longitudinal direction, such as a circular shape having a constant diameter. In other examples, the first portion of the lumen can include a channel or sector formed in the housing, while the transfer tube itself has a substantially circular cross-section. In other examples, the first portion of the lumen can include a channel or sector formed in the housing and a channel or sector formed in the transfer tube, wherein each channel facilitates the flow of sheath fluid through the channel. Preferably, in this example, the channels in the housing and transfer tube are aligned to provide a single channel volume that is greater than the volume of the channel formed in either the housing or the transfer tube alone. This first portion S1 of the lumen 310 is configured to receive the spine 335 of the transfer tube 330. The second portion 310-S2 of the lumen tapers, having a decreasing size as it extends toward the distal end of the transfer tube. The end of the ridge 335 having a larger dimension prevents the transfer tube from extending beyond this point, thereby ensuring accurate and stable longitudinal positioning of the transfer tube outlet 333 within the focusing chamber 370. The end of the ridge can be shaped to complement the internal shape of the cavity 310 as shown to further improve this positioning. In other arrangements, a groove or channel in the wall of the cavity 310 can be used to receive the ridge 335, and the length of the groove can be controlled to control the longitudinal position of the transfer tube outlet 333 within the focusing chamber 370. In this example, the engagement of the transfer tube ridge in the housing provides a rotational engagement to ensure angular alignment, the advantages of which will be further described herein. In contrast, in more conventional arrangements, the transfer needle can be placed in a tapered volume of sheath fluid, however, if the distal end of the needle is not correctly positioned, the sheath flow can turbulently interact with the particle flow, resulting in undesirable mixing, chaotic misalignment of the particle flow, and poor particle orientation and confinement.

[0289] The tapered second portion 310-S2 of the cavity 310 of the housing 305 may include a tapered angle α relative to the longitudinal axis of the housing, and the tapered second portion 330-S2 of the transfer tube 330 may include a tapered angle β relative to the longitudinal axis of the transfer tube 330. The sheath flow channel 360 may be defined by the volume between the inner wall of the housing and the outer wall of the transfer tube. The housing and its inner wall may in turn be defined by the shape and size of the cavity, and the transfer tube may be defined by the shape and size of its outer wall. The tapered angle may be adjusted to achieve control over the acceleration of the sheath flow in the portion of the sheath flow channel. In a preferred example, the tapered angle α is 5°-90°. In other examples, it is 10° to 45°. In a preferred example, the tapered angle β is 5° to 90°. In other examples, 10° to 45°.

[0290] The third portion 310-S3 of the cavity of the housing 305 may include uniform dimensions extending along the longitudinal length. Similarly, the third portion 330-S3 of the transfer tube 330 may include uniform, but smaller, dimensions extending along a similar longitudinal length. The portion of the sheath channel 360 formed between these two portions 310-S3, 330-S3 does not accelerate the sheath flow 365 and allows it to stabilize to ensure laminar flow and reduce turbulence.

[0291] The fourth portion 330-S4 of the transfer tube includes a distal end that includes a transfer tube outlet 333. The end can be shaped to enhance orientation and / or confinement of the particles, as described in more detail below. This end region can be complemented by a further tapered fourth portion 310-S4 of the housing's cavity 310. When the transfer tube outlet 333 is positioned to discharge the particle stream 345 into the sheath stream 365 that enters the focusing chamber 370 formed by the housing, the focusing chamber 370 is formed in the fifth portion 310-S5 of the housing's cavity 310. The focusing chamber and other components of the flow control device 300 are configured to cause a combined laminar flow of the particle stream and the sheath stream through the transfer microchannel 375 and out of the aperture 380, wherein the particles are primarily oriented along one axis and primarily confined to a plane containing the axis.

[0292] Additional portions of the transfer tube and / or sheath flow channel 360 may be included, or some of the depicted portions may be removed from some examples, such as removing the third portion 330-S3 from the transfer tube, as desired to impart targeted characteristics to the particle flow, such as, but not limited to, the sheath flow channel. Sheath flow channel geometries different from those illustrated may alternatively be employed.

[0293] The sheath flow 365 passing through the sheath flow channel 360 can be symmetrical or asymmetrical. For example, the larger volume in the upper half of the sheath flow channel 360 can cause the particle flow 345 to shift downward. The sheath flow can also be rotated around the transmission tube to generate eddies that contribute to particle flow restriction. For example, this is achieved by using angled ridges to limit spiral or "corkscrew" flow, as shown in Figure 7. 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 channel formed between them serve to stabilize the sheath flow because the sheath flow can be introduced from outside the flow control device as turbulence.

[0294] Figure 4A Shown by Figure 3 AA, in which the housing 305 and the input region of the transfer tube 330 are visible. This shows four evenly spaced ridges 335 extending from the transfer tube 330, but any number of ridges may alternatively be used. The transfer tube and various other components of the housing are used in conjunction with the Figure 3335L are denoted by the same reference numerals as those components in the housing. One of the ridges 335L is longer than the other ridges and corresponds to a groove 315 in the outer wall of the housing cavity 310. This arrangement comprises an indexing arrangement that includes an indexing device that ensures that the transfer tube can only be received into the housing in a single orientation, generally exemplified by R. In another example, the transfer tube may have multiple longer ridges, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 longer ridges, wherein the ridges engage in the grooves in the housing wall. Optionally, the transfer tube may include one or more grooves, and the housing may include one or more extended ridges to enable alignment in a manner opposite to that described above. With a single extended ridge aligned with a groove, this arrangement achieves 360° rotational alignment, allowing the transfer tube to be inserted only in a single orientation. In an alternative example, the indexing device may include two extended ridges opposite each other, and the transfer tube may be inserted in one of two orientations 180° apart from each other so that the ridges can be aligned with the corresponding grooves. Similar angular arrangements may be employed, such as three extending ridges aligned with corresponding grooves to achieve three orientations at 120° to each other, or four extending ridges aligned with corresponding grooves to achieve four orientations at 120° to each other. These examples are particularly useful for achieving proper alignment of the transfer tube outlet when the transfer tube outlet 333 comprises a non-circular cross-section (i.e., it is tilted or otherwise asymmetric) or has a non-circular lumen (e.g., an elliptical or rectangular lumen).

[0295] In an example where multiple grooves are provided in the cavity wall 310, each groove being configured to receive a single ridge 335, the groove 315 for the longer ridge 335L is deeper, so that the longer ridge 335L will still fit within only that one groove, thereby ensuring the predetermined rotational alignment of the transfer tube 330 within the housing 305. In an alternative arrangement where grooves are used for all ridges, one of these grooves may be wider than the others to receive a wider, but not necessarily longer, ridge. In yet another alternative, a pin and corresponding hole arrangement may be used to properly guide the transfer tube within the housing. For example, a pin may extend through the housing into the ridge of the transfer tube, or the transfer tube or ridge may include a pin that extends through a hole in the housing. In another arrangement, a magnet in one of the transfer tube or housing may be used in conjunction with another magnet (or metal feature) in the corresponding housing or transfer tube. Various other mechanical rotational alignment features may be used alternatively or additionally.

[0296] External visually accessible surfaces of the housing and transfer tube may be marked to assist a user in aligning the transfer tube when inserting the transfer tube into the housing, thereby ensuring rotational alignment.

[0297] In the above-described example of using an indexing arrangement to determine the rotational alignment of the transfer tube within the housing, the ridge or other indexing device may extend substantially the length of the transfer tube or housing to the terminal location of the lumen 333, or may extend along a portion of the transfer tube or housing. The portion may be defined by the beginning or end of a taper angle of the transfer tube or housing, or may be a portion of a linear region of the transfer tube or housing. For example, the indexing device may extend for a portion (e.g., one-quarter or one-half) of the distance S1, S2, or S3.

[0298] Various other examples of inner housing or cavity cross-sectional shapes and outer transfer tube cross-sectional shapes are described in Figures 4B to 4I Shown in. Figures 4B to 4E The longitudinal passage formed by the difference in cross-sectional shape between the inner housing and the outer transfer tube is illustrated. Figures 4F to 4I Illustrated are longitudinal channels formed by protrusions extending laterally from the exterior of the transfer tube and / or the interior of the housing.

[0299] Figure 5A and Figure 5B A detailed cross section of the flow control device around the focusing chamber 370 is shown according to one example. The rest of the device can be Figure 3 4 , or may be different therefrom. The lumen 540 of the transfer tube 530 includes a plurality of sections 540-U, 540-T, 540-N having different cross-sectional dimensions. The first wide section 540-W carries an unfocused particle stream 545-U that carries particles of various orientations and passes through a relatively large volume. The tapered section 540-T of the lumen reduces the volume through which the particles flow, thereby accelerating the particle flow and improving some particle flow characteristics. The cross-sectional shape of the lumen may also vary along its length, for example, from a circular shape in the first section 540-W to an elliptical shape in the final section 540-N, which orients the particles along the longer axis of the ellipse. This is illustrated in detail in the device below, in which microparticles 577-C in the circular cross-section of the lumen 540-W are oriented to different axes 577-E in the elliptical cross-section of the lumen 540-N. As can be seen, the unfocused particle stream 545-U narrows along at least one axis, where the particles are oriented and discharged from the transfer tube outlet into the focusing chamber 370. The lumen can change cross-sectional shape and / or size along its length within the transfer tube. In some examples, the lumen can include an upper portion and a lower portion having different cross-sectional shapes and / or sizes. Example size ranges include 50 μm-1 mm for the lower portion and 0.5 mm-3 mm for the upper portion.

[0300] In one example suitable for use with any of the flow control devices described herein, the transfer tube immediately adjacent the lumen includes a tapered portion from a smaller cross-sectional area upstream of the microchannel to a larger cross-sectional area downstream of the transfer tube's outlet lumen. It has been found that this flared transfer tube outlet transition minimizes turbulence and promotes laminar flow when the sample flow encounters the sheath flow.

[0301] The transfer tube outlet 645 is designed to have a size suitable for transferring cells and sample fluids. In one example, the inner diameter of the outlet of the transfer tube lumen is 50 μm-1 mm. In another example where a more tightly restricted flow is required, the transfer tube outlet is 50 μm-500 μm.

[0302] exist Figure 5B In one example shown, the transition from the first wide section of lumen 540-U to the narrowed portion 540-N includes a step or steeply tapered portion 541 at an angle of substantially 90° to the flow axis. Steeply tapered portion 541 preferably forms an angle of 45° or greater to the flow axis. The lumen may be adapted to receive an inlet tube 542, which transports a fluid containing particles. In this example, the difference in lumen width between point 540-U and point 540-N substantially corresponds to the thickness of the inlet tube wall. The inlet tube inner diameter substantially corresponds to the inner diameter of the lumen beyond the end of the inlet tube. This allows for a smooth transition between the inlet tube and the transfer tube lumen and reduces turbulence that could affect particles or interfere with downstream orientation or confinement. The step or steeply tapered portion serves as an inlet tube stop at point 540-T, preventing the inlet tube from advancing further down the transfer tube and providing a seal between the inlet tube and the transfer tube. In one example, an O-ring or gasket is included at point 540-T to improve the seal and prevent fluid leakage. The inlet tube stop is also labeled 640 in FIG. 6 .

[0303] The input tube stop 541 can be used in conjunction with the tapered portion 540-T to achieve both a) enhanced flow restriction and / or orientation (e.g., using a non-circular cross-section lumen) and b) a smooth transition from the input tube to the transfer tube lumen at various points along the longitudinal axis of the transfer tube lumen.

[0304] The laminar sheath flow 365 interacts with the partially aggregated particle flow to further optimize the characteristics of the particle flow. For example, this may be suitable for further limiting and / or further orienting particles. The laminar fluid merged from the device output includes an internal focusing particle flow 545-F within the surrounding sheath flow 365. An orifice or hole 580 is formed in the housing that emits the microfluid stream from the transmission microchannel. In one example, the hole 380 may be coupled to a processing microchannel or other conduit for forward transport and downstream processing. In another example, the microfluid stream is transferred to a fluid environment such as a liquid or gaseous environment.

[0305] In this example, the microfluidic stream is emitted from the hole into a flow environment. The microfluidic stream can be emitted in a downward or gravity-based direction or in a direction angled relative to gravity (e.g., vertically or approximately upwardly angled). In one example, the flow environment is defined by an extended microchannel or conduit that allows the microfluidic stream to travel from the hole in a controlled manner and restricts the flow of particles as the particles pass through the particle interrogation device 285 and the sorting device 295. In one example, the flow environment can include a liquid with substantially the same viscosity as the liquid in the microfluidic stream. In some examples, the flow environment is a liquid with a higher viscosity than the microfluidic stream. In some examples, the fluid in the flow environment can move in the direction of movement of the microfluidic stream. The speed of this movement can be the same as or different from the speed of the microfluidic stream. In an alternative example, the flow environment includes a gas environment. In this example, the microfluidic stream does not need to be defined by a processing microchannel or conduit. This approach has many advantages and results in reduced friction with the conduit, thereby resulting in improved laminar flow and achieving higher flow rates. The inventors have discovered that where an aperture is coupled to a processing microchannel or catheter, the aperture size must be aligned with the catheter's internal dimensions to ensure that the microfluidic flow remains laminar with minimal turbulence. The catheter size does not always align with the aperture size, which limits the range of aperture sizes. By exploiting the fluid flow environment, this allows for flexibility in aperture size and shape. Specifically, using smaller apertures than permitted by standardized catheters allows for a reduced sheath flow relative to the particle flow. This reduction in sheath volume therefore increases the concentration of selected cells.

[0306] Figure 6A A side view and an end view of a transfer tube according to one example are illustrated. Transfer tube 630 includes an internal lumen 640 for carrying a particle stream, and a plurality of ridges 635 comprising longitudinally extending mechanical engagement structures. Between ridges 635 are recessed or scalloped portions 637, which also extend longitudinally and form channels with the lumen wall of the housing when received in the housing. These channels can form part of a larger sheath fluid channel that delivers sheath fluid from a sheath fluid inlet adjacent the larger end of the transfer tube and a focusing chamber in an assembled flow control device containing the transfer tube.

[0307] The ridges 635 and depressions 637 may have the same length as shown, or they may have different lengths. The ridges 635 and depressions 637 may have the same width and / or cross-sectional profile as shown, or they may have different widths and / or profiles. The ridges 635 may form hemispherical depressions 637 as shown, but alternative arrangements are possible, including polygonal or complex curvature shapes, triangular or rectangular cross-sections, or shapes with both straight and curved components. The ridges and depressions may be regularly or irregularly spaced.

[0308] In one example, the sheath channel formed between the ridges is tapered such that the cross-sectional area of ​​the channel increases at a downstream location in the housing compared to an upstream location in the housing. Figure 4BA -Upstream and Figure 4BB - downstream illustrates an example of such a tapered channel architecture, but alternatively, a Figure 4A This tapered arrangement allows the sheath fluid flow to hydrodynamically merge with the sample fluid stream at the end of the sheath channel and reduce "dead space" that could increase flow turbulence or bubble formation during initiation.

[0309] The transfer tube 630 can be formed into different sections 630-S1, 630-S2, 630-S3, 630-S4 having different geometries as described herein. Similarly, the lumen 640 can be formed into different sections having different geometries as described herein. The combination of these geometries and the corresponding geometries of the lumen of the housing into which the transfer tube is assembled enables the sheath flow and particle flow to be controlled to optimize different characteristics of the particle flow, such as particle orientation and / or confinement.

[0310] The transfer tube and housing of the present invention can be combined with a method for controlling the flow rate of the particle flow and the sheath flow. In certain examples, the flow rate is selected from the group consisting of 1 mm / s to 20 m / s, greater than 1 mm / s, greater than 10 mm / s, greater than 100 mm / s, greater than 1 m / s, greater than 2 m / s, greater than 10 m / s, or greater than 20 m / s, and ranges therebetween.

[0311] These channels merge into a conical cavity defined by a tapered transfer tube and an inverted tapered housing wall. The inventors have discovered that a smooth transition from the channels to the conical cavity provides a directional sheath flow with minimal turbulence. In this example, the sheath flow enters from each channel in a symmetrical flow. Compared to the flow from, for example, a sheath inlet on one side of the transfer tube or on the opposite side of the transfer tube, the multidirectional flow is enhanced. The multidirectional flow preferably flows from channels arranged at regular intervals around the entire 360° transfer tube. Simultaneous sheath flow from multiple directions minimizes turbulence before and after merging with the sample fluid flowing through the lumen.

[0312] Thus, a single sheath flow from each channel gently converges with the particle flow at an angle with minimal displacement of the particle or sample flow and minimal turbulence, ensuring that the particle and sheath flow components remain laminar and there is little or no mixing between them.

[0313] In one example, the focusing chamber includes an upstream portion and a downstream portion. Figure 6B 、 Figure 6BA 、 Figure 6C 、 Figure 6D and Figure 6EAn example of a dual focusing chamber comprising a transfer tube and a housing that engage and cooperate with one another is provided. An upstream first focusing chamber (P1) can be defined by a frustoconical cavity of the housing. More generally, the upstream first focusing chamber (P1) can be defined by an inner longitudinal tapered portion of an inner wall of the housing, wherein the transverse cross-section of the cavity defined by those tapered walls has a substantially 1:1 aspect ratio. For example, the upstream cross-section can comprise a circle, a rounded square, or a square. In this example, the substantially equal aspect ratio (1:1) is intended to refer to a shape wherein the length of a first axis of the shape is no more than 20% longer than the width of a second axis of the shape perpendicular to the first axis.

[0314] The downstream second focusing chamber P2 may include a cavity defined by the inner wall of the housing, and the cavity has a transverse cross-sectional shape having an unequal aspect ratio of substantially greater than 1:1, wherein the length of one axis is longer than the length of the orthogonal axis of the cavity. For example, the downstream cross-section may include a rectangle, an ellipse, a rounded rectangle, or a stadium shape (a geometric term for a rectangle with a pair of semicircles positioned at either end). In particular examples, the downstream cross-sectional aspect ratio is greater than (i.e., not equal to) 1:10, such as greater than 1:20, greater than 1:30, greater than 1:50, greater than 1:100, greater than 1:150, greater than 1:200, greater than 1:300, greater than 1:500, greater than 1:1000, greater than 1:2000, greater than 1:3000, or greater than 1:5000.

[0315] First reference Figure 6B and Figure 6BA , illustrating an example of a dual focusing chamber. Figure 6B shows a side view of a flow focusing device 600 having dual focusing chambers P1 and P2, and Figure 6BA Shown from Figure 6BFIG2 is an end view of a dual focusing chamber viewed from the right side of FIG3 , looking left. First focusing chamber P1 has a square cross-section that tapers from a maximum side length P1-L1 to a minimum side length P1-L2. Second focusing chamber P2 has a rectangular cross-section with a maximum side length P2-L1 and a minimum side length P2-L2. In this example, the longest side length P2-L1 of second focusing chamber P2 is substantially the same as the minimum side length P1-L2 of first focusing chamber P1. This results in a slit at the leftmost or tapered end of the first focusing chamber, which extends a length L between P2-a and P2-b to form a linear segment of second focusing chamber P2. In this example, the second focusing chamber also includes a tapered segment P3 that tapers from the maximum side length P2-L2 toward the entry point 374 of the transport microchannel 375. This tapered segment P3 is also referred to herein as a confinement chamber. In this example, the width of the transport microchannel 375 is substantially the same as the minimum length P2-L2 of the second focusing chamber P2. The interaction of the particle stream emanating from the particle transfer tube into the first focusing chamber, the sheath stream entering the first focusing chamber through the sheath flow channel, and the combined stream moving through the first and second focusing chambers into the transfer microchannel 375 creates a laminar microfluidic stream 385 that exits the transfer microchannel 375 at the aperture 380 in the fluid focusing device 300. This configuration provides improved orientation and confinement of particles within the microfluidic stream 385.

[0316] exist Figure 6BC and Figure 6BD In the illustrated alternative example, at least the upper surface 692 or lower surface 693 of the second focusing chamber includes an inverted gable shape (i.e., the internal shape of a v-shaped or u-shaped top or ridge-shaped recess). The inverted gable may taper along its length (i.e., to form the internal shape of a hull) such that the height of the chamber along the centerline (Y-axis) is greater at upstream locations than at downstream locations. The inverted gable may include a rounded gable 693 or a pointed gable 692. Such a tapered inverted gable provides a hydrodynamically streamlined shape to orient and confine cells as they pass along the second focusing chamber. In one example, the inverted gable shape extends through the portion formed by Figure 6B 、 Figure 6BC and Figure 6BD The W in φ defines the longer axis of the cross section of the chamber. Thus, the cross section of the second flow focusing chamber may comprise a diamond, pentagon, or hexagon.

[0317] The cross-sectional shape of the second focusing chamber can be said to include two opposing straight lines and one or more of: a curve between the two straight lines; a V-shaped line or a ridge line between the two straight lines.

[0318] Figure 6BB An alternative dual focusing chamber arrangement for flow focusing device 600' is illustrated, wherein the first focusing chamber P1' has a circular cross-sectional shape. Figure 6BFrom right to left in the figure, it tapers from a large diameter P1-D1 to a small diameter P1-D2. This ends in a second focusing chamber P2', which has a rounded rectangular cross-section with a large side length P2-L1' and a small side length P2-L2'.

[0319] Various alternative cross-sectional shapes may be employed for the first and second focusing chambers. More generally, the first focusing chamber comprises a constant lateral cross-sectional shape having a substantially 1:1 aspect ratio and tapering longitudinally from a larger size to a smaller size adjacent to the second focusing chamber. In other words, the lateral dimension of the shape decreases along at least one axis, and in some examples, along two perpendicular axes. The second focusing chamber comprises a constant lateral cross-sectional shape having a higher aspect ratio than the first focusing chamber and extending longitudinally at a substantially constant dimension along one lateral axis, and in some examples, along two perpendicular lateral axes (i.e., without tapering). The second focusing chamber is fluidically coupled to a transport microchannel having a smaller cross-sectional area. The second focusing chamber may include a tapered section that tapers longitudinally along one or more lateral axes toward the transport microchannel. The transport microchannel 375 may include an aspect ratio higher than that of the first focusing chamber.

[0320] Thus, in one example, a flow focusing arrangement includes:

[0321] a. a first focusing chamber comprising a substantially constant lateral cross-sectional shape forming a frusto-cone having an aspect ratio of substantially 1:1 and tapering longitudinally from a larger diameter at an upstream location to a smaller diameter at a downstream location;

[0322] b. A second focusing chamber comprising a substantially planar chamber.

[0323] In this example, the substantially planar chamber can include a substantially constant lateral cross-sectional shape having a higher aspect ratio than the first focusing chamber and extending longitudinally with a substantially constant dimension along a lateral axis. In one example, the second focusing chamber includes a substantially constant lateral cross-sectional shape forming a rectangle that tapers longitudinally along at least one axis through at least a portion of the longitudinal length of the chamber. In other words, at least one perpendicular lateral axis of the rectangle decreases longitudinally from a larger dimension at an upstream location to a smaller dimension at a downstream location. In some examples, both perpendicular lateral axes decrease longitudinally from a larger dimension at an upstream location to a smaller dimension at a downstream location.

[0324] A flow focusing arrangement comprising a flow focusing portion comprising at least first and second focusing chambers as described herein can be fluidically connected to any sheath channel architecture including a sheath channel arrangement comprising a plurality of longitudinally extending channels as described herein. Similarly, a sheath channel architecture comprising a plurality of longitudinally extending channels can be fluidically connected to other flow focusing devices. However, the inventors have discovered that the combination of the flow focusing portion and the sheath channel architecture provides benefits including minimization of bubble generation during initiation, minimization of turbulence, and enhanced cell confinement and orientation.

[0325] In some examples, the downstream confinement chamber depth tapers from the depth of the second focusing chamber to a shallower depth. In one example, the shallower depth is aligned with the height of the entry point of the transfer microchannel 375. In some examples, the fluid sample streams pass through the flow control device along substantially the same plane. This means that the sample fluid is not directed above or below other streams (e.g., sheath fluid streams). In other words, the sheath fluid and the sample fluid are substantially coplanar. The fluid streams travel along a substantially flat trajectory through the center of the device without large deviations in the direction of flow in either of the focusing chambers or before being ejected from the transfer tube. The inventors have discovered that this aspect of minimal deviation from the direction of the emission stream is important for minimizing turbulence and reducing the g-forces experienced by particles within the fluid. These disruptions to the flow and particles can cause stress on cells, which can reduce their biological efficacy or viability in vivo.

[0326] In some examples, the cone angle is the interior angle between the tapered walls of a cone. Figure 6B and Figure 6C Half of the angle 671 is shown. In some examples, the angle 671 is between 5° and 90°. In other examples, 671 is between 10° and 45°.

[0327] Figure 6C An example is shown in which the transfer tube includes an elongated substantially constant transverse cross-sectional area portion 672. In this example, the transfer tube includes two taper angles. The first is the angle relative to the longitudinal Z-axis of the flow of the transfer tube end 673. The second is the angle relative to the Z-axis of the flow of the transfer tube body 674. In some examples, the angle 673 is between 0°-90°, and in other examples, the angle 673 is between 5° and 30°. In some examples, the angle 674 is between 0°-90°, and in other examples, the angle 674 is between 5° and 30°. About Figures 6B to 6E The angles, dimensions, and features described are intended to be combined with other examples described herein (such as those described above with respect to Figure 2 、 Figure 3 、 Figure 5A and Figure 5B device described) to read.

[0328] In certain examples, the downstream second focusing chamber (P2) cross-sectional width "W," or "orientation width" (i.e., the larger of the two cross-sectional dimensions P2-L2) is 0.5 mm to 10 mm. In preferred examples, the width is 2 mm to 8 mm, or 3 mm to 6 mm. Figure 6F Shows the use of Figure 6B The device shown has been measured for orientation efficiency and resolution at different flow rates (V) and different orientation width measurements. It can be observed that bovine sperm cells flowing through the flow focusing device have substantially uniform orientation efficiency and a high level of orientation.

[0329] In certain examples, the downstream second focusing chamber (P2) cross-sectional depth "D" ("orientation depth" (i.e., the smaller of the two cross-sectional dimensions)) can be 0.01 mm to 4 mm. In other examples, the width can be 0.01 mm to 10 mm. In preferred examples, the depth can be 0.025 mm to 0.5 mm, or 0.05 mm to 0.25 mm. Figure 28a An example is shown comprising a second focusing chamber with a width of 2 mm. Figure 28b An example is shown comprising a second focusing chamber with a width of 6 mm.

[0330] In one preferred example, the depth D is equal to the diameter or major dimension of the transport microchannel 375. The inventors have discovered that aligning the depth of the cross-section of the downstream second focusing chamber P2 with the major dimension of the transport microchannel 375 and the aperture 380 allows the microfluidic stream to flow out of the transport microchannel and the aperture with minimal turbulence. The width of the downstream second focusing chamber P2 then tapers to the minor dimension 670 of the transport microchannel 375, as described below. In other examples, the depth D decreases from point P2-a to point P2-b to provide further focusing and orientation vectors over the length of the downstream second focusing chamber P2.

[0331] The aspect ratio of width to height is in the range of 1:1.25 to 1:800 w:h. In a preferred example, the above preferred width and height aspect ratio ranges can be combined in the previously described aspect ratios to define a downstream second focusing chamber having the exemplary dimensions of 1:5 to 1:160 w:h shown in the table.

[0332]

[0333] Table 1 - Exemplary dimensions of downstream cross sections and accompanying aspect ratios

[0334] Example 1 presents experimental data demonstrating that equal aspect ratios of the cross-sectional areas of the upstream first focusing chamber P1 and unequal aspect ratios of the cross-sectional areas of the downstream second focusing chamber P2 provide high orientation efficiency and resolution. In one specific example, the inventors observed that a width of 4 mm and a depth of 0.1 mm for P2 consistently provided high orientation and resolution between X- and Y-bearing sperm cells over a range of flow rates.

[0335] The orientation length "L" of the downstream second focusing chamber P2 may also be important when considering the orientation efficiency of asymmetric particles that have passed through some example devices. In one example, the length L of region P2 from point P2-a, defined as the location where the wall of the upstream first focusing chamber joins the wall of the downstream second focusing chamber, to point P2-b, defined as the location where the downstream flow focusing chamber joins the downstream confinement chamber P3, is at least 3 mm. In other examples, length L is between 3 mm and 10 mm. In some examples, the orientation width W is constant over distance L. The inventors have discovered that such a constant-width channel has a flow stabilizing effect and reduces downstream turbulence. This beneficial effect is observed in increased confinement and orientation efficiency of asymmetric particles measured downstream of the interrogation region. In another example, the downstream second focusing chamber P2 tapers directly to the start of the transport microchannel. In this example, regions P2 and P3 are merged to achieve orientation and confinement in a single region.

[0336] Figure 6B and Figure 6C The third region P3 shown in FIG is referred to herein as the downstream restriction chamber. In this region, the two walls of the housing taper from the width W defined in P2 to the width of the transport microchannel 375.

[0337] Figure 6D and Figure 6E An alternative example is shown in which the second focusing chamber P2 tapers longitudinally along one lateral axis. As in the previous example, the first focusing chamber P1 tapers longitudinally (z-axis) along both lateral axes (x and y axes), such that, for example, the large square cross-section on the right side decreases to a smaller square cross-section as the first focusing chamber extends longitudinally to the left. In this example, the second focusing chamber P2 tapers longitudinally along one lateral axis (x-axis) while maintaining its cross-sectional dimensions along the perpendicular lateral axis (y-axis). The second focusing chamber P2 tapers to the cross-sectional shape and dimensions of the transport microchannel 375.

[0338] Figure 28e An example is shown in which the second flow focusing chamber tapers from a first width W1 to a narrower width downstream of W2. This tapered configuration helps confine and orient cells and provides a smooth flow path for the transport microchannel.

[0339] In one example, the second focusing chamber is adjacent to a downstream confinement region that tapers from a first depth (y-axis) defined by the depth of the second focusing chamber to a shallower second depth. In one example, the second depth is defined by the depth of entry point 374 of transport microchannel 375.

[0340] An upstream first focusing chamber is employed for use with the above-mentioned transfer tube. Specifically, reference is made to Figure 3 、 Figure 4A -I. Figure 6B 、 Figure 6C 、 Figure 6E The example shown in FIG7 depicts a transfer tube. The directed sheath fluid flows around the ridges with minimal turbulence and through the channels / sectors 656. It then cascades over and around the tapered section of the transfer tube 657, which together with the tapered housing forms the cavity. For example, Figure 3 S1-S4. This open cavity and upstream channel minimizes the effects of turbulence caused by uneven flow. For example, if the sheath fluid is introduced into the focusing chamber from opposite sides of the housing, rather than flowing circumferentially as achieved in the present invention, such uneven flow may be caused. It may also be caused by not properly directing the sheath fluid using multiple channels around the circumference of the housing to achieve laminar and non-turbulent flow in the direction of flow. If the sheath fluid is not directed, it can move in a radial direction around the transfer tube, which can negatively impact confinement and orientation efficiency, for example through turbulence or unequal flow rates around the circumference.

[0341] In one example, the end of the transfer tube protrudes into the downstream second focusing chamber. In another example, the end of the transfer tube is positioned within the upstream first focusing chamber.

[0342] Without being bound by theory, it is believed that the consistently high alignment efficiency achieved using the combination of the directed sheath flow, the circular cross-section upstream first focusing chamber, and the unequal aspect ratio downstream second focusing chamber results from the following effects. First, the directed sheath flow 656 enters the upstream first focusing chamber ( Figure 6B 650 in the example shown). This compresses the particle stream 655 in all directions, which has the effect of increasing confinement, i.e., reducing the variance in the distances of individual particles from the nominal center flow vector along the flow axis -Z. This has the effect of increasing the flow velocity. Secondly, the particle stream passes from a substantially circular cross section to a cross section of unequal aspect ratio as described above, and Figure 6BIn this region, the restricted flow is compressed along the Y axis (i.e., the axis of the smaller dimension of the downstream cross-section). This is believed to cause asymmetric particles, such as sperm cells, to preferentially align their substantially "flat" face (defined by the particle's "major dimension") with the X axis, defined by the unequal aspect ratios of the major dimensions. This region is believed to "prime" the particles to achieve their final orientation after the orifice.

[0343] Then, as the fluid flows into the third region P3 (i.e., the downstream confinement chamber, where at least one of the width (X-axis) and height (Y-axis) of the downstream second focusing chamber is reduced), the particles are subjected to a third compressive force. In this region, the fluid flow rate increases and the confinement of the particles increases. The orientation of the particles is believed to flip from the downstream orientation region so that the flat face of the particle is aligned with the Y-axis.

[0344] The combined sheath flow and particle flow or microfluidic stream then flows into the transport microchannel 375 . Figure 6E An alternative arrangement is shown in FIG, wherein the downstream second focusing chamber P2 tapers at a different angle from the upstream first focusing chamber P1 to the transport microchannel 375. Angle of the downstream second focusing chamber confinement area

[0345] In the downstream restriction chamber, the angles β1 and β2 of the two tapered walls 660 and 661 relative to the flow axis Z can vary from about 20° to about 80°. In another example, the angles β1 and β2 can vary from about 10° to about 80°. In a preferred example, these angles are in the range of 30° to 55°. Figure 29 a and Figure 29 In an example shown in b, the downstream second focusing chamber P3 has an angle of β1 = β2 = 45°.

[0346] Figure 28c An example of the invention is shown, wherein β1 = β2 = 67.5°. Figure 28d An example of the invention is shown, wherein β1 = β2 = 22.5°. Figure 28e An example of the present invention is shown, where β1 = β2 = 11.5°. Corner radius

[0347] In some examples, the angles between the three regions P1, P2, and P3 are curved rather than sharp. This concept of having curved transition angles can be described in terms of the corner radii of the P1-P2 transition, the P2-P3 transition, and the P3-exit transition. In one example, the radius of the angle is at least 20 μm. In a preferred example, the corner radius is 20 μm to 1000 μm. Where reference is made herein to the distance between transitions, it will be understood by those skilled in the art that the distance measurement is made from the center point of the corner radius, i.e., half the angle of the full angle defined by the corner radius.

[0348] The orientation efficiency can be measured using a processing unit implementing orientation efficiency estimation functionality that analyzes the output from the interrogation beam 285L via one or more detectors 290. The orientation efficiency can be used to provide sorting system monitoring to the operator and can also be used to control upstream processes, such as shutting down the system if the orientation efficiency drops too low.

[0349] Orientation efficiency is a measure of the proportion of cells that are adequately oriented relative to a reference direction. Sperm cells and other cells such as red blood cells are asymmetric, having a flattened elliptical shape in which the perpendicular dimension defines the face or large surface plane and the short dimension defines the thickness of the edge (and partially defines the orthogonal short surface plane). In some examples, the particle selection / displacement technology can be arranged to achieve optimal performance when its propagation direction is perpendicular to the face or large surface plane. In this case, the reference direction is perpendicular to the laser propagation direction. The reference direction may alternatively correspond to the orientation of the interrogation beam 285L and / or detector 290. Asymmetric cells that are oriented within a certain range of the reference direction can still be oriented well enough for laser-based sorting or other processes. Other cells that fall outside this range, such as asymmetric cells that present their edges to the laser, may result in measurement data points that cannot be classified and / or are suboptimally sorted.

[0350] A low orientation efficiency metric is an indication that the sorting system is suboptimally configured, resulting in wasted cells; for example, because their fluorescence emissions cannot be accurately measured because they are not well oriented relative to the illuminator or detector. A low orientation metric can also indicate that even if accurately sorted, many cells may not be properly sorted due to poor orientation relative to the laser when it is used for sorting.

[0351] Cell orientation efficiency can be determined by comparing the number of data points within the focal region of interest to the total number of data points corresponding to all cell measurements, although a different definition may alternatively be used. This can be calculated by the following formula:

[0352]

[0353] where ∑ A is the number of cells in the focal region of interest, and ∑ B is the number of cells in another region that can correspond to all cell data points or all live cell data points. Regardless of the population of cell data points used, this metric represents the orientation efficiency over a period of time. The orientation efficiency can vary over different time periods. If the orientation efficiency drops below a threshold (e.g., 30%), this can trigger an alarm or other action to reconfigure the sorting system to improve the measured orientation efficiency.

[0354] In some examples, the flow focusing device is modified in response to suboptimal orientation efficiency. In one example, adjusting Figure 6B The distance dZ in the center of the focusing chamber is defined as the distance between the end of the transfer tube and the intersection of the first focusing chamber and the substantially flat second focusing chamber. Figure 27a and Figure 27b In one example, the end of the transfer tube is positioned downstream of the intersection point—that is, dZ is negative, as shown in FIG. Figure 27a In another example, the end of the transfer tube is positioned upstream of the intersection point—that is, dZ is positive, as shown in Figure 27b The inventors have found that negative dZ provides enhanced orientation efficiency compared to positive dZ. In one example, the transfer tube outlet is positioned within the housing at a position corresponding to the orientation of the tube. Figure 27a and Figure 27b The outlet is positioned downstream of the point laterally aligned with the start of the tapered portion of the housing at 2807 in the transfer tube. Positioning the outlet at this point provides significant advantages in terms of flow hydrodynamics due to the constricting and accelerating effect of the taper on the sheath fluid passing around the transfer tube, which also results in beneficial confinement of the sample stream. Alternatively, the transfer tube outlet can be positioned at a point greater than or equal to 10 μm upstream of the end of the tapered portion at negative dZ, at which point the substantially flat second focusing chamber begins. Positioning it near the second focusing chamber requires sufficient flow space to allow the sheath fluid to pass around the end of the transfer tube and advantageously confine the sample fluid from multiple directions. Those skilled in the art will understand that this distance will be determined based on the outer diameter of the end of the transfer tube and the cone angle of the tapered portion. Another beneficial effect of the arrangement described herein is to provide enhanced cell health by minimizing the acceleration of cells when the sample fluid is engaged by the sheath fluid. This reduces shear stress and provides a higher chance of survival while maintaining high yield.

[0355] In another example, the rotational alignment between the sheath channel and the focusing chamber can be adjusted. In another example, the flow rate of the particle flow and / or sheath flow can be adjusted. In another example, the shape and / or size of the aperture 380 can be adjusted. These adjustments can be combined in any suitable manner and can be controlled based on the estimated alignment efficiency.

[0356] Figure 7A and Figure 7B Two alternative examples of transfer tubes are illustrated, wherein longitudinally extending ridges 735 may also be angled relative to the longitudinal axis. As shown in the first example 7A, transfer tube 730 includes a plurality of angled ridges or ribs 735. These can be configured to induce a spiral sheath flow that can be used to enhance orientation and / or confinement. The angle θ of the ridges can be adjusted to optimize this effect.

[0357] like Figure 7BAs shown in the example of , the transfer tube 730-2 includes angled ridges or ribs 735-2 grouped together in groups, which define channels 736 between the groups. In some examples, these ridges can protrude from the interior of the housing to define channels therein.

[0358] Figure 7C The example in shows ridges 740 extending from the transfer tube in two directions to engage the housing. These ridges are optionally tapered in the Z-axis (the flow axis) to present a thin leading edge to the flowing liquid. The thin leading edge can expand into a wider support structure and then taper again to a thin trailing edge. This optimized form of the ridges resembles a wing that reduces turbulence caused by the liquid flowing over it. In this example, the ridges can define an ellipse in its cross-section, where the longer dimension of the ellipse is roughly aligned with the Z-axis direction of the flow. This configuration of the ridges can be applied to any of the other examples described herein, particularly with respect to Figure 3 、 Figure 6B 、 Figure 6C and Figure 6E An example of description.

[0359] exist Figure 7C In the example shown, the transfer tube, ridges, upstream focusing chamber, downstream second focusing chamber, downstream confinement chamber, and transfer microchannel are a unitary flow control device comprised of a single piece of material. The inventors have discovered that fabricating such a unitary piece enables precise engagement of the transfer tube with the focusing chamber to enhance orientation and confinement.

[0360] Figures 8 to 12A Various transfer tip geometries are illustrated that can be used to optimize certain features of the particle flow, such as particle orientation and / or confinement and / or particle integrity. All of these tips can be used in conjunction with other features of the invention described above, including ridges on the transfer tube or housing, flow focusing chambers, and confinement chambers. Figure 8 A conical tip 830C is illustrated at the delivery tube outlet 840 of a delivery tube 830 and can be used with any of the aforementioned examples or variations thereof. The lower figure shows a perspective view of the conical tip, with the middle and upper figures showing an end view and a longitudinal cross-section in vertical planes XZ and XY. As can be seen, the conical tip tapers toward the distal end of the delivery tube in both planes.

[0361] Figure 9An example of a beveled tip 930B at the tube outlet of a transfer tube 930 is shown, which can be used with any of the aforementioned examples or variations thereof. The lower figure shows a perspective view of the beveled tip, with the middle and upper figures showing an end view and a longitudinal cross-section in vertical planes - XZ and XY. As can be seen, the beveled tip tapers toward the distal end of the transfer tube in one plane (XY), but does not taper in the other plane (XZ). The bevel angle δ can be varied to change the orientation characteristics of the particle stream.

[0362] Figure 10 A dual-bevel tip 1030B1, 1030B2 at the delivery tube outlet of a delivery tube 1030 is illustrated and can be used with any of the aforementioned examples or variations thereof. The lower figure shows a perspective view of the dual-bevel tip, with the middle and upper figures showing an end view and a longitudinal cross-section in vertical planes - XZ and XY. As can be seen, the dual-bevel tip has one bevel 1030B2 that tapers toward the distal end of the delivery tube in one plane (XZ), and another bevel 1030B1 that tapers toward the distal end of the delivery tube 1030 in another plane (XY). The taper angles of the two planes can be different or the same. In the illustrated example, the taper in the XZ plane is shorter and shallower than the taper in the XY plane, maintaining more width at the distal end of the delivery tube.

[0363] Figure 11 The double bevel 1130B1, 1130B2 with a notched 1130U end is illustrated and can be used with any of the previous examples or variations thereof. The lower figure shows a perspective view of the double bevel and notched end, with the middle and upper figures showing an end view and a longitudinal cross-section in the vertical planes - XZ and XY. This example is similar to Figure 10 1130B2 and includes a double-beveled tip and additionally includes a notch 1130U that includes the transfer tube material removed from around the transfer tube outlet 1140. The notch can be implemented in any plane, but in this example, it is in the XZ plane, which is the same plane as the smaller bevel 1130B2 and includes some width 1130E of the distal end of the transfer tube 1130. The notch can also be any suitable shape, such as a rectangle as shown in the two upper figures or a semicircle as shown in the lower figure.

[0364] Figure 12 An example of a tip with a notch 1230U is shown, which can be used with any of the previous examples or variations thereof. The right-hand figure shows a perspective view of the tip with the notch, and the left-hand figure shows an end view and a longitudinal cross-section in vertical planes - XZ and XY. This example includes a tip similar to Figure 11 The notch 1230U of the example does not include any inclination of the end. The notch can be implemented in any plane.

[0365] Figure 12A Four additional examples of terminal shapes are illustrated in longitudinal cross-sectional views. The two upper terminals include a convex cross-sectional shape 1230CV (left) and a concave cross-sectional shape 1230CC (right). The two lower terminals each include two tapered sections in cross section. The lower left terminal 1230T1 has a first tapered section that has a larger acute angle than the second distal tapered section. The lower right terminal 1230T2 has a first tapered section that has a smaller acute angle than the second distal tapered section. In another alternative, the convex or concave profile may not be along the entire length of the terminal, but for example only at the end of the terminal. Various other alternatives are possible, in which the profile of the terminal is uneven along its length.

[0366] The tip profile can be used to control the fluid velocity and the angle at which the sheath approaches the sample fluid.

[0367] Figure 13 Four examples of sheath flow channels and lumens of flow control devices are illustrated and can be used with any of the preceding examples or variations thereof. Figure 3 BB and illustrates a cross section through the housing of the flow control device and the third portion S3 of the transfer tube. In the left-hand figure, the housing 1305-1 has a circular cross-section cavity 1310-S3-C, the transfer tube portion 1330-S3-E has an elliptical cross section, and the inner cavity 1345 has a circular cross section.

[0368] In the second figure from the left, the housing 1305-2 has an elliptical cross-section cavity 1310-S3-E, the transfer tube portion 1330-S3-E has a (smaller) elliptical cross-section, and the inner cavity 1345 has a circular cross-section. In the second figure from the right, the housing 1305-3 has a circular cross-section cavity 1310-S3-C, the transfer tube portion 1330-S3-D has a diamond-shaped cross-section, and the inner cavity 1345 1345-E has an elliptical cross-section.

[0369] In the rightmost figure, the outer shell 1305-4 has a circular cross-sectional cavity 1310-S3-C, the transfer tube portion 1330-S3-A has an asymmetrically curved cross-sectional shape, and the inner cavity 1345 1345-E has an elliptical cross-sectional shape and is offset from the center of the transfer tube. The cross-sectional shape may change along the length of the tube.

[0370] By varying the combination of cavity, transfer tube, and lumen geometries, particle and sheath flows through the flow control device can be controlled to optimize various characteristics including, but not limited to: particle orientation and confinement by the flow stream, particle integrity, particle distribution within the combined fluid stream, and particle transport time.

[0371] Various experimental results related to some examples are given in Figures 14 to 21 The axes of the graph are micrometers (x-axis) and the number of pixels representing the number of particles (y-axis). Figure 14 Illustrates particle confinement using a conical tip, with an image of the particle flow at a flow rate of 200 mm / s on the vertical axis (left) and a graph illustrating the distribution of 80% of the particles (right). As can be seen, 80% of the particles are well confined on both axes. Figures 15 to 17 Similar graphs and charts are illustrated for different particle flow rates of 100 mm / s, 50 mm / s and 500 mm / s, respectively. It can be seen that similar tight confinement is maintained for the different flow rates.

[0372] Figure 18 The positions of the particles at various particle flow rates are illustrated. The particles are well confined at all flow rates, although they shift slightly to one side as the flow rate increases. However, this shift is well within the tolerance of downstream investigation, orientation, and / or displacement / selection processes. This compares favorably to conventional needle delivery methods, where the particle stream changes position significantly with varying flow rates and often requires realignment.

[0373] Figure 19 The core flow distribution for 80% of the particles using a transfer tube end with a 40 degree bevel and a transfer tube with a circular outer cross section paired with a shell cavity also having a circular cross section is illustrated to the left of the vertical axis. This may correspond to, for example, the sheath flow chamber arrangement of FIG. 4 and Figure 8 On the right, the core flow distribution for 80% of the particles using a transfer tube with a double-slope tip and an elliptical outer section paired with a shell cavity with a circular section is shown. This may correspond to, for example Figure 13 Sheath flow chamber arrangement (far left) and Figure 10 The graph shows these distributions for two arrangements (left and right) at a flow rate of 200 mm / s. The upper and lower images show the particles scattered in two orthogonally aligned sensors. It can be seen that the double-bevel tip with an elliptical transfer tube provides improved and stable confinement in both axes at this flow rate.

[0374] Figure 20 Corresponding to a transfer tube having a 40 degree single beveled end and a circular cross-section outer surface paired with a circular cross-section housing cavity inner surface. Figure 20 The distribution and position of particles at different particle flow rates are illustrated.

[0375] Figure 21 Corresponding to a transfer tube having a 40 degree single beveled end and an elliptical cross-section outer surface paired with a circular cross-section housing cavity inner surface. Figure 21The positions of the particles at different particle flow rates are illustrated. It can be seen that using an elliptical cross-sectional outer surface of the transfer tube reduces particle distribution and provides a more uniform flow position at different flow rates.

[0376] Using some of these examples, it can be seen that using different combinations of the above features of the flow control device, 80% of the particles can be confined to a range of ranges including 100 microns, 80 microns, 60 microns, 50 microns, 30 microns, and 20 microns. Different ranges can be used for different downstream particle processing.

[0377] Figure 22 A flow chart illustrating a method 2200 of using a flow control device according to one example is shown. At 2205, the method 2200 optionally selects a transfer tube and housing combination and joins them to form a flow control device for use in the remainder of the method. This can be performed manually and can involve selecting different combinations of housings and transfer tubes in order to implement different controls over the particle stream. For example, these can be selected to produce focusing chambers and / or sheath flow channels with certain dimensional characteristics in order to control various characteristics of the sheath flow and particle flow through the device as previously described. The selected housing and transfer tube can be engaged with each other using a simple push fit, where the engagement of the transfer tube within the housing is controlled by one of the engagement structures such as those previously described. In another example, the transfer tube and housing can be a unitary assembly.

[0378] At 2210, the flow control device receives a fluid flow and a particle flow in a sheath fluid flow. As previously described, the particle flow can be received into the lumen of the transfer tube, and the sheath flow can be received into the passage between the transfer tube and the housing.

[0379] At 2215, one or more properties of the particle and sheath flows can be controlled to produce a microfluidic stream having desired properties. For example, the velocities of the particle and sheath flows, as well as the geometry of the selected transfer tube and housing, can be controlled to produce a microfluidic stream having a well-confined particle flow path in which the particles are generally oriented in a predetermined manner.

[0380] At 2220, the microfluidic stream of the particle stream and the sheath stream can be launched into a flow environment including a processing microchannel. Such a processing microchannel can guide the microfluidic stream to the particle processing device. In an alternative arrangement, the flow environment can be a liquid or gas environment through which the microfluidic stream flows.

[0381] The particles can be examined to determine particle characteristics at 2225. For example, a radiation beam can be directed at the particles to determine their type, such as X or Y sperm cells.

[0382] At 2230, a subpopulation of particles can be selected based on the determined particle characteristics. For example, Y sperm cells can be displaced into a different flow path than X sperm cells. This can be accomplished using a laser to displace the Y cells from their current flow path. The particle stream containing X sperm cells can then be directed to a collection container, while the particle stream containing Y sperm cells can be discarded.

[0383] The aperture 380 emits the microfluidic stream generated using the or each focusing chamber into a flow environment and confinement chamber (if used) for downstream processing. The aperture may have a cross-sectional shape having a dimension that is longer along one axis than along a perpendicular axis. The microfluidic stream is emitted from the aperture into a flow environment, such as a process microchannel or a gas atmosphere.

[0384] In some examples, the width 670 (X-axis) of the transport microchannel is between 10 μm and 200 μm. Preferably, the width is between 50 μm and 150 μm. In some examples, the width is equal to the depth (Y-axis).

[0385] In one example suitable for use with any of the flow control devices described herein, a transport microchannel proximate an orifice includes a tapered portion from a smaller transport microchannel cross-sectional area upstream to a larger transport microchannel cross-sectional area downstream, such as at the orifice itself, where the cross-sectional area is defined perpendicular to the flow axis. Figure 29 3001. It has been discovered that this trumpet-shaped transport microchannel to orifice transition produces a more stable microfluidic stream 385 within the downstream flow environment, which provides enhanced confinement, interrogation, and sorting.

[0386] In some examples, the length (Z-axis) of the transport microchannel 675 is at least 10 microns to 10 mm from the outlet of the downstream confinement chamber to the orifice 380, where the orifice is defined as the point on a plane perpendicular to the z-axis of flow aligned with the terminal end of the flow focusing device. In one example, the channel length 675 is in the range of 50 microns to 1 mm. This length is important to allow laminar flow to be reestablished after orientation and confinement of particles in the upstream orientation and confinement chambers.

[0387] exist Figure 6D In one example shown, the width 670 is reduced from width W2 at the downstream aperture 380 compared to the upstream width 670 at the entry point 374 of the transport microchannel 375 at W1. This reduction in width from W1 to W2 shown has the beneficial effect of further increasing particle confinement. The reduction in width can be as follows: Figure 6DThe tapering shown is along the length of the transport microchannel or along a portion thereof. In one example, W2 is between 50% and 95% of W1. In a specific example, the width decreases from 100 μm-150 μm to 50 μm-99 μm. In a specific example, the width decreases from approximately 125 μm to approximately 75 μm. In this case, approximately is intended to mean (+ / - 20%).

[0388] The cross-sectional shape of the transport microchannel 375, its inlet and outlet or aperture 380 may be the same or different and may include: circular, elliptical, triangular, square or rectangular of various aspect ratios. In one example, the transport microchannel 375, its inlet and aperture 380 include identical rectangular cross-sections with an aspect ratio greater than 1:1. In another example, the aperture cross-sectional shape includes a square or a rectangle. Without being bound by theory, the inventors believe that a cross-sectional shape with right-angled corners provides benefits in providing a substantially flat surface for the interrogation beam to enter the stream and for any light emission to exit the stream. This is particularly true where the stream is emitted into a rectangular microfluidic channel, but also applies to embodiments where the microfluidic stream is emitted into a gas free space fluid flow environment.

[0389] Although it should be understood that the various characteristic structures of examples can be combined, they also can be used independently of each other. For example, the end geometry can be used with or without the described joint structure or sheath flow channel and lumen geometry characteristic structure. Similarly, sheath flow and lumen geometry can or can not be used with the joint structure or end geometry. The shell and transfer tube of example can also be used independently of each other, for example, assembled together with different transfer tubes and shells. Shell and transfer tube can be produced as consumable parts for larger particle processing systems and can be sold and installed separately.

[0390] Various channels, chambers, surfaces, and flows are configured as microfluidics, i.e., having dimensions that are geometrically constrained to small scales (such as sub-millimeter) where surface forces dominate. However, some examples can be configured with larger dimensions where microfluidic phenomena are partially or completely absent.

[0391] It will be appreciated that these examples provide numerous advantages, including improved particle control to facilitate downstream processing. Accurate and secure positioning control of some examples ensures that the particle flow is positionally stable, improving the accuracy and efficiency of downstream processing, such as particle interrogation, particle orientation, particle displacement / selection, and particle sorting. Accurate control of sheath and particle flow also helps improve various downstream processing-related properties of the particles, such as orientation and confinement. This, in turn, enables increased particle flow rates while minimizing damage to the particles, thereby accelerating particle processing and processing efficiency. In one example, this can allow for faster separation rates of X and Y chromosome sperm cells while improving separation efficiency, such as retrieving samples with 80% or more sperm cells bearing an X chromosome.

[0392] These examples also provide the ability to repeatably and accurately position the transfer tube within the housing of the flow control device, which reduces setup time and avoids the need for trained operators. Furthermore, the improved mating engagement between the transfer tube and the housing provides mechanical rigidity and isolation from external forces, which ensures that the transfer tube is not only accurately positioned initially, but remains so for extended periods of time, thereby reducing the need for realignment and processing downtime.

[0393] It should be noted that the above examples illustrate rather than limit the present disclosure, and that those skilled in the art will be able to design many alternative examples without departing from the scope of the appended claims. The word "comprising" does not exclude the presence of elements or steps other than those listed in a claim, and "a" or "an" does not exclude a plurality, and a single processor or other unit may perform the functions of several units recited in a claim. Any reference signs in the claims should not be construed as limiting their scope.

[0394] 6. Examples

[0395] Example 1 - Particle Orientation and Resolving Characterization Using a Two-Part Focusing Chamber

[0396] method

[0397] Staining of bovine sperm cells was performed using Hoescht 33342 according to standard protocols as described in Garner et al. 2013 (Garner DL, Evans KM, Seidel GE. Sex-sorting sperm using flow cytometry / cell sorting. Methods Mol Biol. 2013; 927: 279-95).

[0398] Install Figure 6CThe flow focusing device shown in FIG is primed with fluid to remove any bubbles. The emitted microfluidic stream is aligned with the interrogation beam. The stained cells are injected through the sample channel and combined with the sheath fluid within the flow focusing device. The combined sample and sheath fluids are passed through the orientation and confinement chamber and through the aperture.

[0399] Fluorescence intensity data was detected using an orthogonal detector perpendicular to the z-axis of the flow, and the fluorescence integral was determined. A scatter plot corresponding to the integral of individual particles was generated. The alignment efficiency was measured as described in WO2022139597A1.

[0400] Two Gaussian peaks are obtained, corresponding to the two populations of sperm cells carrying X and Y chromosomes, respectively. These peaks reflect the integration of multiple fluorescence pulses. Resolution is calculated as the difference between the peak integration values ​​divided by the sum of the peak standard deviations. This is a standardized method for determining the spacing and width of the peaks, which in turn provides a model for determining the probability of a new fluorescence pulse belonging to the first or second population.

[0401] result

[0402] Figure 6F Results from adjusting the width of the downstream flow focusing chamber in the flow focusing device of the present invention are shown. The flow rate was adjusted between 5 m / s and 9 m / s, and the width of the downstream flow focusing chamber was adjusted between 3 mm and 6 mm. It can be observed that the cells were consistently well-oriented, i.e., all greater than 0.68. A width of 4 mm appears to be optimal for both orientation efficiency and resolution.

[0403] A device with a width of 3 mm will experience PODT alignment issues at a speed of 5 m / s, but will still provide good orientation efficiency.

[0404] in conclusion

[0405] The flow focusing device of the present invention provides excellent orientation efficiency and resolution within a certain range of widths and flow rates.

[0406] Example 2 - Improving Orientation Efficiency by Changing the PODT End Position

[0407] method

[0408] The position of the particle orientation and delivery tube (PODT) end along the flow direction (Z axis) was modified to analyze its effect on the orientation effect. Figure 6C, and the adjusted Z-axis distance is marked dZ. Sheath fluid is received from an inlet above the transfer tube. Sample fluid containing sperm cells is transferred from the stop point through tubing within the transfer tube. The cavity that houses the transfer tube is 8 mm in diameter and has a conical angle of 30° to the Z-axis, entering a downstream second focusing chamber with a cross-section of 130 μm depth (D) by a constant width of 4 mm (L). The transfer tube itself has 4 channels / sectors to allow the sheath fluid to flow and establish a stable flow around its 20° to Z-axis tapered end. The position of the PODT end in the z-direction is varied to the nominal point where the conical end will meet the downstream second focusing chamber ( Figure 6D The final outlet of the fluid into the transport microchannel has a downstream restriction chamber with a gradually tapering outlet and pore from 4 mm wide to 100 μm in diameter.

[0409] A series of PODTs were manufactured with tip positions ranging from 1 mm to 6 mm in the z-axis. At each position, a series of flow rates ranging from 3.3 m / s to 7 m / s were used.

[0410] The orientation efficiency can be seen in Figure 2 A and Figure 23B middle.

[0411] result

[0412] Over a range of flow rates and transfer tube end distances, orientation efficiencies greater than 50% were achieved using this PODT device. For each of the PODT end positions, there was a slight trend in the orientation coefficient (OE) decreasing with increasing flow rate. Furthermore, the OE decreased as the PODT moved forward in the Z axis, closer to where the end of the cone meets the downstream second focusing chamber.

[0413] in conclusion

[0414] The results show that the PODT and shell configurations provide consistently high orientation efficiencies across a range of PODT tip distances and flow rates. This demonstrates that the PODT configurations described herein as part of the present invention can be adapted to varying locations and parameters without sacrificing the high orientation efficiencies required for effective downstream particle resolution and sorting.

[0415] Example 3 - Modeling of Orientation Characteristics of Flow Focusing Devices

[0416] Finite element modeling (FEM) simulations have been performed with the same geometry as described in Example 2. The mechanism of orientation is believed to be caused by asymmetry. Therefore, the following parameters were calculated and compared to understand the fluid dynamics of the proposed device: asymmetric compressibility factor (ACF), velocity component ratio (V r ), velocity gradient ratio (V gr) and vorticity (the curl of the velocity field).

[0417] In the examples provided herein, the particles being analyzed are sperm cells. Bovine sperm cells have flat heads. For maximum fluorescence detection, it is desirable to have the sperm facing flat side up toward the interrogation beam. Therefore, it is desirable to adjust the flow focusing device to orient the cells in a specific orientation relative to the interrogation beam (285L). The simulation data reported in this example are focused on the downstream confinement chamber ( Figure 6B and Figure 6C on P3).

[0418] Method - Velocity Gradient Ratio

[0419] The asymmetry of the velocity gradient is a metric used to determine and optimize orientation. There are a total of 9 velocity gradients, 3 for each velocity component (x, y, z) as shown. x and V y The ratio between is chosen as the key metric for evaluating orientation. It describes the asymmetric velocity component of the fluid field at each point in space, which can reflect the OE preference of the flow field.

[0420] The motivation for observing velocity components stems from the fluid drag Among them F D is the resistance, p is the density of the fluid, v is the relative velocity between the particle and the fluid, c D is the drag coefficient, and A is the particle cross-sectional area. It is the force that acts in opposition to the relative motion between the particle and the fluid. If there is an asymmetry between the x and y velocities, there should also be an asymmetry between the x and y drag forces, which could potentially indicate a preferred orientation within the flow, such as Figure 24 shown.

[0421] result

[0422] like Figure 25A As can be seen in Figure 2, this metric shows a generally high orientation efficiency. There is also a trend that the OE increases as the PODT moves forward in the z direction, and that the orientation efficiency increases as the maximum speed decreases ( Figure 25B ).

[0423] Conclusions from Orientation Efficiency Experiments

[0424] Effect of Flow Rate— When the effect of flow rate was considered, similar trends were observed between experimental observations and modeling, i.e., orientation efficiency decreased with increasing flow rate.

[0425] Effect of the Transfer Tube's dZ Position—A mismatch was observed between simulation and experiment regarding the effect of the PODT tip position on the OE. This was investigated and attributed to experimental deviations in the XY plane of the PODT tip relative to the chip center. Inspection revealed that this was due to incorrect positioning of the PODT within the housing, causing the tip to tilt or shift laterally to one side, leading to a decrease in OE. Due to this observation, further stabilization and alignment of the PODT within the housing was performed. This demonstrates the importance of the engagement structure, including ridge 335, for laterally and longitudinally positioning the PODT tip within the housing.

[0426] Example 4 - Modeling of Misaligned Ends of Transfer Tubes

[0427] method

[0428] To illustrate the effects of misaligned or tilted tips, further simulations were performed, e.g. Figure 26A 、 Figure 26B and Figure 26C shown.

[0429] result

[0430] V x / V y exist Figure 26B and Figure 26C The example in was chosen as the most relevant metric for determining orientation efficiency. Figure 26D A trend is shown in which the orientation efficiency decreases with increasing flow rate.

[0431] in conclusion

[0432] and Figure 23A Compared with the experimental analysis in Figure 26B The graph in clearly shows a similar OE behavior. This indicates that a small change in the XY position (tilt) of the PODT tip leads to a significant drop in OE. Figure 26C The modeling of PODT with lateral displacement shows the same results. The trend of decreasing orientation efficiency with increasing flow rate is consistent with the results from Figure 23B and Figure 25B The results of the experimental analysis are consistent with those in .

[0433] The simulation and experimental results performed showed that the PODT tip position in the z-axis and the flow rate do affect the OE.

[0434] These simulations show that if the tips are misaligned, the sample flow cannot be sustained in the center of the downstream second focusing chamber. This leads to a disruption of the asymmetric compression of the flow and thus adversely affects the OE.

[0435] Simulations have confirmed that the experimentally observed loss of orientation with increasing flow rate ( Figure 23B ) is due to misalignment of the transfer tube end. Simulations also found that as the PODT end gets closer to the downstream second focusing chamber, the tilt / lateral change of the PODT has a negative impact on OE. This effect is believed to be caused by the increased deviation of the particle flow path when the transfer tube end is closer to the downstream second focusing chamber. These experiments illustrate the importance of end positioning when using a PODT with particle flow. The invention described herein is able to accurately position the transfer tube outlet at a predetermined longitudinal, lateral or rotational position within the focusing chamber, and various feature structures exert stabilizing and orienting torques on the cells. Therefore, the present invention provides enhanced orientation efficiency and therefore provides improved downstream resolution and sorting.

Claims

1. A flow control device, comprising: a transfer tube having an inner lumen, the transfer tube extending within the housing along a longitudinal axis to the focusing chamber; one or more channels defined between an outer surface of the transfer tube and an inner surface of the housing, the channels extending within the housing toward an inner lumen outlet of the transfer tube and at least partially aligned with the longitudinal axis of the transfer tube; The focusing chamber is fluidly coupled to an aperture in the flow control device.

2. The device of claim 1, wherein the one or more channels extend substantially parallel to or at an angle relative to the longitudinal axis of the transfer tube.

3. A flow control device according to claim 1 or 2, wherein the one or more channels are defined by corresponding protrusions extending along the transfer tube and / or the housing. 4 . The flow control device of claim 3 , wherein at least some of the protrusions extend from the transfer tube to contact the inner surface of the housing, or extend from the inner surface of the housing to contact the transfer tube.

5. The flow control device of any one of claims 2 to 4, wherein the protrusions are arranged to contact the inner surface of the housing or the transfer tube at multiple longitudinal and lateral locations so as to position the transfer tube outlet at a predetermined position within the focusing chamber.

6. A flow control device according to any preceding claim, wherein the one or more channels: a. comprising a substantially semicircular or triangular cross-section; and / or b. extending from the sheath fluid inlet to the focusing chamber; and / or c. being configured to provide a single flow path or multiple flow paths for sheath fluid between the sheath flow inlet and the focusing chamber over at least a portion of the longitudinal extension of the transfer tube within the housing; and / or d. having a larger cross-sectional area at a downstream location than at an upstream location of the one or more channels.

7. A flow control device according to any preceding claim, wherein the one or more channels are spaced around the entire outer cross-sectional circumference of the transfer tube and merge into the focusing chamber, which is at least partially defined by the tapered transfer tube and the inverted tapered outer shell wall.

8. A flow control device according to any preceding claim, wherein the one or more channels are defined by two or more ridges that define the cross-sectional shape of the channels, and wherein the ridges are sized to provide a friction fit with the housing such that there is essentially zero volume between the ridges and the inner surface of the housing.

9. A flow control device according to any preceding claim, comprising an engagement structure configured to: The transfer tube is secured to the housing during use to form at least one of the one or more channels between the housing and the transfer tube, and the transfer tube outlet is positioned at a predetermined longitudinal, lateral, and / or rotational position within the focusing chamber.

10. The flow control device of claim 9, wherein the housing has a first portion having a longitudinal cross-sectional shape arranged to engage the first portion of the transfer tube at a plurality of longitudinal locations so as to position the transfer tube outlet at a predetermined lateral position within the focusing chamber.

11. A flow control device according to any preceding claim, wherein the engagement structure includes a ridge extending longitudinally within the cavity of the housing, the ridge being configured to position the transfer tube outlet according to a predetermined lateral relationship with the housing when the transfer tube outlet is received in the housing.

12. The flow control device of any one of claims 9 to 11, wherein the engagement structure comprises a rotational alignment feature arranged to secure the transfer tube outlet at a predetermined rotational angle relative to the housing.

13. A flow control device according to any preceding claim, wherein the internal lateral cross-sectional shape of the housing in which the transfer tube is positioned is different from the external lateral cross-sectional shape of the transfer tube at a corresponding longitudinal position, and wherein the one or more channels are defined by the difference in lateral cross-sectional shapes at different longitudinal positions.

14. A flow control device according to any preceding claim, wherein the inner surface of the housing defines a cavity for receiving the transfer tube, the cavity terminating in the focusing chamber, wherein the geometry of the cavity is different from the geometry of the focusing chamber at the terminal end, the geometry including one or more of the following: size, cross-sectional shape, longitudinal taper angle.

15. The flow control device of claim 14, wherein the end of the transfer tube is positioned within the focusing chamber at a predetermined distance from the terminal end.

16. The flow control device of any preceding claim, comprising a second focusing chamber fluidly coupled between the first focusing chamber and the aperture, wherein the geometry of the first focusing chamber is different from the geometry of the second focusing chamber.

17. The flow control device of claim 16, wherein the geometries differ in one or more of the following: size, cross-sectional shape, longitudinal taper angle.

18. The flow control device of claim 16 or 17, wherein the cross-sectional shape of the first focusing chamber has a lower aspect ratio than the cross-sectional shape of the second focusing chamber.

19. The flow control device of claim 18, wherein the cross-sectional shape of the first focusing chamber is circular and the cross-sectional shape of the second focusing chamber is substantially rectangular.

20. The flow control device of any one of claims 15 to 19, wherein the second focusing chamber is fluidly coupled to the aperture by a transport microchannel having a smaller dimension in a lateral axis than the second focusing chamber.

21. The flow control device of any one of claims 15 to 20, comprising a restriction chamber fluidly coupled to the second focusing chamber, the restriction chamber tapering longitudinally from the second focusing chamber in a lateral axis.

22. A flow control device, comprising: a transfer tube having an inner lumen, the transfer tube extending within the housing along the longitudinal axis to the first focusing chamber; the housing including a second focusing chamber fluidly coupled between the first focusing chamber and an aperture in the flow control device; The geometry of the first focusing chamber is different from the geometry of the second focusing chamber.

23. The flow control device of claim 22, wherein the geometries differ in one or more of the following: size, cross-sectional shape, longitudinal taper angle.

24. The flow control device of claim 22 or 23, wherein the cross-sectional shape of the first focusing chamber has a lower aspect ratio than the cross-sectional shape of the second focusing chamber.

25. The flow control device of claim 24, wherein the cross-sectional shape of the first focusing chamber is circular and the cross-sectional shape of the second focusing chamber is rectangular.

26. The flow control device of any one of claims 22 to 25, wherein the second focusing chamber is fluidly coupled to the aperture by a transport microchannel having a smaller dimension in a lateral axis than the second focusing chamber.

27. The flow control device of any one of claims 22 to 26, comprising a restriction chamber fluidly coupled to the second focusing chamber, the restriction chamber tapering longitudinally from the second focusing chamber in a lateral axis.

28. The flow control device of any one of claims 22 to 27, wherein the cross-sectional shape of the second focusing chamber comprises two opposing straight lines and one or more of: a curve between the two straight lines; a V-shaped line or a ridge line between the two straight lines.

29. A particle processing system, comprising: A flow control device according to any preceding claim, and Particle interrogation and / or sorting devices.

30. A method of controlling fluid flow associated with transporting particles, the method comprising: conveying a particle stream of a particle-containing liquid in a lumen of a transfer tube extending along the longitudinal axis within the housing to a focusing chamber; conveying a sheath flow of liquid in one or more channels between the transfer tube and the housing toward the focusing chamber, wherein the channels extend within the housing toward a lumen outlet of the transfer tube and are at least partially aligned with the longitudinal axis of the transfer tube; A microfluidic stream is generated using the focusing chamber and emanates from a well, the microfluidic stream comprising a laminar flow of liquid from the sheath stream surrounding liquid from the particle stream.

31. The method of claim 30, wherein the fluid flow is controlled using a flow control device according to any one of claims 1 to 21.

32. A method of controlling fluid flow associated with transporting particles, the method comprising: transporting a particle flow of a particle-containing liquid in an inner lumen of a transfer tube to a first focusing chamber; delivering a sheath flow of liquid to the first focusing chamber; A microfluidic stream is generated using the first focusing chamber and a second focusing chamber fluidically coupled to the first focusing chamber, wherein the first focusing chamber has a geometry different from a geometry of the second focusing chamber.

33. A method according to claim 32, wherein the fluid flow is controlled using a flow control device according to any one of claims 22 to 29.

34. A method of controlling fluid flow associated with transporting particles, the method comprising: conveying a particle flow of a particle-containing liquid in a lumen of a transfer tube extending along the longitudinal axis within the housing to a first focusing chamber; conveying a sheath flow of liquid in one or more channels between the transfer tube and the housing toward the focusing chamber, wherein the channels extend within the housing toward a lumen outlet of the transfer tube and are at least partially aligned with the longitudinal axis of the transfer tube; generating a microfluidic stream using the first focusing chamber and a second focusing chamber fluidically coupled to the first focusing chamber, wherein the first focusing chamber has a geometry different from a geometry of the second focusing chamber; The microfluidic stream emanates from a well, the microfluidic stream comprising a laminar flow of liquid from the sheath stream surrounding liquid from the particle stream.

35. The method of claim 33, wherein the fluid flow is controlled using a flow control device according to any one of claims 1 to 29.

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