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WO2024102006A1 - Flow control apparatus, parts and methods therefore - Google Patents

Flow control apparatus, parts and methods therefore Download PDF

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Publication number
WO2024102006A1
WO2024102006A1 PCT/NZ2023/050128 NZ2023050128W WO2024102006A1 WO 2024102006 A1 WO2024102006 A1 WO 2024102006A1 NZ 2023050128 W NZ2023050128 W NZ 2023050128W WO 2024102006 A1 WO2024102006 A1 WO 2024102006A1
Authority
WO
WIPO (PCT)
Prior art keywords
delivery tube
flow
chamber
focussing
housing
Prior art date
Application number
PCT/NZ2023/050128
Other languages
French (fr)
Inventor
Matheu BROOM
Peter Anthony Greenwood HOSKING
Simon Andrew ASHFORTH
Fan Hong
Liam Jay BARBER
Miriam Cather SIMPSON
Aidan MASON-MACKAY
Gregory George BARKER
Original Assignee
Engender Technologies Limited
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from AU2022903397A external-priority patent/AU2022903397A0/en
Application filed by Engender Technologies Limited filed Critical Engender Technologies Limited
Priority to AU2023378289A priority Critical patent/AU2023378289A1/en
Publication of WO2024102006A1 publication Critical patent/WO2024102006A1/en
Priority to IL320793A priority patent/IL320793A/en

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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B01L3/502746—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by the means for controlling flow resistance, e.g. flow controllers, baffles
    • B—PERFORMING OPERATIONS; TRANSPORTING
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    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B01L3/502761—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip specially adapted for handling suspended solids or molecules independently from the bulk fluid flow, e.g. for trapping or sorting beads, for physically stretching molecules
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    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B01L3/502769—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by multiphase flow arrangements
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    • B01L3/502769—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by multiphase flow arrangements
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    • C12N5/06—Animal cells or tissues; Human cells or tissues
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    • B01L2300/0816—Cards, e.g. flat sample carriers usually with flow in two horizontal directions
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    • B01L2400/0403—Moving fluids with specific forces or mechanical means specific forces
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Definitions

  • the present disclosure relates to the manipulation of particles in a fluid flow for downstream interrogation, orientation, displacement and/or sorting or other processes.
  • this relates to manipulation of biological cells such as sperm cells in laminar liquid flows.
  • Microfluidic laminar flows have been used to control the flow of particles such as biological cells in order to enable processing, analysis or sorting of these particles.
  • the orientation of asymmetric particles such as sperm cells can be useful for optimal processing.
  • a laser beam may be focussed at passing cells which emit various wavelengths of light which may be detected and interpreted by interrogation equipment. Therefore, it may be desirable to orient these cells to optimise the performance of the processing, analysis or sorting system.
  • Such arrangements may have application in identifying which types of cells are contained in a particle flow, for example whether they contain X or Y chromosomes. This can then be used to sort the cells into different containers. In the dairy industry this may be used to ensure that breeding cows are inseminated with X-chromosome sperm cells to provide female calves which are more valuable than male calves.
  • a flow control apparatus comprising a delivery tube having a lumen, the delivery tube extending along a longitudinal axis within a housing to a focussing chamber; one or more channels defined between an external surface of the delivery tube and an internal surface of the housing, the channels extending towards a lumen exit of the delivery tube within the housing_and at least partially aligned with the longitudinal axis of the delivery tube; the focussing chamber fluidically coupled to an aperture in the flow control apparatus.
  • the one or more channels extend substantially parallel to the longitudinal axis of the delivery tube or are angled with respect to the longitudinal axis of the delivery tube.
  • the one or more channels may be defined by corresponding projections which extend along the delivery tube and/or the housing. At least some of the projections may extend from the delivery tube to contact with the inner surface of the housing or from the inner surface of the housing to contact the delivery tube.
  • the projections may be arranged to contact the delivery tube or inner surface of the housing at multiple longitudinal and lateral locations in order to position the delivery tube outlet at a predetermined position within the focussing chamber.
  • the one or more channels a comprise a substantially semi-circular or triangular cross-section; and/or b. extend from a sheath fluid inlet to the focussing chamber; and/or c. are configured to provide the only flow path or paths for the sheath fluid between a sheath flow inlet and the focussing chamber over at least a portion of the longitudinal extension of the delivery tube within the housing; and/or d. comprise a larger cross-sectional area at a downstream position compared with a cross-sectional area at an upstream position of said one or more channels.
  • the one or more channels may be spaced around the entirety of the external sectional circumference of the delivery tube and merge into the focussing chamber which is at least partially defined by a tapered delivery tube and an inversely tapered housing wall.
  • the one or more channels may be defined by two or more ridges defining the cross- sectional shape of the channels and wherein said ridges are dimensioned to provide a friction fit with the housing such that there is substantially zero volume between the ridge and an interior surface of the housing.
  • the flow control apparatus comprises an engagement structure configured to secure the delivery tube during use with the housing to form at least one of the one or more channels between the housing and the delivery tube, and position the delivery tube outlet at a predetermined longitudinal, lateral, and/or rotational location within the focusing chamber.
  • the housing may have a first portion with a longitudinal cross-sectional shape arranged to engage at multiple longitudinal locations with a first portion of the delivery tube in order to position the delivery tube outlet at a predetermined lateral location within the focussing chamber.
  • the engagement structure may comprise longitudinally extending ridges within the cavity of the housing, the ridges configured to position the delivery tube outlet according to the predetermined lateral relationship with the housing when received therein.
  • the engagement structure comprises a rotational alignment feature arranged to secure the delivery tube outlet at a predetermined rotational angle with respect to the housing.
  • an interior lateral cross-sectional shape of the housing within which the delivery tube is positioned is different to an exterior lateral cross-sectional shape of the delivery tube at a corresponding longitudinal position, and wherein the one or more channels are defined by said difference in lateral cross-sectional shape at different longitudinal positions.
  • the inner surface of the housing defines a cavity for receiving the delivery tube, the cavity terminating in the focussing chamber, wherein the geometry of the cavity is different than the geometry of the focussing chamber at the termination; the geometries comprising one or more of the following: dimensions; cross-sectional shape; longitudinal tapering angle.
  • An end of the delivery tube may be positioned within the focussing chamber a predetermined distance from the termination.
  • the flow control apparatus comprises a second focussing chamber fluidically coupled between the first focussing chamber and the aperture, wherein the geometry of the first focussing chamber is different to the geometry of the second focussing chamber.
  • the geometries may differ in one or more of the following: dimensions; cross-sectional shape; longitudinal tapering angle.
  • the cross-sectional shape of the first focussing chamber may have a lower aspect ratio that the cross- sectional shape of the second focussing chamber.
  • the cross-sectional shape of the first focussing chamber may be circular and the cross-sectional shape of the second focussing chamber may be substantially rectangular.
  • the second focussing chamber may be fluidically coupled to the aperture by a delivery microchannel with a dimension in one lateral axis smaller than the second focussing chamber.
  • the flow control apparatus comprises a confinement chamber fluidically coupled to the second focussing chamber, the confinement chamber tapering longitudinally in one lateral axis from the second focussing chamber.
  • a flow control apparatus comprising a delivery tube having a lumen, the delivery tube extending along a longitudinal axis within a housing to a first focussing chamber; the housing comprising a second focussing chamber fluidically coupled between the first focussing chamber and an aperture in the flow control apparatus; wherein the geometry of the first focussing chamber is different to the geometry of the second focussing chamber.
  • the geometries differ in one or more of the following: dimensions; cross- sectional shape; longitudinal tapering angle.
  • the cross-sectional shape of the first focussing chamber may have a lower aspect ratio that the cross-sectional shape of the second focussing chamber.
  • the cross-sectional shape of the first focussing chamber may be circular and the cross- sectional shape of the second focussing chamber may be rectangular.
  • the second focussing chamber is fluidically coupled to the aperture by a delivery microchannel with a dimension in one lateral axis smaller than the second focussing chamber.
  • the flow control apparatus comprises a confinement chamber fluidically coupled to the second focussing chamber, the confinement chamber tapering longitudinally in one lateral axis from the second focussing chamber.
  • the cross-sectional shape of the second focussing chamber comprises two opposing straight lines and one or more of: a curved line between the two straight lines; a V-shaped or chined line between the two straight lines.
  • a particle processing system comprising a flow control apparatus as defined above and a particle interrogation apparatus and/or a sorting apparatus.
  • a method of controlling fluid flows associated with carrying particles comprises carrying a particle flow of liquid containing particles in a lumen of a delivery tube extending along a longitudinal axis within a housing to a focussing chamber; carrying a sheath flow of liquid in one or more channels between the delivery tube and the housing towards the focussing chamber, wherein the channels extend towards a lumen exit of the delivery tube within the housing and at least partially aligned with the longitudinal axis of the delivery tube; generating a microfluidic stream using the focussing chamber and issuing the microfluidic stream from an aperture, the microfluidic stream comprising a laminar flow of liquid from the sheath flow surrounding liquid from the particle flow.
  • a method of controlling fluid flows associated with carrying particles comprises carrying a particle flow of liquid containing particles in a lumen of a delivery tube to a first focussing chamber; carrying a sheath flow of liquid to the first focussing chamber; generating a microfluidic stream using the first focussing chamber and a second focussing chamber flu id ica I ly coupled to the first focussing chamber, wherein the geometry of the first focussing chamber is different to the geometry of the second focussing chamber.
  • a method of controlling fluid flows associated with carrying particles comprises carrying a particle flow of liquid containing particles in a lumen of a delivery tube extending along a longitudinal axis within a housing to a first focussing chamber; carrying a sheath flow of liquid in one or more channels between the delivery tube and the housing towards the focussing chamber, wherein the channels extend towards a lumen exit of the delivery tube within the housing_and at least partially aligned with the longitudinal axis of the delivery tube; generating a microfluidic stream using the first focussing chamber and a second focussing chamber fluidically coupled to the first focussing chamber, wherein the geometry of the first focussing chamber is different to the geometry of the second focussing chamber; issuing the microfluidic stream from an aperture, the microfluidic stream comprising a laminar flow of liquid from the sheath flow surrounding liquid from the particle flow.
  • one or more of these methods use a flow control apparatus as defined above.
  • a flow control apparatus for controlling fluid flows associated with carrying particles.
  • the flow control apparatus comprises a delivery tube having a lumen for carrying a particle flow of liquid containing particles from a delivery tube inlet to a delivery tube outlet, the delivery tube extending along a longitudinal axis within a housing to a focussing chamber; one or more channels for carrying a sheath flow of liquid towards the focussing chamber, the one or more channels defined between an external surface of the delivery tube and an internal surface of the housing, the channels extending in a direction substantially aligned with the longitudinal axis of the delivery tube within the housing; the focussing chamber configured to combine the particle flow and the sheath flow to generate and issue a microfluidic stream from an aperture in the flow control apparatus, the microfluidic stream comprising a laminar flow of liquid from the sheath flow surrounding liquid from the particle flow.
  • the housing comprises a cavity for receiving the delivery tube, the cavity terminating in the focussing chamber, wherein the geometry of the cavity is different than the geometry of the focussing chamber at the termination; the geometries comprising one or more of the following: dimensions; cross-sectional shape; longitudinal tapering angle.
  • an end of the delivery tube is positioned within the cavity a predetermined distance from the termination.
  • an end of the delivery tube is positioned within the focussing chamber a predetermined distance from the termination.
  • the one or more channels extend in a direction parallel to the extension of the delivery tube within the housing, or parallel to a direction of flow of the particle flow.
  • the one or more channels extend along a screw axis wherein the longitudinal axis of the delivery tube within the housing or the direction of flow of the particle flow is the axis of rotation of the screw axis.
  • the flow control apparatus comprises a plurality of channels for carrying a sheath flow of liquid between the delivery tube and the housing towards the focussing chamber, wherein a downstream outlet of each said channel is regularly spaced around the delivery tube with respect to a flow path of the particle flow.
  • the one or more channels are defined by corresponding projections which extend along the delivery tube and/or the housing.
  • the projections extend from the delivery tube towards an internal surface of the housing or from the internal surface of the housing towards the delivery tube in a direction substantially perpendicular to the direction in which the channels defined by said projections extend.
  • the projections extend from the delivery tube to engage with the inner surface of the housing or from the inner surface of the housing to engage with the delivery tube.
  • an interior lateral cross-sectional shape of the housing within which the delivery tube is positioned is different to an exterior lateral cross-sectional shape of the delivery tube at a corresponding longitudinal position, and wherein the one or more channels are defined by said difference in lateral cross-sectional shape at different longitudinal positions.
  • the one or more channels : a. comprise a substantially semi-circular or triangular cross-section; and/or b. extend from a sheath fluid inlet to the focussing chamber; and/or c. are configured to provide the only flow path or paths for the sheath fluid between a sheath flow inlet and the focussing chamber over at least a portion of the longitudinal extension of the delivery tube within the housing; and/or d. comprise a larger cross-sectional area at a downstream position compared with a cross-sectional area at an upstream position of said one or more channels.
  • the one or more channels are defined by two or more ridges defining the cross-sectional shape of the channels and wherein said ridges are dimensioned to smoothly engage with the housing such that there is substantially zero volume between the ridge and an interior surface of the housing.
  • the one or more channels are angled with respect to the longitudinal axis of the delivery tube.
  • the one or more channels are arranged at regular intervals around the entirety of the external sectional circumference of the delivery tube and merge into the focussing chamber which is at least partially defined by a tapered delivery tube and an inversely tapered housing wall.
  • a longitudinal section of the exterior of the delivery tube adjacent the delivery tube outlet comprises one or more of the following: a conical shape; a hemispherical shape; a cylindrical shape; bevelling in one lateral axis; bevelling at two angles in one lateral axis; partial bevelling in a second perpendicular lateral axis; a notch in a lateral axis.
  • the interior shape of a longitudinal section of the housing corresponding to the longitudinal section of the exterior of the delivery tube comprises a constant or a tapering lateral cross-sectional shape and/or dimensions.
  • the delivery tube and the housing define multiple longitudinal cavity sections between them for carrying the sheath flow, the longitudinal cavity sections having different cross-sectional shapes and/or dimensions from each other.
  • the interior shape of the housing or the exterior shape of the delivery tube of any one of the longitudinal sections comprises one or more of the following: longitudinal tapering; a cross-sectional shape that is circular or elliptical or rectangular.
  • the interior shape of the housing has a first portion with a longitudinal cross-sectional shape arranged to engage at multiple longitudinal locations with a first portion of the delivery tube in order to position the delivery tube outlet at a predetermined lateral location within the focussing chamber.
  • the first portion of the interior shape of the housing has a rectangular longitudinal cross-sectional shape
  • the first portion of the delivery tube has a rectangular longitudinal cross-sectional shape
  • the interior shape of the housing has a second cross-section shape with a reducing transverse width arranged to position the delivery tube outlet at a predetermined longitudinal location within the focussing chamber.
  • the delivery tube has an delivery tube inlet portion having delivery tube inlet cross-sectional dimensions, a delivery tube tapering portion and a delivery tube distal portion having delivery tube distal cross-sectional dimensions which are smaller than the delivery tube inlet cross-sectional dimensions, and wherein the interior shape of the housing has a cavity inlet portion having cavity inlet cross-sectional dimensions, a cavity tapering portion and a cavity distal portion having cavity distal portion dimensions.
  • the delivery tube distal cross-sectional dimensions and the cavity distal cross-sectional dimensions are both uniform in the longitudinal direction.
  • the cross-sectional shape of any one of the delivery tube inlet portion, the delivery tube tapering portion, the delivery tube distal portion is: circular; noncircular; elliptical; rectangular.
  • the cross-sectional shape of any one of the cavity inlet portion, the cavity tapering portion, the cavity distal portion is: circular; non-circular; elliptical; rectangular.
  • the cross-sectional shape of any one of the delivery tube inlet portion, the delivery tube tapering portion, the delivery tube distal portion is different to the cross-sectional shape of the corresponding cavity inlet portion, cavity tapering portion, cavity distal portion.
  • a delivery tube tapering angle of the delivery tube tapering portion is the same as a cavity tapering angle of the cavity tapering portion.
  • a delivery tube tapering angle of the delivery tube tapering portion is less than a cavity tapering angle of the cavity tapering portion.
  • the delivery tube at the delivery tube outlet has a conical shape.
  • the delivery tube at the delivery tube outlet is bevelled in one axis.
  • the delivery tube at the delivery tube outlet is partially bevelled in a second perpendicular axes.
  • the delivery tube at the delivery tube outlet has a notch in a notch axis.
  • the lumen comprises one or more of the following: a non-circular cross- sectional shape along at least part of its length; a plurality of portions having different cross-sectional dimensions.
  • the lumen comprises a plurality of portions having different cross- sectional dimensions.
  • the focussing chamber comprises a substantially constant lateral cross- sectional shape along a longitudinal axis.
  • the lateral cross-sectional shape may have an aspect ratio of substantially 1 : 1.
  • the downstream second focussing chamber comprises a longitudinally extending projection.
  • the height of the longitudinally extending projection tapers along its length.
  • the longitudinally extending projection forms a V-shaped inner surface of the focussing chamber.
  • the V-shaped inner surface forms at least one side of a lateral rectangular cross-sectional shape of the focussing chamber.
  • a second V-shaped inner surface forms a second side of the lateral rectangular cross-sectional shape of the focussing chamber.
  • the focussing chamber tapers longitudinally such that the height of the focussing chamber in at least one axis perpendicular to the longitudinal axis varies.
  • the focussing chamber tapers longitudinally such that the height and width of the focussing chamber in both axes perpendicular to the longitudinal axis both vary.
  • the flow control apparatus comprises a second focussing chamber fluidically coupled between the first focussing chamber and the aperture, wherein the geometry of the first focussing chamber is different than the geometry of the second focussing chamber; the geometries comprising one or more of the following: dimensions; cross-sectional shape; longitudinal tapering angle; a longitudinally extending projection.
  • the second focussing chamber comprising a lateral cross-sectional shape having a higher aspect ratio than a lateral cross-sectional shape of the first focussing chamber.
  • the second focussing chamber comprises a longitudinally extending projection.
  • the height of the longitudinally extending projection tapers along its length.
  • the longitudinally extending projection forms a V-shaped inner surface of the second focussing chamber.
  • the V-shaped inner surface forms one side of a lateral cross-sectional shape of the second focussing chamber.
  • a second V-shaped inner surface forms a second side of the lateral rectangular cross-sectional shape of the second focussing chamber.
  • the second focussing chamber extends longitudinally with a substantially constant cross-sectional dimension in a first lateral axis.
  • the second focussing chamber extends longitudinally with a substantially constant cross-sectional dimension in a second lateral axis perpendicular to the first lateral axis.
  • the focussing chamber tapers longitudinally such that the height of the focussing chamber in at least one axis perpendicular to the longitudinal axis varies.
  • the focussing chamber tapers longitudinally such that the height and width of the focussing chamber in both axes perpendicular to the longitudinal axis both vary.
  • the second focussing chamber is fluidically coupled to the aperture by a delivery microchannel.
  • the delivery microchannel has a cross sectional shape with an aspect ratio higher than the first focussing chamber and with a dimension in one lateral axis smaller than the second focussing chamber.
  • the delivery microchannel has one of the following cross-sectional shapes: circular; elliptical; triangular; square; rectangular.
  • the delivery microchannel increases cross-sectional area towards the aperture.
  • the delivery microchannel forms an inverse conical shape towards the aperture.
  • the flow control apparatus comprises a confinement chamber fluidically coupled to the second focussing chamber, the confinement chamber tapering longitudinally in at least one lateral axis from the second focussing chamber.
  • the lateral axis comprises the same axis that the second focusing chamber has as it's longest lateral axis dimension.
  • the flow control apparatus comprises an engagement structure configured to secure the delivery tube during use with the housing to form at least one of the one or more channel between the housing and the delivery tube, and position the delivery tube outlet at a predetermined longitudinal, lateral, and/or rotational location within the focusing chamber.
  • the housing has a first portion with a longitudinal cross-sectional shape arranged to engage at multiple longitudinal locations with a first portion of the delivery tube in order to position the delivery tube outlet at a predetermined lateral location within the focussing chamber.
  • the engagement structure comprises longitudinally extending ridges within the cavity of the housing, the ridges configured to position the delivery tube outlet according to the predetermined lateral relationship with the housing when received therein.
  • the ridges are formed with the housing and/or the delivery tube.
  • the engagement structure comprises a rotational alignment feature arranged to secure the delivery tube outlet at a predetermined rotational angle with respect to the housing.
  • the rotational alignment feature is a projection from one of the delivery tube or housing and a corresponding recess in one of the housing or delivery tube.
  • a flow control apparatus a delivery tube having a lumen for carrying a particle flow of liquid containing particles from a delivery tube inlet to a delivery tube outlet; the delivery tube extending longitudinally within a housing to a focussing chamber adjacent the delivery tube outlet; the flow control apparatus configured to carry a sheath flow of liquid towards the focussing chamber; the focussing chamber configured to join the particle flow and the sheath flow in order to issue a microfluidic stream from an aperture in the flow control apparatus, the microfluidic stream comprising a laminar flow of liquid from the sheath flow surrounding liquid from the particle flow; wherein the focussing chamber comprises a substantially constant lateral cross-sectional shape along a longitudinal axis and has an aspect ratio of substantially 1: 1.
  • the delivery tube and the housing together define one or more longitudinally extending channels for carrying the sheath flow.
  • the one or more longitudinally extending channels are defined by corresponding longitudinally extending projections extending laterally from the delivery tube portion and/or the housing portion.
  • an interior lateral cross-sectional shape of the housing portion within which the delivery tube portion is positioned is different to an exterior lateral cross- sectional shape of the delivery tube portion at a corresponding longitudinal position, and wherein the one or more longitudinally extending channels are defined by said difference in lateral cross-sectional shape at different longitudinal positions.
  • the longitudinally extending channels have a semi-circular or triangular cross-section.
  • the longitudinally extending channels comprise at least one of: a. a semi-circular or triangular cross-section; b. extend from a sheath fluid inlet to the focussing chamber; c. configuration to provide the only flow path for the sheath fluid between a sheath flow inlet and the focussing chamber over at least a portion of the longitudinal extension of the delivery tube within the housing.
  • the longitudinally extending channels are angled with respect to the longitudinal axis of the delivery tube.
  • a longitudinal section of the exterior of the delivery tube adjacent the delivery tube outlet comprises one or more of the following: a conical shape; a hemispherical shape; a cylindrical shape; bevelling in one lateral axis; bevelling at two angles in one lateral axis; partial bevelling in a second perpendicular lateral axis; a notch in a lateral axis.
  • the interior shape of a longitudinal section of the housing corresponding to the longitudinal section of the exterior of the delivery tube comprises a constant or a tapering lateral cross-sectional shape and/or dimensions.
  • the delivery tube and the housing define multiple longitudinal cavity sections between them for carrying the sheath flow, the longitudinal cavity sections having different cross-sectional shapes and/or dimensions from each other.
  • the interior shape of the housing or the exterior shape of the delivery tube of any one of the longitudinal sections comprises one or more of the following: longitudinal tapering; a cross-sectional shape that is circular or elliptical or rectangular.
  • the lumen comprises one or more of the following: a non-circular shape along at least part of its length; a plurality of portions having different cross-sectional dimensions.
  • the focussing chamber tapers longitudinally.
  • the flow control apparatus comprises a second focussing chamber fluidically coupled between the first focussing chamber and the aperture, the second focussing chamber comprising a lateral cross-sectional shape having a higher aspect ratio than the first focussing chamber.
  • the second focussing chamber extends longitudinally with a substantially constant cross-sectional dimension in a first lateral axis.
  • the second focussing chamber extends longitudinally with a substantially constant cross-sectional dimension in a second lateral axis perpendicular to the first lateral axis.
  • the second focussing chamber is fluidically coupled to the aperture by a delivery microchannel having a cross sectional shape with an aspect ratio higher than the first focussing chamber and with a dimension in one lateral axis smaller than the second focussing chamber.
  • the flow control apparatus comprises a confinement chamber fluidically coupled to the second focussing chamber, the confinement chamber tapering longitudinally in one lateral axis from the second focussing chamber.
  • a particle processing system comprises a flow control apparatus as defined above, an interrogation apparatus and/or a sorting apparatus arranged to direct electromagnetic radiation at particles in a microfluidic stream issued by the flow control apparatus.
  • the flow control apparatus is arranged to orient the asymmetric particles such that the electromagnetic radiation is directed at a predetermined facet of the asymmetric particles.
  • the delivery tube and the housing form are formed together as a single unified material unit or separately as respective material units.
  • the flow control apparatus comprises an engagement structure configured to: secure the delivery tube during use with the housing to form at least one longitudinally extending channel between the housing and the delivery tube, and position the delivery tube outlet at a predetermined longitudinal, lateral, and/or rotational location within the focusing chamber.
  • a cavity of the housing has a first portion with a longitudinal cross- sectional shape arranged to engage at multiple longitudinal locations with a first portion of the delivery tube in order to position the delivery tube outlet at a predetermined lateral location within the focussing chamber.
  • the engagement structure comprises longitudinally extending ridges within the cavity of the housing, the ridges configured to position the delivery tube outlet according to the predetermined lateral relationship with the housing when received therein.
  • the engagement structure comprises a rotational alignment feature arranged to secure the delivery tube outlet at a predetermined rotational angle with respect to the housing.
  • a method of processing a stream of particles emitted from a delivery tube comprising receiving a first fluid stream comprising one or more particles and passing the stream through at least one of: a flow control apparatus as defined above or herein; a particle processing system as defined above or herein; a housing as defined above or herein; and a delivery tube as defined above or herein.
  • the method of processing comprises one or more of the following: inspecting the particles; displacing the particles into a different flow path within the processing channel; orienting the particles.
  • the method comprises exposing particles to one or more radiation sources.
  • the method comprises directing electromagnetic radiation at the particles to expose the particles to electromagnetic radiation to displace the particles by a predetermined amount and/or to orient the particles to a predetermined orientation and/or to ablate the particles.
  • the method comprises inspecting the particles and further comprises the steps of determining one or more characteristics of the particle within the stream to yield a particle characteristic; and selecting a subpopulation of the particles based on the particle characteristic.
  • the particle comprises a cell or a sperm cell.
  • the step of determining one or more characteristics comprises detecting fluorescence emitted from a particle.
  • the step of selecting a subpopulation of the particles comprises sorting the particles using an electromagnetic sorting method.
  • the electromagnetic sorting method comprises a pulse or continuous flow of energy to displace a charged or uncharged particle.
  • the sorting method comprises a radiation source configured to direct radiation on to the particle to effect at least one of a force and torque on each particle so as to induce at least one of displacing and orienting each particle relative to an axis defined by the direction of the fluid flow along the microfluidic channel.
  • a flow control apparatus comprises a housing having a particle flow outlet in fluid communication with a cavity for receiving a delivery tube, the delivery tube having a lumen for carrying a source fluid containing particles from a delivery tube inlet to a delivery tube outlet; an engagement structure arranged to secure the delivery tube in the cavity to form a channel between the housing and the delivery tube, and a focusing chamber in fluid communication with the particle flow outlet, the channel for carrying a sheath fluid to the focussing chamber; the engagement structure arranged to position the delivery tube outlet at a predetermined longitudinal, lateral or rotational location within the focusing chamber.
  • the cavity of the housing has a first portion with a longitudinal cross- sectional shape arranged to engage at multiple longitudinal locations with a first portion of the delivery tube in order to position the delivery tube outlet at a predetermined lateral location within the focussing chamber.
  • the first portion of the cavity has a rectangular longitudinal cross- sectional shape
  • the first portion of the delivery tube has a rectangular longitudinal cross-sectional shape
  • the cavity has a second cross-section shape with a reducing transverse width arranged to position the delivery tube outlet at a predetermined longitudinal location within the focussing chamber.
  • the engagement structure comprises longitudinally extending ridges on at least one of the delivery tube or within the cavity of the housing, the ridges configured to position the delivery tube outlet according to the predetermined lateral relationship with the housing when received therein.
  • the ridges are configured to form a plurality of channels between the housing and delivery tube for carrying the sheath fluid to the focussing chamber.
  • the channels have a semi-circular or triangular cross-section.
  • the ridges are angled with respect to the longitudinal axis of the delivery tube.
  • the engagement structure comprises a rotational alignment feature arranged to secure the delivery tube outlet at a predetermined rotational angle with respect to the housing.
  • the rotational alignment feature is a projection from one of the delivery tube or housing and a corresponding recess in one of the housing or delivery tube.
  • the delivery tube at the delivery tube outlet has a conical shape.
  • the delivery tube at the delivery tube outlet is bevelled in one axis.
  • the delivery tube at the delivery tube outlet is partially bevelled in a second perpendicular axes.
  • the delivery tube at the delivery tube outlet has a notch in a notch axis.
  • the lumen has a non-circular shape along at least part of its length.
  • the lumen comprises a plurality of portions having different cross- sectional dimensions.
  • the delivery tube has an delivery tube inlet portion having delivery tube inlet cross-sectional dimensions, a delivery tube tapering portion and a delivery tube distal portion having delivery tube distal cross-sectional dimensions which are smaller than the delivery tube inlet cross-sectional dimensions, and wherein the cavity of the housing has a cavity inlet portion having cavity inlet cross-sectional dimensions, a cavity tapering portion and a cavity distal portion having cavity distal portion dimensions.
  • the delivery tube distal cross-sectional dimensions and the cavity distal cross-sectional dimensions are both uniform in the longitudinal direction.
  • a delivery tube tapering angle of the delivery tube tapering portion is the same as a cavity tapering angle of the cavity tapering portion.
  • a delivery tube tapering angle of the delivery tube tapering portion is less than a cavity tapering angle of the cavity tapering portion.
  • the cross-sectional shape of any one of the delivery tube inlet portion, the delivery tube tapering portion, the delivery tube distal portion is: circular; noncircular; elliptical; rectangular.
  • the cross-sectional shape of any one of the cavity inlet portion, the cavity tapering portion, the cavity distal portion is: circular; non-circular; elliptical; rectangular.
  • the cross-sectional shape of any one of the delivery tube inlet portion, the delivery tube tapering portion, the delivery tube distal portion is different to the cross-sectional shape of the corresponding cavity inlet portion, cavity tapering portion, cavity distal portion.
  • a flow control apparatus as defined above or herein, a combined flow microchannel connected to the particle flow outlet and a downstream module are arranged to direct electromagnetic radiation at particles carried by the combined flow microchannel.
  • the flow control apparatus is arranged to orient the asymmetric particles such that the electromagnetic radiation is directed at a predetermined facet of the asymmetric particles.
  • the delivery tube and the direction of the electromagnetic radiation are rotationally aligned about the longitudinal axis of the particle flow.
  • the downstream module comprises one or more of the following: a particle inspection module; a particle displacement module arranged to displace particles into a different flow path within the combined flow microchannel; a particle orientation module arranged to expose particles to one or more radiation sources to orient particles.
  • a housing for a flow control apparatus comprises a cavity in fluid communication with a particle flow outlet in fluid communication and configured to receive a delivery tube for carrying a source fluid containing particles to a delivery tube outlet; an engagement structure arranged to secure the delivery tube in use in the cavity to form a channel between the housing and the delivery tube, and to form a focusing chamber in fluid communication with the particle flow outlet, the channel for carrying a sheath fluid to the focussing chamber; the engagement structure arranged to position the delivery tube outlet at a predetermined longitudinal, lateral or rotational location within the focusing chamber.
  • the cavity has a first portion with a longitudinal cross-sectional shape arranged to engage at multiple longitudinal locations with a first portion of the delivery tube in order to position the delivery tube outlet at a predetermined lateral location within the focussing chamber.
  • the first portion of the cavity has a rectangular longitudinal cross- sectional shape, configured to engage with the first portion of the delivery tube with a rectangular longitudinal cross-sectional shape, and wherein the cavity has a second cross-section shape with a reducing transverse width arranged to position the delivery tube outlet at a predetermined longitudinal location within the focussing chamber.
  • the engagement structure comprises longitudinally extending ridges within the cavity of the housing, the ridges configured to position the delivery tube outlet according to the predetermined lateral relationship with the housing when received therein.
  • the ridges are configured to form a plurality of channels between the housing and delivery tube for carrying the sheath fluid to the focussing chamber.
  • the engagement structure comprises a rotational alignment feature arranged to secure the delivery tube outlet at a predetermined rotational angle with respect to the housing.
  • a delivery tube for a flow control apparatus for controlling fluid flows associated with carrying particles comprises a lumen for carrying a source fluid containing particles from a delivery tube inlet to a delivery tube outlet; an engagement structure arranged to secure the delivery tube in a cavity of a housing in use to form a channel between the housing and the delivery tube, and a focusing chamber in fluid communication with a particle flow outlet, the channel for carrying a sheath fluid to the focussing chamber; the engagement structure arranged to position the delivery tube outlet at a predetermined longitudinal, lateral or rotational location within the focusing chamber.
  • the delivery tube comprises a first portion with a longitudinal cross- sectional shape arranged to engage at multiple longitudinal locations with a first portion of the cavity of the housing in order to position the delivery tube outlet at a predetermined lateral location within the focussing chamber.
  • the first portion of the delivery tube has a rectangular longitudinal cross- sectional shape which is configured to engage with a second portion of the cavity with a reducing transverse width in order to position the delivery tube outlet at a predetermined longitudinal location within the focussing chamber.
  • the engagement structure comprises longitudinally extending ridges on the delivery tube, the ridges configured to position the delivery tube outlet according to the predetermined lateral relationship with the housing when received therein.
  • the ridges are configured to form a plurality of channels between the housing and delivery tube for carrying the sheath fluid to the focussing chamber.
  • a delivery tube for a flow control apparatus for controlling fluid flows associated with carrying particles.
  • the delivery tube comprises a lumen for carrying a source fluid containing particles from a delivery tube inlet to a delivery tube outlet; wherein the delivery tube at the delivery tube outlet is bevelled in one axis and is partially bevelled in a second perpendicular axis and/or has a notch in a notch axis.
  • the delivery tube at the delivery tube outlet is both partially bevelled in a second perpendicular axis and has a notch in a notch axis, wherein the notch axis is the same as the second perpendicular axis.
  • a flow control apparatus for controlling fluid flows associated with carrying particles.
  • the flow control apparatus comprises a housing having a particle flow outlet in fluid communication with a cavity for receiving a delivery tube, the delivery tube having a lumen for carrying a source fluid containing particles from a delivery tube inlet to a delivery tube outlet; wherein the delivery tube has an delivery tube inlet portion having delivery tube inlet cross-sectional dimensions, a delivery tube tapering portion and a delivery tube distal portion having delivery tube distal cross- sectional dimensions which are smaller than the delivery tube inlet cross-sectional dimensions, and wherein the cavity of the housing has a cavity inlet portion having cavity inlet cross-sectional dimensions, a cavity tapering portion and a cavity distal portion having cavity distal portion dimensions.
  • the delivery tube distal cross-sectional dimensions and the cavity distal cross-sectional dimensions are both uniform in the longitudinal direction.
  • a delivery tube tapering angle of the delivery tube tapering portion is the same as a cavity tapering angle of the cavity tapering portion.
  • a delivery tube tapering angle of the delivery tube tapering portion is less than a cavity tapering angle of the cavity tapering portion.
  • the cross-sectional shape of any one of the delivery tube inlet portion, the delivery tube tapering portion, the delivery tube distal portion is: circular; noncircular; elliptical; rectangular.
  • the cross-sectional shape of any one of the cavity inlet portion, the cavity tapering portion, the cavity distal portion is: circular; non-circular; elliptical; rectangular.
  • the cross-sectional shape of any one of the delivery tube inlet portion, the delivery tube tapering portion, the delivery tube distal portion is different to the cross-sectional shape of the corresponding cavity inlet portion, cavity tapering portion, cavity distal portion.
  • the lumen has a non-circular shape along at least part of its length.
  • the lumen comprises a plurality of portions having different cross- sectional dimensions.
  • Figure 1 shows a perspective view of a flow control apparatus according to an example
  • Figure 2 shows a side and end view of a system having the flow control apparatus of Figure 1;
  • Figure 3 shows a longitudinal section of a flow control apparatus according to an example
  • Figure 4A shows a cross-section view through section line AA of the flow control apparatus of Figure 3;
  • Figures 4B - 41 show alternative cross-sections through section line AA of the flow control apparatus of Figure 3;
  • FIG. 5A and 5B show detailed longitudinal sections of the control chamber area of the flow control apparatus of Figure 3;
  • Figure 6A shows side and end views of a delivery tube according to an example
  • Figure 6B shows a longitudinal section of a flow control apparatus according to an example
  • Figure 6BA and 6BB show lateral section views of the flow control apparatus of Figure 6B according to examples
  • Figure 6C shows a longitudinal section of a flow control apparatus according to an example
  • Figure 6D and 6E show longitudinal sections of a flow control apparatus according to examples;
  • Figure 6F shows orientation efficiency and discrimination measurements at differing flow velocities for the example of Figure 6B;
  • Figure 7A, 7B and 7C show side views of a delivery tube according to other examples
  • Figure 8 shows end views and longitudinal sections through different axes and a perspective view of a conical tip of a delivery tube according to an example
  • Figure 9 shows end views and longitudinal sections through different axes and a perspective view of a tip of a delivery tube which is bevelled in one axis, and according to an example
  • Figure 10 shows end views and longitudinal sections through different of a tip of a delivery tube which is bevelled in two axes , and according to an example
  • Figure 11 shows end views and longitudinal sections through different axes and a perspective view of a tip of a delivery tube which is bevelled in one axis, partially bevelled in a second perpendicular axis and includes a notch through the second axis, and according to an example;
  • Figure 12 shows end views and longitudinal sections through different axes and a perspective view of a tip of a delivery tube which includes a notch through one axis, and according to an example
  • Figure 12A shows longitudinal sections through one axis for a number of other tapered tips of a delivery tube according to some examples
  • Figure 13 shows section views through the flow control apparatus having different combinations of housing cavity and delivery tube geometries according to some examples
  • Figure 14 illustrates experimental results of particle flow confinement at a particle velocity of 200mm/s according to one example
  • Figure 15 illustrates experimental results of particle flow confinement at a particle velocity of lOOmm/s according to one example
  • Figure 16 illustrates experimental results of particle flow confinement at a particle velocity of 50mm/s according to one example
  • Figure 17 illustrates experimental results of particle flow confinement at a particle velocity of 500mm/s according to one example
  • Figure 18 illustrates experimental results of particle flow position under different particle velocity conditions according to one example
  • Figure 19 illustrates experimental results of particle flow confinement results using different delivery tube tips according to one example
  • Figure 20 illustrates experimental results of particle flow position in a minor axis under different particle velocity conditions according to one example
  • Figure 21 illustrates experimental results of particle flow position for an elliptical delivery tube outlet cross-section under different particle velocity conditions according to one example.
  • Figure 22 illustrates a method of controlling flows associated with particles and according to an example.
  • Figures 23A and 23B show respectively a summary plot of experimental orientation efficiencies (y axis) vs PODT tip position in z (bottom x axis, [mm]) and max cell speed (top x axis, [m/s]),
  • Figure 24 illustrates how velocity components induce drag forces and can lead to a preferred orientation for aspheric particles in the focusing apparatus geometry described herein.
  • Figure 25A shows simulation v x /v y (y axis) vs max cell speed (top x axis, [m/s]) and PODT tip position in z (bottom x axis, [m]).
  • Figure 25B shows simulation v xx /v yy (y axis) vs max cell speed (top x axis, [m/s]) and PODT tip position in z (bottom x axis, [m]).
  • Figure 26A, 26B and 26C illustrate the effect of a mis-aligned or tilted tip
  • Figure 27a and 27b illustrate example longitudinal positions of the delivery tube in the second focussing chamber
  • Figure 28a-e illustrate example configurations of a second focussing chamber
  • Figure 29 illustrates a cross-section of an aperture according to an example.
  • the phrase "at least one,” in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements.
  • This definition also allows that elements may optionally be present 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 elements specifically identified.
  • “at least one of A and B" can refer, in one example, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another example, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another example, 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); etc.
  • Hardware implementation may include or encompass, without limitation, digital signal processor (DSP) hardware, a reduced instruction set processor, hardware (e.g., digital or analogue) circuitry including but not limited to application specific integrated circuit(s) (ASIC) and/or field programmable gate array(s) (FPGA(s)), and (where appropriate) state machines capable of performing such functions.
  • DSP digital signal processor
  • ASIC application specific integrated circuit
  • FPGA field programmable gate array
  • Memory may be employed to storing temporary variables, holding and transfer of data between processes, nonvolatile configuration settings, standard messaging formats and the like. Any suitable form of volatile memory and non-volatile storage may be employed including Random Access Memory (RAM) implemented as Metal Oxide Semiconductors (MOS) or Integrated Circuits (IC), and storage implemented as hard disk drives and flash memory.
  • RAM Random Access Memory
  • MOS Metal Oxide Semiconductors
  • IC Integrated Circuits
  • Some or all of the described apparatus or functionality may be instantiated in cloud environments such as Docker, Kubenetes or Spark.
  • This cloud functionality may be instantiated in the network edge, apparatus edge, in the local premises or on a remote server coupled via a network such as 4G or 5G.
  • this functionality may be implemented in dedicated hardware.
  • confinement refers to the restriction of the cross- sectional shape and size of a flow of particles in a sample stream.
  • the diameter of a circular section of the flow may be restricted or the dimensions of the major and minor axes of an elliptical section flow may be restricted which may result in a single narrow trajectory with minimal deviation in any polar axis of particles from a defined central longitudinal axis of the flow.
  • This constriction of flow simultaneously has the effect of reducing the average distance of individual particles from a nominal central flow vector along the axis of flow. It is generally desirable to increase confinement to result in an accurate focussing of an interrogation beam and a sorting beam for downstream interrogation and sorting. If the particles are not accurately confined, measurements may be less accurate which can negatively affect the yield of selected cells and the speed with which cells can be accurately detected.
  • orientation of asymmetric particles means the predominant angle of a face of a representative sample of said particles with respect to an axis substantially perpendicular to the axis of flow of the particles. Without any features imparting an orienting torque on the particles, it is expected that the orientation of said face will be randomly distributed and facing any angle around 360°.
  • a sample of cells that have had an orienting torque applied via an orienting feature will have a nonrandom angular orientation that preferentially directs the face of the particle in a particular angle so that a predominant angle can be determined or observed.
  • Orientation efficiency of a method or apparatus for orienting asymmetric particles corresponds to the percentage or proportion of particles in a sample of particles which are oriented with predominant angle, within a predetermined tolerance.
  • Cells and X-cells are referred to herein as examples of particular types of particles that may be desirable to retain within a microfluidic sorting arrangement. Where the term cell is used herein, it may be substituted with the term "particle” and there is no requirement for the cell/pa rticle to be a living cell. Those of skill in the art will readily appreciate that the mention of X-cells is intended to be indicative of any other cells or particles that have characteristics suitable for interrogation and sorting according to the present invention. In particular, X-cells may be substituted herein for any type of particle or cell, including substantially symmetric and asymmetric cells, neurons, red blood cells, tagged cells, viruses, or microbiota as will be known to those of skill in the art.
  • microfluidic stream refers to a flow of liquid having at least one geometrically constrained dimension at which surface forces dominate volumetric forces. In an example this may include a liquid stream having a submillimetre diameter or other cross-sectional dimension.
  • the microfluidic stream may be a continuous phase flow of liquid such as an unbroken stream of one or more aqueous solutions. This could be a laminar flow having a particle flow comprising particles and a sheath flow surrounding the particle flow.
  • the microfluidic stream may alternatively or additionally comprise a dispersed flow of liquid drops.
  • the microfluidic stream may be associated with one or more performance metrics such as flow rate, cross-sectional diameter and/or dimensions, distance to droplet formation.
  • flow environment refers to an environment in which the microfluidic stream may flow through.
  • An example includes a microchannel which may comprise a material such as glass forming an elongate lumen or pathway through which the microfluidic stream flows.
  • the pathway may be fully encompassed by the material between each end of the pathway; or the pathway may have at least one boundary exposing the microfluidic stream to a fluid environment with the material forming a substrate interfacing with the other boundary(s) of the microfluidic stream.
  • the flow environment may be a fluid environment or volume which may be substantially static or which itself may be flowing.
  • the microfluidic stream may not interface with a material substrate but be fully encompassed by the fluid environment.
  • the fluid environment may be a liquid such as an aqueous solution or a gas such as air.
  • FIG. 1 is a perspective view of a flow control apparatus according to an example.
  • the flow control apparatus 100 comprises a delivery tube 130 which in use is installed within a housing 105.
  • the figure shows these two components 105, 130 coupled together for use as well as separated.
  • the two components 105, 130 may be replaceable separately of each other and may be consumable parts of a larger system. Alternatively, the components may be formed as part of a single monolithic structure.
  • the housing 105 comprises a cavity 110 into which the delivery tube 130 is located, and in use may be coupled to a processing microchannel 180 at an aperture from the flow control apparatus.
  • the delivery tube 130 comprises a lumen 145 extending from an inlet end as shown to a distal end within the cavity 110.
  • the housing 105 and delivery tube 130 may be constructed of various materials including: plastics, polymers, metal, glass, ceramic and composites.
  • the flow control apparatus comprises both the delivery microchannel and focusing and/or confinement chambers.
  • At least one of the flow control apparatus and the microfluidic channel may be formed from any one or more of a polymer, glass, ceramic, or other solid substrate, or may be pre-formed components such as PTFE tubing or glass capillaries.
  • a variety of materials and methods can be used to form any of the above-described components of the present disclosure.
  • the flow focusing apparatus, tube, channels and chambers of the present disclosure can be formed from solid materials, in which the channels can be formed via micromachining, film deposition processes such as spin coating and chemical vapor deposition, laser fabrication, photolithographic techniques, etching methods including wet chemical or plasma processes, and the like.
  • the microfluidic channel or flow control apparatus is formed of silicon by etching features in a silicon chip. Technologies for precise and efficient fabrication of various fluidic systems and devices of the present disclosure from silicon are known.
  • various components of the systems and devices of the present disclosure can be formed of a polymer, for example, an elastomeric polymer such as polydimethylsiloxane (PDMS), polytetrafluoroethylene (PTFE), or the like.
  • the channels of the present disclosure can be formed from a polymer, glass, ceramic or other solid substrate, or may be pre-formed components such as PTFE tubing or glass capillaries. Different components can be fabricated of different materials.
  • at least one of the flow control apparatus and the microfluidic channel can be fabricated from an opaque material such as silicon, or from a transparent or at least partially transparent material, such as glass or a transparent polymer, for observation and/or control of the flow process.
  • Components can be coated so as to expose a desired chemical functionality to fluids that contact interior channel walls.
  • components can be fabricated with interior channel walls coated with another material.
  • Material used to fabricate various components of the systems and devices of the present disclosure e.g., materials used to coat interior walls of fluid channels
  • various components of the present disclosure are fabricated from polymeric and/or flexible and/or elastomeric materials, and can be conveniently formed of a hardenable fluid, facilitating fabrication via moulding (e.g., replica moulding, injection moulding, cast moulding, etc.).
  • the hardenable fluid can be essentially any fluid that can be induced to solidify, or that spontaneously solidifies, into a solid capable of containing and/or transporting fluids contemplated for use in and with the described microfluidic system.
  • the hardenable fluid comprises a polymeric liquid or a liquid polymeric precursor (i.e. a "prepolymer").
  • Suitable polymeric liquids can include, for example, thermoplastic polymers, thermoset polymers, or a mixture of such polymers heated above their melting point.
  • a suitable polymeric liquid may include a solution of one or more polymers in a suitable solvent, which solution forms a solid polymeric material upon removal of the solvent, for example, by evaporation.
  • Such polymeric materials which can be solidified from, for example, a melt state or by solvent evaporation, are well known to those of ordinary skill in the art.
  • a non-limiting list of examples of such polymers includes polymers of the general classes of silicone polymers, epoxy polymers, and acrylate polymers. Silicone polymers including PDMS have several beneficial properties simplifying fabrication of the microfluidic structures of the present disclosure.
  • such materials are inexpensive, readily available, and can be solidified from a prepolymeric liquid via curing with heat.
  • PDMSs are typically curable by exposure of the prepolymeric liquid to temperatures of about, for example, about 65 °C to about 75 °C for exposure times of, for example, about an hour.
  • silicone polymers, such as PDMS can be elastomeric and thus may be useful for forming very small features with relatively high aspect ratios, necessary in certain embodiments of the present disclosure.
  • Flexible (e.g., elastomeric) moulds or masters can be advantageous in this regard.
  • the components of the present invention may be formed from recycled polymers or biodegradable polymers such as polylactic acid (PLA) and polyhydroxyalkanoates (PHAs).
  • the material is pre- or post- processed to increase smoothness.
  • This example has surprisingly been found to substantially improve some of the flow properties of the material, for example reducing turbulence or drag.
  • stainless steel may be electropolished for improved smoothness and 3D printed plastics may be sanded, surface melted and/or coated with a smooth surface material.
  • the delivery tube 130 comprises a plurality of ridges or projections which extend longitudinally and in use engage with the inside wall of the cavity. In practice this secures the delivery tube within the housing such that the lumen 145 at the distal end of delivery tube is securely and accurately positioned within the housing. In some examples this feature also influences the fluid flow characteristics.
  • accurate positioning enables a flow of particles from the delivery tube to be carefully controlled within a microfluidic stream issued into the processing microchannel 180 for downstream processing.
  • the flow of particles in the microfluidic stream may benefit from improved characteristics selected from the group consisting of: enhanced positional stability, reduced mechanical stresses on the particles, reduced clumping or more uniform distribution of particles, increased or more accurate confinement, and/or more effective particle orientation.
  • the microfluidic stream comprises a laminar combination of a particle flow from inside the delivery tube and a sheath flow from around the outside of the delivery tube, with the sheath flow surrounding the particle flow in the microfluidic stream.
  • the generation of a stable laminar flow from the flow control apparatus in which particles are desirably arranged enables more effective downstream processing as described in more detail below.
  • the microfluidic stream may be issued into a processing microchannel 180 as shown or into another flow environment such as a gas or liquid.
  • the microfluidic stream issued from an aperture of a delivery microchannel within the flow control apparatus.
  • the other end of the delivery microchannel couples to one or more focussing chambers within the flow control apparatus which contribute to the desired arrangement of the particles within the stable laminar flow of the microfluidic stream.
  • the focussing chamber may comprise the upstream focusing chamber, the downstream focusing chamber or the downstream confinement chamber.
  • downstream processing may include detection of particles (e.g. sperm) characteristics, particle orientation, particle displacement and/or particle sorting according to said characteristics.
  • An advantage of some examples is the controlled and stable confinement of particles within a narrow stream emitted from the delivery tube.
  • this confined stream passes into a processing microchannel 180 and the uninterrupted fluid stream is subjected to a sorting step to sort particles according to certain detected particle characteristics.
  • the controlled confinement achieved enables enhanced sorting efficiency.
  • the processing microchannel 180 may have a width in the range 10 to 500 microns, or 100 to 400 microns, and a depth in the range 5 to 250 microns, for example.
  • the width and depth are identical, and the cross-section of the processing microchannel may be of any shape. Particularly preferred shapes of the cross-sectional shape are circular or square.
  • the dimensions of the processing microchannel supports laminar flow, with minimal turbulence.
  • the processing microchannel has a planar form with in-plane length and width greater than depth transverse to the plane.
  • the depth may be greater than the length and/or width of the microchannel.
  • the processing microchannels may comprise one or more capillaries, and may feature curved segments as well as angles.
  • the microfluidic stream from the flow control apparatus 100 is ejected into free space or another flow environment rather than the processing microchannel 180. Particles within the microfluidic stream may then be subjected to interrogation and sorting, absent processing microchannel 180. This may provide some advantages including faster flow rate and reduced diffraction of interrogation and sorting beams. This may in turn lead to greater accuracy in the focussing of these beams and a reduction in their power, reducing their impact on wanted particles.
  • a sheath flow of liquid is passed through channels formed between the ridges 135 and the inside surface of the cavity 110.
  • the ridges may be formed on the inside surface of the cavity to create channels when engaged with the delivery tube.
  • These channels stabilise the sheath flow, for example by reducing turbulence which may be present in the introduced fluid and/or by homogenising the flow.
  • turbulence is introduced by having one or more sheath flow channels which enter the flow control apparatus orthogonally with respect to the longitudinal or Z- axis of flow of the particle flow or sample stream/fluid.
  • the flow control apparatus 100 is configured such that a combined particle and sheath flow having the desired characteristics noted above emerges as a microfluidic stream into the processing microchannel, with a central particle flow and a surrounding sheath flow in a coaxial arrangement stabilised into a single laminar flow.
  • the particle and sheath flows may be independently formed to control a number of particle flow properties, and/or combined flow properties.
  • asymmetric or non-spherical particles are substantially oriented in a common axis or plane within the microfluidic stream and/or the particles are focussed or confined such that the particles flow more evenly.
  • This enables the particles to be more easily investigated and/or manipulated downstream, for example in the processing microchannel or a free space flow environment.
  • Some examples also allow maintaining orientation and confinement properties of particles across many flow rate ranges.
  • Other particle and/or flow properties may additionally or alternatively be adjusted using the flow control apparatus.
  • examples provide more accurate lateral and longitudinal positioning of the delivery tube 130 within the housing 105 resulting in more effective focussing of the particles within a microfluidic stream. This also results in enhanced positional stability of the particles within the microfluidic stream which improves downstream interrogation and sorting performance.
  • Examples also reduce particle direction changes or shearing forces as well as flow turbulence thereby reducing the mechanical stresses on the particles. This in turn can improve the viability of biological cells such as sperm cells.
  • Examples also more evenly distribute particles within the microfluidic stream, resulting in less clumping and therefore better downstream interrogation and sorting performance.
  • Examples provide improved and more accurate confinement by reducing the displacement of particles from an axis of the microfluidic stream. This also results in better downstream interrogation and sorting performance.
  • Examples provide improved orientation efficiency, increasing the proportion of particles that are optimally oriented for downstream interrogation and sorting processes.
  • FIG. 2 illustrates a system 200 according to an example and including a flow control apparatus 205, 230 which has an aperture 235 coupled to a processing microchannel 280.
  • a side view of the system 200 is shown in an XY plane on the upper left.
  • the flow control apparatus 205 controls separate sheath and particle flows which are combined to generate a microfluidic stream with desired properties.
  • the microfluidic stream is emitted into the processing microchannel 280 as described in more detail below.
  • the processing microchannel terminates prior to downstream interrogation or sorting.
  • the microfluidic stream is emitted from an aperture 380 of the flow control apparatus 300 into a different flow environment which may comprise a free space volume with a static or moving gaseous atmosphere.
  • the processing microchannel terminates at an aperture 380 of a delivery microchannel 375 within the flow control apparatus.
  • the microfluidic stream is stabilised into a combined single laminar flow 260.
  • a detail on the lower left of Figure 2 illustrates the flow of particles 277 within a particle flow path 275 within a sheath flow path 265 in the XY plane.
  • An end view of the system 200 is shown in a YZ plane on the upper right of Figure 2 with a detail on the lower right illustrating the flow of particles 277 within the particle flow path 275, within the sheath flow path 265 of the combined flow 260 in the YZ plane.
  • the external surface of the processing microchannel 280 is transparent along at least part of its length and may be comprised of plastic or glass or other suitable substrates for example.
  • a particle interrogation apparatus 285 is positioned to direct a beam of electromagnetic radiation 285L at particles 277 passing within the particle flow path 275.
  • the asymmetric particles may be oriented to present a desired face to the incident beam 285L.
  • the lower left figure shows the particles side-on and having a narrow ellipse shape which in some examples may be a less optimal surface shape onto which the beam may impinge due to its reduced presentation area. Alternative orientations of ellipsoid particles and angles of incident beam may be preferred.
  • the beam 285L may be a laser of predetermined wavelength and intensity and which interacts with a particle to generate a number of secondary fluorescent wavelengths 285S which may be detected by a detector 290.
  • the intensity and/or spread of the detected fluorescent wavelengths may be used to infer the type of particle under interrogation.
  • the particles may be identified as sperm cells having X or Y chromosomes.
  • the interrogation beam 285L is directed to interact with particles within a flow environment so that the interrogation beam intersects the microfluidic stream 260 directly and not through an processing microchannel 280.
  • the processing microchannel 280 may be omitted, with the microfluidic stream 260 emerging from the aperture 380.
  • a shortened processing microchannel 280 may be employed which is coupled to the aperture 380 of the delivery microchannel 375 but terminates prior to the interrogation beam 285L.
  • the interrogation beam 285L may be located close to the aperture 235, 375 or shortened processing microchannel termination which enables the interrogation event to occur close to the focussing chamber where asymmetric particles are oriented. This may ensure maximal orientation and reduces possible turbulence.
  • a sorting apparatus 295 Downstream of the particle interrogation apparatus 285 is a sorting apparatus 295 which directs a second beam ("sorting beam") of electromagnetic radiation 295L at some particles depending on their type as identified by the particle interrogation module 285 and detector 290.
  • the second beam 295L may be a laser of predetermined wavelength and intensity and which is directed at one type of cell but not another.
  • the second beam 295L may be directed at sperm cells having Y chromosomes but not sperm cells having X chromosomes.
  • the second beam 295L may be directed at sperm cells having X chromosomes but not sperm cells having Y chromosomes.
  • the second beam 295L may cause particles upon which it impinges to be displaced along the optical axis of the incoming beam. This is configured such that these particles are displaced out of a flow path 275 corresponding to an original particle flow and into one or more different flow paths with positions which may correspond to an original sheath streams and/or into one or more different particle trajectories.
  • the two or more types of particles may then be separated by separating the two or more particle flow paths.
  • the flows of differently selected particles may be collected in different vessels to create discrete cell populations.
  • the particles 277 may be controlled to orient themselves at a preferred angle with respect to the beams 285L, 295L.
  • the particles may also, or alternatively, be laterally aligned such that they intersect the beams.
  • the particles 277 are also controlled to be largely within the same plane or distance from the interrogation module 285 or displacement/selection module 295.
  • the delivery tube 230 is located accurately within the housing 205 of the flow control apparatus. Accurate longitudinal, lateral and/or rotational location of the delivery tube with respect to the housing, and accurate rotational location of the housing with respect to the direction of the beams may enhance the accuracy and efficiency of these downstream processes.
  • the delivery tube and/or housing comprise an engagement structure arranged to improve the longitudinal, lateral and/or rotational positioning of the delivery tube within the housing, and by extension the delivery tube's longitudinal, lateral and/or rotational positioning with the rest of the system 200.
  • the particle flow control apparatus may be employed in a system which does not use an extended microchannel, and instead may be used with alternative cell sorting approaches that employ any method of displacing or selecting a particle in space, whether in fluid, aerosol droplet or in the gas phase.
  • displacement/selection methods include, but are not limited to, forces derived from electric fields, electrostatics, electromagnetics, acoustic pulses or pneumatic pulses.
  • the flow control apparatus also may be used with selection/sorting approaches that employ damaging or destroying the unwanted population of cells rather than physical displacement. This is referred to herein as "ablation".
  • such selection/sorting methods may include a radiation source (RS) configured to direct radiation on to the particle to effect at least one of a force and torque on each particle so as to induce ablation or at least one of displacing and orienting each particle relative to an axis defined by the direction of the fluid flow.
  • RS radiation source
  • Ablation may be considered the process of transferring energy to the particle sufficient to permanently inactivate the particle. In the context of biological cells, this may include rendering the cell unviable for its normal function or purpose.
  • sperm cells may be ablated to rapidly induce permanent immotility, or they may be ablated to "prime" them to be incapable of surviving downstream processes such as freezing and thawing. In the former, the ablation may involve rupturing the cell surface membrane which destroys cell integrity. During "priming", the cell surface membrane remains substantially intact, even though motility may be reduced or cease.
  • the methods and apparatus of some examples may have utility in achieving orientation of non-spherical particles such as sperm cells.
  • the angle of the non- spherical particles is controlled to achieve a desired angle with respect to one or more radiation beams that may be used in downstream processing of the particles.
  • the particles are oriented to optimise downstream processes interacting with beams of radiation where the absorption and emission of radiation may be highly orientation dependent in asymmetric particles such as sperm cells. Therefore it may be desired to orient a preferred facet of an asymmetric particle towards an incoming radiation beam, the facet having a maximum or minimum surface area or some other property.
  • downstream processing may be implemented using an orienting module or stage employed downstream of the flow control apparatus 230, 250 in order to orient particles.
  • an orienting module may be located to direct electromagnetic radiation at particles in the combined flow 260. This may expose the particles to radiation pressure causing asymmetric particles to adopt a preferred orientation. Details of operation of an orientation stage are described in published PCT patent document WO2014/017929 which is incorporated herein by reference. However other particle orientation technologies may alternatively or additionally be employed with the flow control apparatus 205, 230 of the examples.
  • downstream processes may be performed by installing corresponding modules or stages downstream of the flow control apparatus 205, 230, for example as described above.
  • These downstream processes may include particle orienting and/or particle interrogation and/or particle displacement/selection; and may be performed separately or in any combination.
  • FIGS. 3 and 4 illustrate longitudinal section and transverse cross-section views respectively of a flow control apparatus according to an example.
  • the transverse cross- section of Figure 4 is through section line AA in the longitudinal cross-section of Figure 3.
  • the flow control apparatus 300 comprises a delivery tube 330 fitting within a housing 305.
  • the delivery tube 330 is securely and accurately received within a cavity 310 of the housing 305.
  • the delivery tube 330 comprises a lumen 340 for carrying a particle flow 345 which is a moving liquid such as an aqueous solution containing particles such as sperm cells.
  • the lumen 340 is open at an input end of the delivery tube 330 to a delivery tube inlet 332 and is open at a distal end of the delivery tube to a delivery tube outlet 333.
  • the delivery tube 330 also comprises ridges, fins, or projections 335 which extend longitudinally along the delivery tube or the inside surface of the housing.
  • the ridges 335 engage with the internal surface of the cavity 310 of the housing 305 in order to secure the delivery tube within the housing.
  • the housing 305 comprises ridges, fins or projections which extend longitudinally along the housing and also engage with the external surface of the delivery tube.
  • the ridges 335 may be dimensioned to ensure a friction fit with the featureless walls of the cavity 310 or the walls of the cavity may comprise corresponding grooves into which the ridges locate.
  • Various other mechanical fixing mechanisms may alternatively be used.
  • the ridges 335 improve the lateral positioning of the distal end of the delivery tube 330 so that the delivery tube outlet 333 is securely and accurately located within the housing 305. Further, the channels formed result in the sheath flow being stabilised and aligned with the Z-axis of flow prior to intersecting with the sample fluid containing particles.
  • ridges 335 are longitudinally extending, they may also be angled with respect to the longitudinal axis to form a spiral shape along the outside of the delivery tube. Furthermore, whilst the ridges have been shown as continuous, they may be discontinuous with parts engaging with the cavity walls at different longitudinal locations. In a further alternative arrangement, longitudinally extending ridges may extend from the cavity to engage with the delivery tube. In this alternative, the delivery tube 330 may or may not also have ridges 335 extending to the cavity wall. In a yet further alternative, the outer circumference of the delivery tube 330 may be dimensioned to mate directly with the inner wall of the cavity to ensure a friction fit. The outer surface of the delivery tube and/or the inner surface of the cavity 310 may include recesses to form channels between the housing 305 and delivery tube 330.
  • the ridges on the housing and/or delivery tube are arranged so as to provide channels through which sheath fluid flows.
  • the sheath flow is homogenous. Homogenous sheath flow means that the flow distance through each channel is substantially the same. This ensures that when the ridges end and the sheath fluid from one channel combines with sheath flow from a different channel, there is minimal turbulence caused.
  • the sheath flow from each channel flows at the same speed and along the same distance as sheath flows from the other channels. This homogenous sheath flow is achieved by having the cross-sectional arrangement of channels have at least one line of symmetry.
  • This configuration improves 360° confinement and focusing of the fluid flows and obviates the need for multiple sheath intersections. If multiple sheath intersections interact with each other or the sample flow, this can cause turbulence and different directions of sheath flow.
  • Figures 4B-4I show non-limiting examples of channels having at least one line of symmetry where the hatched area comprises solid material and the open areas comprise flow channels.
  • the channels may have a scalloped shape where the channel is formed at the delivery tube or housing.
  • a scalloped shape means that one or more edges of the channel has a concave or convex profile as shown in figure 4F-H and 6A.
  • the scallop forms channels between one or more vertices of the shape of the delivery tube when viewed in cross-section.
  • one or more vertices of the shape of the delivery tube is rounded or otherwise shaped to smoothly engage with the housing.
  • Figure 4H and 6A show the smooth engagement of rounded ridges or vertices 635. Smooth engagement means that there is substantially zero volume between the smoothed ridge/vertex and the housing wall.
  • the engagement of at least one vertex, preferably all vertices, with the housing is preferably for a portion of at least 1% of the internal circumference of the housing.
  • the smooth engagement of the smoother vertex with the housing is for a portion of at least 5%, at least 10%, at least 20%, or at least 50%.
  • the housing comprises channels formed in the internal surface to enable sheath fluid to flow over and past the delivery tube.
  • the delivery tube may be of a circular cross-section or may have channels as described above.
  • Figure 41 shows a housing with channels and a circular cross section delivery tube.
  • both the housing and delivery tube may have channels, for example opposing channels where vertices engage to form elliptical or circular channels, or staggered channels formed in the housing and the delivery tube. The inventors have found that this scalloped profile minimises hydrodynamic turbulence when the sheath flows combine in the focussing chamber or prior to the focussing region, for example in region S2, S3 or S4 in figure 3.
  • the scalloped profile also assists with maintaining a hydrodynamically smooth and non-turbulent flow when the sheath fluid combines with the sample fluid (particle fluid) in the focussing and/or confinement chamber(s)).
  • the scalloped profile also enables easier manufacturing and facilitates cleaning of the delivery tube and housing without removal and the use of special tools to clean corners.
  • the number of channels may vary. However, in some examples at least 3 channels, preferably 4, 5, 6, 7, 8, 9, 10, 11, or 12 channels provides enhanced throughput and secure location of the delivery tube in the housing.
  • the engagement structure may extend for a portion of the length of the delivery tube.
  • Figure 7C indicates an example where the engagement structure only extends for a portion of the length of the delivery tube.
  • the flow through the channels may be asymmetric, for example with more flow on one side of the delivery tube 330 than the other. This may be used to displace the particle flow within the sheath flow to one side of a central axis. Having a non-concentric particle stream with the sheath flow may be useful in some downstream interrogation and/or sorting processes.
  • the channels may be angled with respect to a longitudinal direction such that the flows within the channels circle around the delivery tube, forming helical or partially helical flows. This may result in a swirling sheath flow encompassing the particle flow which may provide some additional confinement.
  • one or more sheath flow channels 360 are formed between the delivery tube 330 and housing 305 to carry a sheath flow 365 such as an aqueous solution.
  • the sheath flow channel(s) 360 may extend from a sheath flow inlet 362 at the input end of the delivery tube and which includes the channels formed between the ridges 335.
  • the sheath flow channel(s) extends along the outside of the delivery tube 330 to a focussing chamber 370 defined by a volume formed within the housing 305 at the end of the delivery tube 330 and into which the particle flow 345 is discharged from the delivery tube outlet 333.
  • the focussing chamber 370 is also fluidly coupled to a delivery microchannel 375 from which a microfluidic stream is output via an aperture 380 into a processing microchannel or into a flow environment for downstream processing.
  • the sheath flow inlet 362 is configured to receive pumped sheath fluid from a sheath reservoir.
  • the inlet may feed sheath fluid at substantially a 90° angle to the direction of flow Z-axis. The inventors have found that receiving the sheath flow at this angle reduces bubbles in the system and enables the sheath channel to fill completely before the fluid flows down the channels.
  • the sheath inlet is positioned to inject sheath fluid in a downstream direction at an angle of between 90° to 0° (as illustrated) with respect to the Z-axis direction of sample flow.
  • the sheath fluid is received via the sheath inlet into a sheath chamber upstream of the sheath flow channel(s).
  • This sheath chamber receives the sheath fluid and allows the flow to stabilise prior to the fluid moving down the channels.
  • a purge outlet is positioned in fluid communication with the sheath chamber to receive air and excess sheath fluid and extract it from the system.
  • the purge outlet is positioned upstream of the sheath inlet.
  • a central particle flow 345 is surrounded by one or more sheath flows 365 in a coaxial arrangement.
  • the shape and size of the focussing chamber 370, the geometry and dimensions of the sheath flow channel(s) 360 and the lumen 340, together with the flow rates of the particle flow 345 and sheath flow 365 all contribute to the control of the combined fluid flows from the delivery microchannel 375.
  • Example use cases include controlling orientation and confinement of particles within the microfluidic stream 385.
  • the invention provides a flow control apparatus for controlling fluid flows associated with carrying particles, the flow control apparatus comprising : a. a delivery tube comprising a lumen for carrying a flow of liquid, the delivery tube extending longitudinally within a housing which together define a focussing chamber adjacent the delivery tube outlet; and b. two, three, four or more longitudinally extending channels for carrying a sheath flow of liquid between the delivery tube and the housing towards the focussing chamber, wherein a downstream outlet of each channel is regularly spaced around the delivery tube at a single longitudinal location with respect to a flow path.
  • the longitudinal axis described in this example is substantially aligned with the direction of flow of the sample fluid through the delivery tube.
  • the focusing chamber is an area where the sheath fluid flow meets the sample fluid flow.
  • the regular spacing of the channel exits around the delivery tube exit results in a non- turbulent flow which compresses and focuses the sample fluid flow from multiple directions simultaneously. This reduces turbulence and enhances confinement.
  • This multi-directional flow focusing arrangement also avoids the need for multiple sheath flow intersections along the chip.
  • the delivery microchannel 375 fluidically couples the focussing chamber 370 with an external surface of the flow control apparatus 300.
  • the delivery microchannel 375 is a conduit which has an inlet from the focussing chamber 370 and an aperture 380 which may be in the form of an external aperture or may be connected to a processing microchannel 280.
  • the cross-sectional shape of the delivery 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.
  • the delivery microchannel 375, its inlet and aperture 380 comprise the same rectangular cross-section with an aspect ratio of greater than 1: 1.
  • the longitudinally extending ridges or other engagement structure ensures accurate and stable lateral positioning of the delivery tube outlet 333 within the focussing chamber 370.
  • a delivery needle is introduced into a tapering volume of sheath fluid however the distal end of the needle is buffeted by the fluid flows and moves laterally causing the resulting particle flow to move within the surrounding sheath fluid flow or even to partially mix with the sheath fluid flow resulting in poorly oriented and poorly confined sample fluid (particle fluid) flows. This may make downstream processing difficult, inaccurate and inefficient.
  • accurate longitudinal positioning of the delivery tube outlet 333 within the focussing chamber 370 helps to optimise the control and stability of orientation and/or confinement of particles or other flow properties of the microfluidic stream 385 delivered through the delivery microchannel 375 and from the aperture 380. In the example of Figure 3 and 4, this is achieved by dimensioning the ridges 335 of the delivery tube 330 to complement the dimensions of the cavity 310 of the housing 305 to prevent the delivery tube 330 from being inserted into the cavity beyond a predetermined longitudinal position.
  • Example 2 indicates that the PODT and housing configurations described herein provide consistently high orientation efficiency across a range of PODT tip distances and flow velocities.
  • the cavity 310 can be divided into several longitudinal sections or portions including a first portion 310-S1 which has a longitudinal cross-sectional shape, such as rectangular, which is arranged to engage at multiple longitudinal locations with a corresponding first portion of the delivery tube 330-S1.
  • the first portion of the cavity may be substantially uniform transverse cross section along the longitudinal direction, for example of circular shape having a fixed diameter.
  • the first portion of the cavity may include channels or scallops formed in the housing while the delivery tube itself is of substantially circular cross-section.
  • the first portion of the cavity may include channels or scallops formed in the housing and channels or scallops formed in the delivery tube where each channel facilitates flow of sheath fluid through the channel.
  • the channels in the housing and delivery tube in this example are aligned to provide a single larger channel volume versus the volume of the channel formed in the housing or delivery tube alone.
  • This first portion SI of the cavity 310 is used to receive the ridges 335 of the delivery tube 330.
  • a second portion of the cavity 310-S2 tapers, having reducing dimensions when extending towards the distal end of the delivery tube.
  • the ends of the ridges 335 having larger dimensions, prevent the delivery tube extending beyond this point, thereby ensuring accurate and stable longitudinal positioning of the delivery tube outlet 333 within the focussing chamber 370.
  • the ends of the ridges may be shaped as shown to complement the internal shape of the cavity 310 to further improve this positioning.
  • grooves or channels in the wall of the cavity 310 may be used to receive the ridges 335 and the length of the grooves controlled to control the longitudinal position of the delivery tube outlet 333 within the focussing chamber 370.
  • the engagement of the delivery tube ridges in the housing provide rotational engagement to ensure angular alignment, the advantages of which are described further herein.
  • a delivery needle may be placed into a tapering volume of sheath fluid however if the distal end of the needle is not correctly positioned the sheath flows may turbulently interact with the particle flow causing unwanted mixing, chaotic misalignment of the particle flow and poor particle orientation and confinement.
  • the tapering second portion 310-S2 of the cavity 310 of the housing 305 may comprise a tapering angle a to the longitudinal axis of the housing, and the tapering second portion 330-S2 of the delivery tube 330 may comprise a tapering angle P to the longitudinal axis of the delivery tube 330.
  • the sheath flow channel(s) 360 can be defined by the volume between the inner walls of the housing and the outer walls of the delivery tube.
  • the housing and its inner walls in turn can be defined by the cavity shape and dimensions and the delivery tube can be defined by the shape and dimensions of its outer walls.
  • the tapering angles can be modulated to achieve control of the acceleration of the sheath flow in portions of the sheath flow channels.
  • the tapering angle a is from 5-90°. In other examples is from 10 to 45°.
  • the tapering angle 3 is from 5 to 90°. In other examples q>/2 is from 10 to 45°.
  • the third portion 310-S3 of the cavity of the housing 305 may comprise uniform dimensions extending over a longitudinal length.
  • the third portion 330-S3 of the delivery tube 330 may comprise uniform, although smaller, dimensions extending over 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 stabilise to ensure laminar flow and reduce turbulence.
  • a fourth portion 330-S4 of the delivery tube includes a distal tip containing the delivery tube outlet 333.
  • the tip may be shaped to enhance orientation and/or confinement of particles as described in more detail below.
  • This tip area may be complemented by a further tapering fourth portion 310-S4 of the cavity 310 of the housing.
  • the focussing chamber 370 is formed in a fifth portion 310-S5 of the cavity 310 of the housing when the delivery tube outlet 333 is positioned to discharge the particle flow 345 into the sheath flow 365 entering the focussing chamber 370 formed by the housing.
  • the focussing chamber and other components of the flow control apparatus 300 are configured to cause a combined laminar flow of the particle and sheath flows through the delivery microchannel 375 and out of the aperture 380, in which the particles are largely oriented in one axis and largely confined to a plane containing that axis.
  • Additional portions of the delivery tube and/or sheath flow channel 360 may be included, or some described portions may be removed from some examples, such as the third portion 330-S3 from delivery tube as needed to impart targeted characteristics, such as but not limited to confinement, to the particle flow.
  • targeted characteristics such as but not limited to confinement, to the particle flow.
  • Different geometries of the sheath flow channel to those illustrated may alternatively be employed.
  • the sheath flow 365 through the sheath flow channel 360 may be symmetric or asymmetric.
  • a larger volume in the upper half of the sheath flow channel 360 may cause the particle flow 345 to be displaced downwards.
  • the sheath flow may also be caused to rotate about the delivery tube to generate a vortex flow which may assist with particle flow confinement. For example this is achieved using angled ridges to define a spiral or "corkscrew" flow as shown in figure 7.
  • the different cross-sectional volumes of the sheath flow channel 360 along its length enables fine control of the sheath flow, including acceleration and stabilisation of the flows.
  • the volumes of the sheath flow channels 360 also control the flow rates of the sheath flow through the channel.
  • the ridges 335 and the channels formed between them act to stabilise the sheath flow as this may be introduced as a turbulent flow from outside the flow control apparatus.
  • Figure 4A shows a cross-section through section line AA of Figure 3, where the input area of the housing 305 and delivery tube 330 can be seen.
  • This shows four evenly spaced ridges 335 extending from the delivery tube 330, although any number of ridges could alternatively be used.
  • Various other parts of the delivery tube and housing are illustrated with the same reference numerals as used for those part in Figure 3.
  • One of the ridges 335L is longer than the others and corresponds with a groove 315 in the outer wall of the cavity 310 of the housing.
  • This arrangement comprises an indexing arrangement comprising an indexing means which ensures that the delivery tube can only be received into the housing in a single orientation illustrated generally by R.
  • the delivery tube may have multiple longer ridges for example 2,3,4,5,6,7,8,9,10,11 or 12 longer ridges where the ridges engage in a groove in the housing wall.
  • the delivery tube may comprise one or more grooves and the housing comprises one or more extending ridges to enable alignment in an inverse manner to that previously described. Where a single extending ridge aligns with a groove, this arrangement enables 360° rotational alignment so that the delivery tube can only be inserted in a single orientation.
  • the indexing means may comprise two extended ridges that are opposite each other, the delivery tube may be inserted in one of two orientations at 180° to each other to enable alignment of the ridges with the corresponding grooves.
  • Similar angular arrangements may be employed, for example three extended ridges which align with corresponding grooves to enable three orientations at 120° to each other, or four extended ridges which align with corresponding grooves to enable four orientations at 120° to each other.
  • These examples are particularly useful to enable correct alignment of the delivery tube outlet 333 when it comprises a non-circular cross-section i.e. it is bevelled or otherwise asymmetric, or has a non-circular lumen, e.g. an elliptical or rectangular lumen.
  • the groove 315 for the longer ridge 335L is deeper such that the longer ridge 335L will still only fit within that one groove in order to ensure a predetermined rotational alignment of the delivery tube 330 within the housing 305.
  • one of these grooves may be wider than the others to receive a wider, though not necessarily longer, ridge.
  • a pin and corresponding hole arrangement may be used to correctly index the delivery tube within the housing.
  • the pin may extend through the housing into a ridge of the delivery tube, or the delivery tube or a ridge may include a pin which extends through a hole in the housing.
  • a magnet in one of the delivery tube or housing may be used with another magnet (or metallic feature) within the corresponding housing or delivery tube.
  • Various other mechanical rotational alignment features may alternatively or additionally be used.
  • the external visually accessible surfaces of the housing and delivery tube may be marked to assist a user to align the delivery tube when inserting this into the housing to ensure rotational alignment.
  • the ridges or other indexing means may extend substantially the length of the delivery tube or housing to the position of the termination of the lumen 333, or may extend along a portion of the delivery tube or housing. This portion may be defined by the beginning or end of a taper angle of the delivery tube or housing, or may be a portion of a linear region of the delivery tube or housing.
  • the indexing means may extend for a portion (e.g. a quarter or half) of the distance SI, S2, or S3.
  • Figures 4B to 41 Various other examples of internal housing or cavity cross-sectional shapes and external delivery tube cross-sectional shapes are shown in Figures 4B to 41.
  • Figures 4B - 4E illustrate longitudinal channels formed by differences in cross-sectional shape between the inner housing and the outer delivery tube.
  • Figures 4F - 41 illustrate longitudinal channels formed by laterally extending projections from the exterior of the delivery tube and/or the interior of the housing.
  • Figure 5A and 5B show a detailed section of a flow control apparatus around the focussing chamber 370 according to an example.
  • the rest of the apparatus may be the same as the example of Figures 3 and 4 or may vary from this.
  • the lumen 540 of the delivery tube 530 comprises multiple portions 540-U, 540-T, 540-N of different cross- sectional dimensions.
  • a first wide section 540-W carries an unfocussed particle flow 545- U which carries particles in various orientations and through a relatively large volume.
  • a tapering portion 540-T of the lumen reduces the volume through which the particles flow, accelerating the particle flow and improving some particle flow properties.
  • the cross-sectional shape of the lumen may also change along its length, for example going from circular in the first portion 540-W to elliptical in a final portion 540-N which causes the particles to orient along the longer axis of the ellipse.
  • This is illustrated in the detail below the apparatus in which a particle 577-C in the circular section of the lumen 540-W is oriented into a different axis 577-E in the elliptical section of the lumen 540-N.
  • the unfocussed particle flow 545-U is narrowed in at least one axis with the particle oriented and discharged from the delivery tube outlet into the focussing chamber 370.
  • the lumen may change cross-sectional shape and/or dimensions along its length within the delivery tube.
  • the lumen may comprise an upper portion and a lower portion which have different cross-sectional shape and/or dimensions.
  • Example dimensional ranges include 50um - 1mm for the lower portion and 0.5 - 3mm for the upper portion.
  • the delivery tube immediately adjacent the lumen comprises a taper from a smaller-cross- sectional area upstream in the microchannel, to a larger cross-sectional area downstream at the exit lumen of the delivery tube.
  • This trumpet-shaped delivery tube exit transition has been found to minimise turbulence and assist laminar flow as the sample flow meets the sheath flow.
  • the delivery tube exit 645 is designed to be of a size appropriate to deliver cells and sample fluid.
  • the internal diameter of the exit of the delivery tube lumen is 50pm-lmm. In another example where a more tightly confined stream is required, the delivery tube exit is from 50-500pm.
  • the transition from a first wide section of the lumen 540-U to the narrowed portion 540-N comprises a step of substantially 90° to the axis of flow or a steeply tapered portion 541.
  • the steeply tapered portion 541 is preferably at an angle of 45° or more to the axis of flow.
  • the lumen may be adapted to receive an input tube 542 which delivers the fluid containing the particles.
  • the difference in width of the lumen at point 540-U compared to point 540-N substantially corresponds to the thickness of the input tube walls.
  • the input tube internal diameter substantially corresponds to the lumen internal diameter beyond the end of the input tube.
  • the step or steeply tapered portion is an input tube arresting means at point 540-T that stops the input tube from travelling any further down the delivery tube and provides a seal between the input tube and delivery tube.
  • an o-ring or gasket is included at point 540-T to improve the seal and prevent leakage of the fluid.
  • the input tube arresting means is also labelled 640 in figure 6.
  • the input tube arresting means 541 may be used in conjunction with the tapered portion 540-T a different points along the longitudinal axis of the delivery tube lumen to achieve both a) enhancements in flow confinement and/or orientation (for example using non circular cross-sectional lumens) and b) a smooth transition from the input tube to the delivery tube lumen.
  • Laminar sheath flows 365 interact with the partly focussed particle flow to further optimise properties of the particle flow. For example, this may be adapted to further confine and/or further orient the particles.
  • a combined laminar fluid is output from the apparatus which comprises an inner focussed particle flow 545-F within a surrounding sheath flow 365.
  • An orifice or aperture 580 is formed in the housing which issues this microfluidic stream from the delivery microchannel.
  • the aperture 380 may be coupled to a processing microchannel or other conduit for onward transport and downstream processing.
  • the microfluidic stream is delivered into a fluid environment such as a liquid or gaseous environment.
  • the microfluidic stream issues from the aperture into a flow environment.
  • the microfluidic stream may issue in a downwards or gravity-based direction or in a direction at an angle with respect to gravity, for example perpendicular or angled generally upwards.
  • the flow environment is bounded by an extended microchannel or conduit which allows the microfluidic stream to travel in a controlled way from the aperture and confines the flow of particles as they pass the particle interrogation apparatus 285 and sorting apparatus 295.
  • the flow environment may comprise a liquid of substantially the same viscosity as the liquid in the microfluidic stream.
  • the flow environment is a liquid of higher viscosity than the microfluidic stream.
  • a fluid in the flow environment may be moving in the direction of movement of the microfluidic stream.
  • the velocity of this movement may be the same or different from the velocity of the microfluidic stream.
  • the flow environment comprises a gaseous environment.
  • the microfluidic stream is not required to be bounded by a processing microchannel or conduit. This approach has a number of advantages and results in reduced friction with the conduit resulting in improved laminar flow and enabling higher flow rates.
  • the inventors have found that where the aperture engages with a processing microchannel or conduit, the aperture size must be aligned with the conduit internal dimensions to ensure that the microfluidic stream maintains laminar flow and has minimal turbulence.
  • Conduit dimensions do not always align with the aperture dimensions which limits the scope of aperture dimensions. By employing a fluid flow environment, this enables flexibility in the size and shape of the aperture. In particular, using a smaller aperture than standardised conduits allow, a reduced proportion of sheath flow compared with particle flow can be achieved. This reduction in sheath volume thereby increases selected cell concentration.
  • FIG. 6A illustrates side and end views of a delivery tube according to an example.
  • the delivery tube 630 comprises a lumen 640 for carrying a particle flow, a plurality of ridges 635 which comprise mechanical engagement structures which extend longitudinally. Between the ridges 635 are recesses or scallops 637 which also run longitudinally and which form channels with the cavity walls of a housing when received therein. These channels may form part of a larger sheath fluid channel which transports sheath fluid from a sheath fluid inlet adjacent the larger end of the delivery tube and a focussing chamber in an assembled flow control apparatus containing the delivery tube.
  • the ridges 635 and recesses 637 may be the same length as illustrated, or they may be of different lengths.
  • the ridges 635 and recesses 637 may be the same width and/or cross-sectional profile as illustrated, or they may be of different widths and/or profiles.
  • the ridges 635 may form hemispherical recesses 637 as illustrated, although alternative arrangements are possible including polygon or complex curvature shapes, triangular or rectangular shaped cross-sections, or shapes with straight and curved components.
  • the ridges and recesses may be regularly or irregularly spaced.
  • the sheath channels formed between the ridges are tapered such that a cross-sectional area of a channel is increased at a downstream position in the housing compared to an upstream position in the housing.
  • Figures 4BA - upstream and 4BB downstream illustrate an example of this tapered channel architecture although alternative channel architectures such as those shown in figure 4A or described elsewhere herein may also be used.
  • This tapered arrangement allows sheath fluid flow hydrodynamically combine with a sample fluid steam at the end of the sheath channels and reduce "dead space" that may increase turbulence of flow or bubble formation during priming.
  • the delivery tube 630 may be formed in different portions 630-S1, 630-S2, 630-S3, 630-S4 having different geometries as described herein.
  • the lumen 640 may be formed into different portions having different geometries as described herein. The combination of these geometries and the corresponding geometries of the cavity of the housing into which the delivery tube is assembled enable the sheath flow and particle flow to be controlled to optimise different characteristics of the flow of particles such as particle orientation and/or confinement.
  • the delivery tube and housing of the present invention may be combined with controlling the flow rates of the particle flow and the sheath flow.
  • the flow rate is selected from the group comprising Imm/s - 20m/s, greater than Imm/s, greater than lOmm/s, greater than lOOmm/s, greater than Im/s, greater than 2m/s, greater than lOm/s or greater than 20m/s and ranges therebetween.
  • the channels merge into the conical cavity defined by the tapered delivery tube and the inversely tapered housing wall.
  • the inventors have found that the smooth transition from the channels to the conical cavity provides a directional sheath flow with minimal turbulence.
  • the sheath flow in this example enters from each channel in a symmetrical flow.
  • the multi-directional flow is enhanced compared to a flow from, for example a sheath inlet on one side of the delivery tube, or on opposing sides of the delivery tube.
  • the multi-directional flow preferably flows from channels arranged at regular intervals around the entirety of the 360° delivery tube.
  • the simultaneous sheath flow from multiple directions minimises turbulent flow prior to and after combination with the sample fluid flowing through the lumen.
  • the individual sheath flows from the various channels therefore converge gently with the particle flow at an angle with minimal displacement of the particle or sample flow and minimal turbulent flow ensuring the particle and sheath flow components remain laminar and there is little or no mixing between them.
  • the focusing chamber comprises an upstream part and a downstream part.
  • Figure 6B, 6BA, 6C, 6D and 6E provide examples of a double focussing chamber comprising a delivery tube and housing which engage and synergise with each other.
  • the upstream first focussing chamber (Pl) may be defined by a truncated conical cavity of the housing. More generally, the upstream first focussing chamber (Pl) may be defined by an internal longitudinal taper of the inner walls of the housing in which the transverse cross-section of a cavity defined by those tapering walls has substantially an aspect ratio of 1 : 1.
  • the upstream cross-section may comprise a circle, a rounded square, or a square.
  • the substantially equal aspect ratio (1 : 1) is intended to mean a shape in which the length of a first axis of said shape is not more than 20% longer than a width of a second axis of said shape perpendicular to the first axis.
  • the downstream second focussing chamber P2 may comprise a cavity defined by the inner walls of the housing and having a transverse cross-sectional shape with an unequal aspect ratio, that is substantially greater than 1 : 1 with the length of one axis being longer than the length of an orthogonal axis of the cavity.
  • the downstream cross-section may comprise a rectangle, an ellipse, a rounded rectangle, or a stadium (a geometric term for a rectangle with a pair of semi-circles positioned at either end).
  • the downstream cross-section aspect ratio is greater than (i.e.
  • more unequal than) 1 : 10 for example 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.
  • Figure 6B shows a side view of a flow focussing apparatus 600 with a double focussing chamber Pl and P2
  • Figure 6BA shows an end view of the double focussing chamber looking from the right of Figure 6B to the left.
  • the first focussing chamber Pl has a square cross section which tapers from a largest side length Pl-Ll to a smallest side length P1-L2.
  • the second focussing chamber P2 has a rectangular cross-section with a largest side length P2-L1 and a smallest side length of P2-L2.
  • the longest side length P2-L1 of the second focussing chamber P2 is substantially the same as the smallest side length P1-L2 of the first focussing chamber Pl. This results in a slit at the left most or tapered end of the first focussing chamber, this slit extending a length L between P2-a and P2-b to form a linear section of the second focussing chamber P2.
  • the second focussing chamber also comprises a tapering section P3 which tapers from the largest side length P2-L2 towards the entry point 374 of a delivery microchannel 375. This tapering section P3 is also referred to herein as a confinement chamber.
  • the width of the delivery microchannel 375 is substantially the same as the smallest length P2-L2 of the second focussing chamber P2.
  • At least an upper or lower surface 692, 693 of the second focussing chamber comprises a reverse gable shape (i.e. the internal shape of a v- or u-shaped roof or a chined recess).
  • the reverse gable may taper along its length (i.e. to form the internal shape of a boat hull) such that the height (Y-axis) of the chamber along a centre-line is greater at an upstream position compared to a downstream position.
  • the reverse gable may comprise a rounded gable 693 or a sharp gable 692.
  • This tapered reverse gable provides a hydrodynamically streamlined shape to orient and confine the cells as they pass along the second focussing chamber.
  • the reverse gable shape extends across the longer axis of a crosssection of the chamber defined by W in figure 6B, 6BC and 6BD.
  • the crosssection of the second flow focusing chamber may comprise a diamond, a pentagon or a hexagon.
  • the cross-sectional shape of the second focussing chamber may be said to comprise two opposing straight lines and one or more of: a curved line between the two straight lines; a a V-shaped or chined line between the two straight lines.
  • Figure 6BB illustrates an alternative double focussing chamber arrangement for the flow focussing apparatus 600' in which the first focussing chamber Pl' has a circular cross- sectional shape, tapering from a large diameter Pl-Dl to a small diameter P1-D2 from right to left in Figure 6B. This terminates in a second focussing chamber P2' having a rounded rectangular cross section with a large side length P2-L1' and a small side length P2-L2'.
  • a first focussing chamber comprises a constant lateral cross-sectional shape having an aspect ratio of substantially 1: 1 and which tapers longitudinally from a larger size to a smaller size adjacent a second focussing chamber.
  • the lateral dimensions of the shape reduce in at least one axis, and in some examples in both perpendicular axes.
  • the second focussing chamber comprises a constant lateral cross-sectional shape having a higher aspect ratio than the first focussing chamber and which extends longitudinally with a substantially constant dimension in one lateral axis, and in some examples both perpendicular lateral axes (ie without tapering).
  • the second focussing chamber is fluidically coupled to a delivery microchannel having a smaller cross-sectional area.
  • the second focussing chamber may include a tapering section which tapers longitudinally towards the delivery microchannel in one or more lateral axes.
  • the delivery microchannel 375 may comprise an aspect ratio which is higher than the first focussing chamber.
  • the flow focusing arrangement comprises: a. a first focussing chamber comprising a substantially constant lateral cross- sectional shape forming a truncated cone having an aspect ratio of substantially 1 : 1 and which tapers longitudinally from a larger diameter at an upstream position, to a smaller diameter at a downstream position; b. a second focussing chamber comprising a substantially planar chamber.
  • the substantially planar chamber may comprise a substantially constant lateral cross-sectional shape having a higher aspect ratio than the first focussing chamber and which extends longitudinally with a substantially constant dimension in one lateral axis.
  • the second focussing chamber comprises a substantially constant lateral cross-sectional shape forming a rectangle that tapers longitudinally in at least one axis across at least a portion of the longitudinal length of the chamber.
  • at least one perpendicular lateral axis of the rectangle reduces longitudinally from a larger size at an upstream position, to a smaller size at a downstream position.
  • both perpendicular lateral axes reduce longitudinally from a larger size at an upstream position, to a smaller size at a downstream position.
  • the flow focusing arrangement comprising a flow focusing portion comprising at least a first and second and focussing chambers as described herein may be fluidically connected to any sheath channel architecture including the sheath channel arrangements described herein comprising a plurality of longitudinally extending channels.
  • the sheath channel architecture comprising a plurality of longitudinally extending channels may be fluidically connected to other flow focusing apparatus.
  • the combination of the flow focusing portion and the sheath channel architecture provides benefits including minimisation of bubble production during priming, minimisation of turbulence, and enhanced confinement and orientation of cells.
  • a downstream confinement chamber depth tapers from a depth of the second focussing chamber to a shallower depth.
  • the shallower depth aligns with the height of the entry point to the delivery microchannel 375.
  • the fluid sample stream passes through the flow control apparatus 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 and sample fluids are substantially co-planar. The fluid stream travels along a substantially flat trajectory through the centre of the apparatus without major deviations of flow direction at any of the focusing chambers, or prior to the ejection from the delivery tube.
  • the cone angle q> is the internal angle between the tapering walls of the cone.
  • Figure 6B and 6C show half of this angle 671. In some examples this angle 671 is from 5-90°. In other examples 671 is from 10 to 45°.
  • Figure 6C shows an example in which the delivery tube comprises an elongated substantially constant transverse cross-sectional area portion 672.
  • the delivery tube comprises two angles of taper. The first being the angle with respect to the longitudinal Z axis of flow of the delivery tube tip 673. The second being the angle with respect to the Z axis of flow of the delivery tube body 674.
  • the angle 673 is between 0-90°, in other examples angle 673 is between 5 and 30°.
  • the angle 674 is between 0-90°, in other examples, angle 674 is between 5 and 30°.
  • the size of the downstream second focussing chamber (P2) cross-section width "W” the "orientation width” (i.e. the greater of the two cross- sectional dimensions, P2-L2) comprises 0.5mm to 10mm.
  • the width comprises 2mm to 8mm, or 3mm to 6mm.
  • Figure 6F shows orientation efficiency and discrimination measurements at differing flow velocities (V) and with different orientation width measurements using apparatus as shown in figure 6B. It can be observed that there is substantially consistent orientation efficiency with a high level of orientation of the bovine sperm cells flowed through the flow focusing apparatus.
  • the size of the downstream second focussing chamber (P2) cross-section depth "D" (the "orientation depth” (i.e. the lesser of the two cross- sectional dimensions) comprises 0.01mm to 4mm.
  • the width may be from 0.01mm to 10mm.
  • the depth comprises 0.025mm to 0.5mm, or 0.05mm to 0.25mm.
  • Figure 28a shows one example of a second focussing chamber comprising a width of 2mm.
  • Figure 28b shows one example of a second focussing chamber comprising a width of 6mm.
  • the depth D is equal to the diameter or major dimension of the delivery microchannel 375.
  • the inventors have found that aligning the depth of the cross-section of the downstream second focussing chamber P2 to a major dimension of the delivery microchannel 375 and the aperture 380 enables the microfluidic stream to flow with minimal turbulence from into the delivery microchannel and from the aperture.
  • the width of the downstream second focussing chamber P2 then tapers to the minor dimension 670 of the delivery microchannel 375 as described below.
  • the depth D decreases from the point P2-a to point P2-bto provide a further focusing and orientation vector over the length of the downstream second focussing chamber P2.
  • the aspect ratio of width to height ranges from 1: 1.25 to 1 :800 w:h. In preferred examples, the aspect ratio ranges from The preferred width and height described above may be combined in the aspect ratios previously described to define a downstream second focussing chamber with the exemplary dimensions shown in Table 1 : 5 to 1 : 160 w:h.
  • Example 1 shows experimental data which indicates that an equal aspect ratio for the upstream first focussing chamber Pl cross-sectional area and an unequal aspect ratio for the downstream second focussing chamber P2 cross-sectional area provides high orientation efficiency and discrimination.
  • the inventors observed that a width of 4mm and depth of 0.1mm for P2 gives consistently high orientation and discrimination between X and Y-bearing sperm cells at a range of flow rates.
  • the orientation length "L" of the downstream second focussing chamber P2 may also be important when considering the orientation efficiency of asymmetric particles that have passed through the apparatus of some examples.
  • the length L of region P2 from point P2-a defined as where a wall of the upstream first focussing chamber joins a wall of the downstream second focussing chamber, to point P2-b defined as where the downstream flow focusing chamber joins the downstream confinement chamber P3, is at least 3mm. In other examples, length L is between 3 to 10mm.
  • the orientation width W is constant for a distance L. The inventors have found that this constant width channel has flow stabilising effects and reduces downstream turbulence.
  • downstream second focussing chamber P2 tapers directly to the start of the delivery microchannel.
  • region P2 and P3 are combined to achieve orientation and confinement in a single region.
  • a third region P3 shown in figure 6B and 6C is referred to herein as a downstream confinement chamber.
  • the two walls of the housing taper from the width W defined in P2 to the width of the delivery microchannel 375.
  • Figures 6D and 6E show an alternative example in which the second focussing chamber P2 tapers longitudinally in one lateral axis.
  • the first focussing chamber Pl tapers longitudinally (z-axis) in both lateral axes (x and y axes) so that, for example, a large square cross-section on the right reduces to a smaller square crosssection as the first focussing chamber extends longitudinally to the left.
  • the second focussing chamber P2 tapers longitudinally in one lateral axis (x axis) whilst maintaining its cross-sectional dimension in the perpendicular lateral axis (y axis).
  • the second focussing chamber P2 tapers to the cross-sectional shape and dimensions of the delivery microchannel 375.
  • Figure 28e shows an example in which the second flow focussing chamber tapers from a first width Wi to a narrower width downstream of W2. This tapered configuration assists in confining and orienting cells and provides a smooth flow-route to the delivery microchannel.
  • the second focussing chamber is adjoined by a downstream confinement region which tapers from a first depth (y axis) defined by the depth of the second focussing chamber, to a shallower second depth.
  • the second depth is defined by the depth of the entry point 374 of the delivery microchannel 375.
  • the upstream first focussing chamber is employed in use with the delivery tube described above.
  • the delivery tube described with reference to the examples shown in Figures 3, 4A-I, 6B, 6C, 6E and 7.
  • the channelled sheath fluid flows with minimal turbulence around the ridges and through the channels/scallops 656. It then cascades over and combines around the tapered section of the delivery tube 657 which forms a cavity with the tapered housing.
  • S1-S4 This open cavity and the upstream channels minimise turbulence effects caused by unequal flow. For example this unequal flow may be caused if sheath fluid is introduced to the focussing chamber from opposing sides of the housing rather than the circumferential flow achieved in the present invention.
  • sheath fluid may also be caused by sheath fluid not being appropriately channelled using multiple channels around the circumference of the housing to obtain laminar and non-turbulent flow in the direction of flow. If the sheath fluid is not channelled it can move in a radial direction around the delivery tube which negatively affects confinement and orientation efficiency, for example by turbulent flow or unequal flow velocity around the circumference.
  • the tip of the delivery tube projects into the downstream second focussing chamber. In another example, the tip of the delivery tube is positioned within the upstream first focussing chamber.
  • the consistently high orientation efficiency achieved using the combination of channelled sheath flow, a circular cross-section upstream first focussing chamber and an unequal aspect ratio downstream second focussing chamber results from the following effect.
  • the channelled sheath fluid 656 enters the upstream first focussing chamber (650 in the example shown in Figure 6B) from 360°. This compresses the particle flow 655 from all directions which has the effect of increasing confinement, i.e. reducing the distance variance of individual particles from a nominal central flow vector along the axis of flow - Z. This has the effect of increasing the speed of the flow.
  • the particle flow passes from a substantially circular cross section to a cross-section of unequal aspect ratio as described above and labelled P2 in fig. 6B.
  • the confined stream is compressed in the Y-axis, i.e. the axis of the smaller dimension of the downstream cross-section.
  • asymmetric particles such as sperm cells to preferentially align their substantially "flattened" face (which is defined by a "major dimension” of the particle) with an axis defined by the major dimension of the unequal aspect ratio - the X-axis.
  • This region is believed to "prime" the particles to attain their eventual orientation post-aperture.
  • the particles are then subjected to a third compressive force as the fluid flows into the third region P3 - the downstream confinement chamber - in which at least one of the width (X-axis) and the height (Y-axis) of the downstream second focussing chamber reduces.
  • the fluid flow rate increases and confinement of the particles increases.
  • the orientation of the particles is believed to flip from the downstream orientation region so that the flattened face of the particles is aligned with the Y axis.
  • the combined sheath and particle flow or microfluidic stream then flows into the delivery microchannel 375.
  • An alternative arrangement is shown in Figure 6E in which the downstream second focussing chamber P2 tapers, at different angles, from the upstream first focussing chamber Pl to the delivery microchannel 375. Angle of downstream second focussing chamber confinement region
  • angles between the three regions Pl, P2 and P3 are curved instead of sharp angles.
  • This concept of having a curved transition angle can be described in terms of the corner radius of the P1-P2 transition, the P2-P3 transition, and the P3-outlet transition.
  • the radius of the angles is at least 20pm.
  • the corner radius is from 20pm to 1000pm.
  • Orientation efficiency may be measured using a processing unit which implements an orientation efficiency estimation function which analyses output from the interrogation beam 285L via one or more detectors 290.
  • the orientation efficiency may be used to provide sorting system monitoring for an operator and may also be used to control upstream processes such as shutting down the system if the orientation efficiency falls too low.
  • the orientation efficiency is a measure of the proportion of cells which are adequately oriented with respect to a reference direction.
  • Sperm cells and other cells such as red blood cells are asymmetric having a flat oval shape, with perpendicular dimensions defining a face or large surface plane and a short dimension defining a thickness of an edge (as well as partially defining an orthogonal short surface plane).
  • the particle selection/displacement technologies may be arranged to achieve optimal performance when its direction of propagation is perpendicular to the face or large surface plane.
  • the reference direction is perpendicular to the laser propagation direction.
  • the reference direction may alternatively correspond with the orientation of the interrogation beam 285L and/or detector(s) 290.
  • Asymmetric cells oriented within a certain range of the reference direction may still be sufficiently well oriented for laser-based sorting or other processes.
  • Other cells which fall outside this range for example asymmetric cells presenting their edge to the lasers, may result in measurement datapoints that cannot be classified and/or sub-optimal sorting.
  • a low orientation efficiency metric is an indication that the sorting system is configured sub-optimally resulting in the wastage of cells; for example because their fluorescence emissions cannot be accurately measured due to not being well oriented with respect to the illuminators or detectors.
  • a low orientation metric may also indicate that even if accurately classified, many cells may not be properly sorted due to poor orientation with respect to a laser when this is used for sorting.
  • Cell orientation efficiency can be determined by comparing a number of datapoints within a focussed region of interest with the number of datapoints overall corresponding to all cell measurements, although different definitions could alternatively be used. This may be calculated by: wherein ZA is the number of cells in a focussed region of interest, and ZB is the number of cells in another region which may correspond to all cell datapoints, or all viable cell datapoints. Whichever populations of cell datapoints are used, this metric represents the orientation efficiency over a period of time. Over different time periods, the orientation efficiency may vary. If the orientation efficiency falls below a threshold, for example 30%, this may trigger an alarm or other measure to prompt reconfiguration of the sorting system to be undertaken in order to improve the measured orientation efficiency.
  • the flow focusing apparatus is modified in response to a sub-optimal orientation efficiency.
  • the distance dZ in figure 6B is adjusted to enhance orientation efficiency.
  • dZ is defined as the distance between the tip of the delivery tube, and the intersection point of the first focussing chamber and the substantially planar second focussing chamber. This is shown in figure 27a and b.
  • the tip of the delivery tube is positioned downstream of the intersection point - i.e. dZ is negative as shown in figure 27a.
  • the tip of the delivery tube is positioned upstream of the intersection point - i.e. dZ is positive as shown in figure 27b.
  • the delivery tube exit is positioned within the housing downstream of a point in lateral alignment with the start of the tapered portion of the housing at 2807 in Figure 27a and 27b. Positioning of the exit at this point provides significant advantages in flow hydrodynamics due to the constricting and accelerating effect of the taper on sheath fluid passing around the delivery tube which also results in beneficial confinement of the sample stream.
  • the delivery tube exit may be positioned at a negative dZ at a point greater than or equal to 10pm upstream of the end of the tapered portion where the substantially planar second focussing chamber starts.
  • Positioning this close to the second focussing chamber requires there to be sufficient flow space for sheath fluid to pass around the tip of the delivery tube and beneficially confine the sample fluid from multiple directions. It will be appreciated by those of skill in the art that this distance will be determined based on the outer diameter of the delivery tube tip, and the cone angle of the tapered portion.
  • a further beneficial effect of the arrangements described herein is in providing enhanced cell health by minimising 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 throughput.
  • the rotational alignment between the sheath channels and the focussing chambers may be adjusted.
  • the flow rates of the particle flow and/or the sheath flow may be adjusted.
  • the shape and/or size of the aperture 380 may be adjusted. These adjustments may be combined in any suitable manner and may be controlled depending on estimated orientation efficiency.
  • Figures 7A and 7B illustrate delivery tubes according to two alternative examples in which the longitudinally extending ridges 735 can also be angled with respect to the longitudinal axis.
  • the delivery tube 730 comprises a number of angled ridges or ribs 735. These may be configured to cause a spiralling sheath flow which may be used to enhance orientation and/or confinement.
  • the angle 9 of the ridges may be adjusted to optimise this effect.
  • the delivery tube 730-2 comprises angled ridges or ribs 735-2 grouped together in sets which define channels 736 between the sets.
  • the ridges may protrude from the inside of the housing to define channels therein.
  • the example in 7C shows the ridges 740 extending in two directions from the delivery tube to engage with the housing.
  • the ridges are optionally tapered in the Z-axis (axis of flow) to present a thin leading edge to the flowing liquid.
  • the thin leading edge may expand into a wider support structure then taper again to thin trailing edge.
  • This optimised version of the ridges is akin to a wing which reduces the turbulence caused by liquid flowing over it.
  • the ridges may define an ellipse in their cross- section with the longer dimension of the ellipse being generally aligned with the Z-axis direction of flow. This configuration of ridges may be applied to any of the other examples described herein, in particular the examples described in relation to figures 3, 6B, 6C and 6E.
  • the delivery tube, ridges, upstream focusing chamber, downstream second focussing chamber, downstream confinement chamber and delivery microchannel are a monolithic flow control apparatus comprising a single piece of material.
  • the inventors have found that fabricating this monolith enables accurate engagement of the delivery tube with the focussing chambers to enhance orientation and confinement.
  • Figures 8 - 12A illustrate various delivery tip geometries which may be used to optimise certain characteristics of the particle flow, such as features of the particle orientation and/or confinement and/or particle integrity. All of these tips may be used in combination with the other features of the invention described above including the ridges on said delivery tube or housing, the flow focussing chambers and confinement chamber.
  • Figure 8 illustrates a conical tip 830C at the delivery tube outlet 840 of the delivery tube 830 and which may be used with any of the previous described example or with variations of these.
  • the lower figure shows a perspective view of the conical tip, with the middle and upper figures show end and longitudinal cross-section views in perpendicular planes - XZ and XY. As can be seen, the conical tip tapers towards the distal end of the delivery tube in both planes.
  • Figure 9 illustrates a bevelled tip 930B at the delivery tube outlet of the delivery tube 930 which may be used with any of the previous described example or with variations of these.
  • the lower figure shows a perspective view of the bevelled tip, with the middle and upper figures showing end and longitudinal cross-section views in perpendicular planes - XZ and XY.
  • the bevelled tip tapers towards the distal end of the delivery tube in one plane (XY) but not the other plane (XZ).
  • the bevel angle 6 may be changed to alter the orientation characteristics of the particle flow.
  • Figure 10 illustrates a double bevelled tip 1030B1, 1030B2, at the delivery tube outlet of the delivery tube 1030 and which may be used with any of the previous described examples or with variations of these.
  • the lower figure shows a perspective view of the double bevelled tip, with the middle and upper figures show end and longitudinal crosssection views in perpendicular planes - XZ and XY.
  • the double bevelled tip has one bevel 1030B2 which tapers towards the distal end of the delivery tube in one plane (XZ) and also another bevel 1030B1 which tapers towards the distal end of the delivery tube 1030 in the other plane (XY).
  • the tapering angles of the two planes may be different or the same. In the illustrated example, the tapering in the XZ plane is shorter and shallower and retains more width of the distal end of the delivery tube than the tapering in the XY plane.
  • Figure 11 illustrates a double bevelled 1130B1, 1130B2 with notch 1130U tip which may be used with any of the previous described example or with variations of these.
  • the lower figure shows a perspective view of the double bevelled and notched tip, with the middle and upper figures showing end and longitudinal cross-section views in perpendicular planes - XZ and XY.
  • This example is similar to the double bevelled tip of Figure 10 and additionally comprises a notch 1130U which comprises material delivery tube material removed from around the delivery tube outlet 1140.
  • This notch may be achieved in any plane, although in the example this is in the XZ plane which is the same plane as the smaller bevel 1130B2 and in which some width 1130E of the distal end of the delivery tube 1130.
  • the notch may also be any suitable shape, for example rectangular as shown in the two upper figures or semi-circular as shown in the lower figure.
  • Figure 12 illustrates a tip with notch 1230U which may be used with any of the previous described example or with variations of these.
  • the right hand figure shows a perspective view of the tip with notch, with the left hand figures showing end and longitudinal crosssection views in perpendicular planes - XZ and XY.
  • This example is comprises a notch 1230U similar to the example of Figure 11 but does not comprise any bevelling of the tip. This notch may be achieved in any plane.
  • Figure 12A illustrates four further tip shape examples in longitudinal cross-section views.
  • the two upper tips comprise a convex cross-sectional shape 1230CV (left) and a concave cross-sectional shape 1230CC (right).
  • the lower two tips each include two tapering sections in cross-section.
  • the left lower tip 1230T1 has a first tapering section with a more acute angle than a second distal tapering section.
  • the right lower tip 1230T2 has a first tapering section with a less acute angle that a second distal tapering section.
  • the convex or concave profiles may not be along the entire length of the tip, but for example only at the end of the tip.
  • Various other alternatives are possible in which the profile of the tip is not uniform along its length.
  • Tip profiles may be used to control both the fluid velocity and the angle at which the sheath approaches the sample fluid.
  • Figure 13 illustrates four examples of the sheath flow channel and lumen of a flow control apparatus and which may be used with any of the previous described example or with variations of these.
  • the three figures are cross-sections through section line BB of Figure 3 and illustrate a cross-section through a third portion S3 of the housing and delivery tube of the flow control apparatus.
  • the housing 1305-1 has a circular cross-section cavity 1310-S3-C
  • the delivery tube portion 1330-S3-E has an elliptical cross-section
  • the lumen 1345 has a circular cross section.
  • the housing 1305-2 has an elliptical cross-section cavity 1310-S3-E
  • the delivery tube portion 1330-S3-E has an (smaller) elliptical crosssection
  • the lumen 1345 has a circular cross section.
  • the housing 1305-3 has a circular cross-section cavity 1310-S3-C
  • the delivery tube portion 1330-S3-D has a diamond shaped cross-section
  • the lumen 1345 1345- E has an elliptical cross section.
  • the housing 1305-4 has a circular cross-section cavity 1310-S3- C
  • the delivery tube portion 1330-S3-A has an asymmetric curved shaped cross-section
  • the lumen 1345 1345-E has an elliptical cross-section and which is offset from the centre of the delivery tube.
  • the cross-section shape can change down the length of the tube.
  • the particle flow and sheath flow through the flow control apparatus can be controlled to optimise various properties including, but not limited to, particle orientation and confinement with the flow stream, particle integrity, particle distribution within the combined fluid flow, particle transport time.
  • Figures 14 - 21 Various experimental results associated with some examples are illustrated in Figures 14 - 21.
  • the axes of the graphs are micron (x-axis) and number of pixels (y-axis) which representative of the number of particles.
  • Figure 14 illustrates particle confinement using a conical tip and showing particle flow images at a flow rate of 200mm/s in perpendicular axes (left) together with graphs illustrating the distribution of 80% of particles (right). It can be seen that 80% of the particles are well confined in both axes.
  • Figures 15 - 17 illustrate similar plots and graphs for different particle flow rates, respectively lOOmm/s, 50mm/s, and 500mm/s. It can be seen that a similar tight confinement is maintained for different flow rates.
  • Figure 18 illustrates the position of the particles at different particle flow rates.
  • the particles are well confined at all flow rates, though are displaced slightly to one side as the flow rate increases. However, this displacement is well within the tolerance of the downstream investigation, orientation and/or displacement/selection processes. This also compares favourably with traditional needle delivery approaches in which the particle flow changes position significantly with different flow rates and typically needs to be realigned.
  • Figure 19 illustrates on the left for perpendicular axes the core stream distribution for 80% of particles using a delivery tube tip with a 40 degrees bevel together with the delivery tube having a circular outer section paired with a housing cavity also having a circular section. This may correspond for example to the sheath flow chamber arrangement of Figure 4 and the tip of Figure 8.
  • Figure 20 corresponds to a delivery tube having a 40-degree single bevelled tip together with circular section outer surface paired with a circular section housing cavity inner surface.
  • Figure 20 illustrates the distribution and position of the particles at different particle flow rates.
  • Figure 21 corresponds to a delivery tube having a 40-degree single bevelled tip together with an elliptical section outer surface paired with a circular section housing cavity inner surface.
  • Figure 21 illustrates the position of the particles at different particle flow rates. It can be seen that using the elliptical section outer surface of the delivery tube reduces particle distribution and provides a more uniform flow position across different flow rates.
  • 80% of the particles can be confined within a series of ranges, including 100, 80, 60, 50, 30 and 20 microns using different combinations of the above-described features of the flow control devices. Different ranges may be useful for different downstream particle processing.
  • Figure 22 illustrates a flow diagram of a method 2200 of using a flow control apparatus according to an example.
  • the method 2200 optionally selects a delivery tube and housing combination and engages these to form a flow control apparatus to use in the rest of the method. This may be performed manually and may involve selecting different combinations of housing and delivery tube in order to implement different controls on a stream of particles. For example, these may be selected to create a focussing chamber and/or sheath flow channels having certain dimensional properties in order to control the various properties of sheath and particle flows through the apparatus as previously described.
  • the selected housing and delivery tube may be engaged with each other using a simple push fit with engagement of the delivery tube within the housing being controlled by an engagement structure, such as one of those previously described.
  • the delivery tube and housing may be a monolithic component.
  • the flow control apparatus receives a stream of particles in a fluid flow as well as a sheath fluid flow.
  • the particle flow may be received into a lumen of the delivery tube and the sheath flow into channels between the delivery tube and housing as previously described.
  • one or more properties of the particle flow and sheath flow may be controlled to produce a microfluidic stream having a desired property.
  • the speed of the particle and sheath flows, as well as the geometries of the selected delivery tube and housing may 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.
  • the microfluidic stream of particles and sheath flow may be emitted into a flow environment comprising a processing microchannel.
  • a processing microchannel may direct the microfluidic stream to a particle processing apparatus.
  • the flow environment may be a liquid or gaseous environment through which the microfluidic stream flows.
  • the particles may be inspected to determine a particle characteristic.
  • a beam of radiation may be directed at the particles in order to determine their type, such as X or Y sperm cells.
  • a sub-population of the particles may be selected based on the determined particle characteristics. For example, Y sperm cells may be displaced into a different flow path compared with X sperm cells. This could be achieved using a laser to nudge the Y cells out of their current flow path. The particle flow containing the X sperm cells may then be directed to a collection vessel and the particle flow containing the Y sperm cells discarded.
  • the aperture 380 emits the microfluidic stream generated using the or each focussing chamber(s) into the flow environment, and confinement chambers if used, for downstream processing.
  • the aperture may have a cross-sectional shape having a dimension which is longer in one axis than in a perpendicular axis.
  • the microfluidic stream issues from the aperture into a flow environment such as a processing microchannel or a gaseous atmosphere.
  • the width 670 (X-axis) of the delivery microchannel is 10pm to 200pm.
  • the width is between 50pm and 150pm.
  • the width is equal to the depth (Y-axis).
  • the delivery microchannel immediately adjacent the aperture comprises a taper from a smaller delivery microchannel cross-sectional area upstream to a larger delivery microchannel cross-sectional area downstream, for example at the aperture itself, where the cross-sectional area is defined perpendicular to the axis of flow.
  • Figure 29 3001 This trumpet-shaped delivery microchannel-to-aperture transition has been found to produce a more stable microfluidic stream 385 within the downstream flow environment which provides enhanced confinement, interrogation and sorting.
  • the length of the delivery microchannel 675 (Z-axis) is at least 10 microns to 10mm from an exit of a downstream confinement chamber to the aperture 380, wherein the aperture is defined as a point on a plane perpendicular to the z-axis of flow aligned with the terminal end of the flow focusing apparatus.
  • the channel length 675 is in the range of 50 microns to 1mm. This length is important to allow laminar flow to re-establish following orientation and confinement of particles in the orientation and confinement chambers upstream.
  • the width 670 is reduced at the downstream aperture 380 from a width W2 compared to the width 670 upstream at the entry point 374 of the delivery microchannel 375 at Wi.
  • This reduction in width from Wi to W2 shown has the beneficial effect of further increasing confinement of the particles.
  • the reduction in width may be a taper along the length of the delivery microchannel as shown in Figure 6D or along a portion of it.
  • W2 is between 50% to 95% of Wi.
  • the width reduces from between 100-150 pm to 50- 99pm.
  • the width reduces from approximately 125pm to approximately 75pm. Approximately in this instance is intended to mean (+/-20%).
  • the cross-sectional shape of the delivery 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.
  • the delivery microchannel 375, its inlet and aperture 380 comprise the same rectangular crosssection with an aspect ratio of greater than 1: 1.
  • the aperture cross-sectional shape comprises a square or a rectangle.
  • the tip geometries may be used with or without the described engagement structures or the sheath flow channel and lumen geometric features.
  • the sheath flow and lumen geometries may or may not be used with the engagement structures or the tip geometries.
  • the housings and delivery tubes of the examples may also be used independently of each other, for example being assembled with different delivery tubes and housings.
  • the housings and delivery tubes may be produced as consumable parts for a larger particle processing system and may be sold and installed separately.
  • the various channels, chambers, surfaces and flows are configured as microfluidic, that is having dimensions that are geometrically constrained to a small scale such as submillimetre, at which surface forces dominate. However, some examples may be configured with larger dimensions in which microfluidic phenomena are partly or wholly absent.
  • the examples provide many advantages including improved control of particles to facilitate downstream processing.
  • the accurate and secure positioning control of some examples ensures that the particle flow is positionally stable to improve the accuracy and efficiency of downstream processing such as particle interrogation, particle orientation, particle displacement/selection and particle sorting.
  • Accurate control of the sheath and particle flows also help to improve various downstream processing related properties of the particles such as orientation and confinement. This in turn enables the ability to increase the particle flow rate whilst minimising damage to the particles, thereby speeding up particle processing and the efficiency of the processing. In an example, this may allow for a faster rate of separating X and Y chromosome sperm cells whilst improving separation efficiency such as retrieving a sample with 80% or more sperm cells with X-chromosomes.
  • Examples also provide the ability to repeatably and accurately position a delivery tube within a housing of a flow control apparatus which reduces setup time and avoids the need for a highly trained operator. Further, the improved engagement of fitting between the delivery tube and housing provides mechanical rigidity and isolation from external forces, this ensures that the delivery tube is not only accurately positioned initially but also remains so over an extended period of time, reducing need for realignment and downtime of processing.
  • Example 1 Particle orientation and discrimination characteristics using two- part focussing chamber
  • the flow focussing apparatus shown in figure 6C was mounted and primed with fluid to remove any bubbles.
  • the emitted microfluidic stream was aligned to the interrogation beam.
  • Stained cells were injected through a sample channel and combined within the flow focusing apparatus with sheath fluid.
  • the combined sample sheath fluid is passed through the orientation and confinement chambers and through the aperture.
  • Fluorescence intensity data was detected using orthogonal detectors perpendicular to the Z axis of flow and a fluorescence integral was determined. A scatterplot of integrals corresponding to independent particles was created. Orientation efficiency was measured as described in WO2022139597A1.
  • Figure 6F shows results from the downstream flow focusing chamber width adjustment in a flow focusing apparatus of the invention. Flow velocity was adjusted between 5 and 9m/s and width of the downstream flow focusing chamber was adjusted from 3 to 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 best for orientation efficiency and discrimination.
  • the width 3 mm apparatus suffered from a PODT alignment issue at speed 5m/s. but still provided good orientation efficiency.
  • the flow focussing apparatus of the invention provides excellent orientation efficiency and discrimination across a range of widths and flow velocities.
  • the position of the particle orientation and delivery tube (PODT) tip along the flow direction (Z axis) was modified to analyse its effect on orientation efficiency.
  • the flow control apparatus is shown in Figure 6C and the Z-axis distance being adjusted is labelled dZ.
  • the sheath fluid is received from an inlet above the delivery tube.
  • the sample fluid including sperm cells was delivered through the tubing inside the delivery tube from the arresting point.
  • the cavity which holds the delivery tube is 8mm in diameter with a cone angle of 30° to the Z-axis into a downstream second focussing chamber with 130pm depth (D) by 4 mm (L) constant width cross-section.
  • the delivery tube itself had 4 channels/scallops to allow sheath fluid to flow and establish into a stable flow around its 20° to Z-axis conical tip.
  • the positional variation of the PODT tip in the z direction was referenced to a nominal point where the tip of the cone would meet the downstream second focussing chamber (dZ in figure 6D).
  • the final exit of the fluid into the delivery microchannel has the downstream confinement chamber tapering down from 4 mm wide to a 100 pm diameter outlet and aperture.
  • a range of PODTs with tip position in z axis from 1 mm to 6 mm were manufactured. At each position a range of flow velocities were used ranging from 3.3 m/s up to 7 m/s.
  • Orientation efficiencies of greater than 50% were obtained using this PODT arrangement over a range of flow speeds and delivery tube tip distances. There was a slight trend in which orientation efficiency (OE) decreases as flow velocity increases for each of the PODT tip positions. In addition, OE decreases as PODT moves forward in Z axis getting closer to where the tip of the cone joins with the downstream second focussing chamber.
  • OE orientation efficiency
  • Finite Element Modelling simulations have been performed with the same geometry as described in example 2. The mechanism for orientation is believed to arise from asymmetry. As such, the following parameters were calculated and compared to understand the flow dynamics of the proposed apparatus: asymmetric compression factor (ACF), ratios of velocity components ( -), ratios of velocity gradient V gr , and vorticity (curl of the velocity field).
  • ACF asymmetric compression factor
  • - ratios of velocity components
  • V gr ratios of velocity gradient
  • vorticity curve of the velocity field
  • the particles analysed are sperm cells.
  • Bovine sperm cells have a flattened head.
  • For maximal fluorescence detection it is desirable to have the sperm flat side facing to the interrogation beam. Therefore it is desirable to tune the flow focussing apparatus to orient cells in a particular orientation relative to the interrogation beam (285L).
  • the simulation data reported in this example is focused on the downstream confinement chamber (P3 in figure 6B and 6C).
  • the asymmetry in velocity gradients is a metric for determining and optimising orientation.
  • the ratio between V x and V y was chosen as a key metric for assessing orientation. It describes the asymmetric velocity components of the fluid field at each point in space, which can reflect OE preference of the flow field.
  • V x /V y was selected as the most relevant metric for determining orientation efficiency an example in figure 26B and C.
  • Figure 26D shows a trend of decreasing orientation efficiency as flow speed increases.

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Abstract

There is provided a flow control apparatus comprising a delivery tube having a lumen, the delivery tube extending along a longitudinal axis within a housing to a focussing chamber; one or more channels defined between an external surface of the delivery tube and an internal surface of the housing, the channels extending towards a lumen exit of the delivery tube within the housing and at least partially aligned with the longitudinal axis of the delivery tube; the focussing chamber fluidically coupled to an aperture in the flow control apparatus.

Description

FLOW CONTROL APPARATUS, PARTS AND METHODS THEREFORE
1. TECHNICAL FIELD
The present disclosure relates to the manipulation of particles in a fluid flow for downstream interrogation, orientation, displacement and/or sorting or other processes. In particular, though not exclusively, this relates to manipulation of biological cells such as sperm cells in laminar liquid flows.
2. BACKGROUND
Microfluidic laminar flows have been used to control the flow of particles such as biological cells in order to enable processing, analysis or sorting of these particles. The orientation of asymmetric particles such as sperm cells can be useful for optimal processing. For example, a laser beam may be focussed at passing cells which emit various wavelengths of light which may be detected and interpreted by interrogation equipment. Therefore, it may be desirable to orient these cells to optimise the performance of the processing, analysis or sorting system.
Such arrangements may have application in identifying which types of cells are contained in a particle flow, for example whether they contain X or Y chromosomes. This can then be used to sort the cells into different containers. In the dairy industry this may be used to ensure that breeding cows are inseminated with X-chromosome sperm cells to provide female calves which are more valuable than male calves.
Increasing the accuracy with which particles can be identified as well as the speed at which this can be achieved whilst avoiding damage to the particles are of ongoing interest.
In this specification where reference has been made to patent specifications, other external documents, or other sources of information, this is generally 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, in any jurisdiction, are prior art, or form part of the common general knowledge in the art.
3. SUMMARY
In one aspect there is provided a flow control apparatus comprising a delivery tube having a lumen, the delivery tube extending along a longitudinal axis within a housing to a focussing chamber; one or more channels defined between an external surface of the delivery tube and an internal surface of the housing, the channels extending towards a lumen exit of the delivery tube within the housing_and at least partially aligned with the longitudinal axis of the delivery tube; the focussing chamber fluidically coupled to an aperture in the flow control apparatus.
In an example, the one or more channels extend substantially parallel to the longitudinal axis of the delivery tube or are angled with respect to the longitudinal axis of the delivery tube. The one or more channels may be defined by corresponding projections which extend along the delivery tube and/or the housing. At least some of the projections may extend from the delivery tube to contact with the inner surface of the housing or from the inner surface of the housing to contact the delivery tube. The projections may be arranged to contact the delivery tube or inner surface of the housing at multiple longitudinal and lateral locations in order to position the delivery tube outlet at a predetermined position within the focussing chamber.
In an example, the one or more channels a. comprise a substantially semi-circular or triangular cross-section; and/or b. extend from a sheath fluid inlet to the focussing chamber; and/or c. are configured to provide the only flow path or paths for the sheath fluid between a sheath flow inlet and the focussing chamber over at least a portion of the longitudinal extension of the delivery tube within the housing; and/or d. comprise a larger cross-sectional area at a downstream position compared with a cross-sectional area at an upstream position of said one or more channels.
The one or more channels may be spaced around the entirety of the external sectional circumference of the delivery tube and merge into the focussing chamber which is at least partially defined by a tapered delivery tube and an inversely tapered housing wall.
The one or more channels may be defined by two or more ridges defining the cross- sectional shape of the channels and wherein said ridges are dimensioned to provide a friction fit with the housing such that there is substantially zero volume between the ridge and an interior surface of the housing.
In an example, the flow control apparatus comprises an engagement structure configured to secure the delivery tube during use with the housing to form at least one of the one or more channels between the housing and the delivery tube, and position the delivery tube outlet at a predetermined longitudinal, lateral, and/or rotational location within the focusing chamber.
The housing may have a first portion with a longitudinal cross-sectional shape arranged to engage at multiple longitudinal locations with a first portion of the delivery tube in order to position the delivery tube outlet at a predetermined lateral location within the focussing chamber.
The engagement structure may comprise longitudinally extending ridges within the cavity of the housing, the ridges configured to position the delivery tube outlet according to the predetermined lateral relationship with the housing when received therein.
In an example, the engagement structure comprises a rotational alignment feature arranged to secure the delivery tube outlet at a predetermined rotational angle with respect to the housing.
In an example, an interior lateral cross-sectional shape of the housing within which the delivery tube is positioned is different to an exterior lateral cross-sectional shape of the delivery tube at a corresponding longitudinal position, and wherein the one or more channels are defined by said difference in lateral cross-sectional shape at different longitudinal positions.
In an example, the inner surface of the housing defines a cavity for receiving the delivery tube, the cavity terminating in the focussing chamber, wherein the geometry of the cavity is different than the geometry of the focussing chamber at the termination; the geometries comprising one or more of the following: dimensions; cross-sectional shape; longitudinal tapering angle. An end of the delivery tube may be positioned within the focussing chamber a predetermined distance from the termination.
In an example, the flow control apparatus comprises a second focussing chamber fluidically coupled between the first focussing chamber and the aperture, wherein the geometry of the first focussing chamber is different to the geometry of the second focussing chamber. The geometries may differ in one or more of the following: dimensions; cross-sectional shape; longitudinal tapering angle. The cross-sectional shape of the first focussing chamber may have a lower aspect ratio that the cross- sectional shape of the second focussing chamber. The cross-sectional shape of the first focussing chamber may be circular and the cross-sectional shape of the second focussing chamber may be substantially rectangular. The second focussing chamber may be fluidically coupled to the aperture by a delivery microchannel with a dimension in one lateral axis smaller than the second focussing chamber.
In an example, the flow control apparatus comprises a confinement chamber fluidically coupled to the second focussing chamber, the confinement chamber tapering longitudinally in one lateral axis from the second focussing chamber. In one aspect there is provided a flow control apparatus comprising a delivery tube having a lumen, the delivery tube extending along a longitudinal axis within a housing to a first focussing chamber; the housing comprising a second focussing chamber fluidically coupled between the first focussing chamber and an aperture in the flow control apparatus; wherein the geometry of the first focussing chamber is different to the geometry of the second focussing chamber.
In an example, the geometries differ in one or more of the following: dimensions; cross- sectional shape; longitudinal tapering angle.
In an example, the cross-sectional shape of the first focussing chamber may have a lower aspect ratio that the cross-sectional shape of the second focussing chamber. The cross-sectional shape of the first focussing chamber may be circular and the cross- sectional shape of the second focussing chamber may be rectangular.
In an example, the second focussing chamber is fluidically coupled to the aperture by a delivery microchannel with a dimension in one lateral axis smaller than the second focussing chamber.
In an example, the flow control apparatus comprises a confinement chamber fluidically coupled to the second focussing chamber, the confinement chamber tapering longitudinally in one lateral axis from the second focussing chamber.
In an example, the cross-sectional shape of the second focussing chamber comprises two opposing straight lines and one or more of: a curved line between the two straight lines; a V-shaped or chined line between the two straight lines.
In another aspect, there is provided a particle processing system comprising a flow control apparatus as defined above and a particle interrogation apparatus and/or a sorting apparatus.
In another aspect, there is provided a method of controlling fluid flows associated with carrying particles. The method comprises carrying a particle flow of liquid containing particles in a lumen of a delivery tube extending along a longitudinal axis within a housing to a focussing chamber; carrying a sheath flow of liquid in one or more channels between the delivery tube and the housing towards the focussing chamber, wherein the channels extend towards a lumen exit of the delivery tube within the housing and at least partially aligned with the longitudinal axis of the delivery tube; generating a microfluidic stream using the focussing chamber and issuing the microfluidic stream from an aperture, the microfluidic stream comprising a laminar flow of liquid from the sheath flow surrounding liquid from the particle flow. In another aspect, there is provide a method of controlling fluid flows associated with carrying particles. The method comprises carrying a particle flow of liquid containing particles in a lumen of a delivery tube to a first focussing chamber; carrying a sheath flow of liquid to the first focussing chamber; generating a microfluidic stream using the first focussing chamber and a second focussing chamber flu id ica I ly coupled to the first focussing chamber, wherein the geometry of the first focussing chamber is different to the geometry of the second focussing chamber.
In another aspect, there is provided a method of controlling fluid flows associated with carrying particles. The method comprises carrying a particle flow of liquid containing particles in a lumen of a delivery tube extending along a longitudinal axis within a housing to a first focussing chamber; carrying a sheath flow of liquid in one or more channels between the delivery tube and the housing towards the focussing chamber, wherein the channels extend towards a lumen exit of the delivery tube within the housing_and at least partially aligned with the longitudinal axis of the delivery tube; generating a microfluidic stream using the first focussing chamber and a second focussing chamber fluidically coupled to the first focussing chamber, wherein the geometry of the first focussing chamber is different to the geometry of the second focussing chamber; issuing the microfluidic stream from an aperture, the microfluidic stream comprising a laminar flow of liquid from the sheath flow surrounding liquid from the particle flow.
In an example, one or more of these methods use a flow control apparatus as defined above.
In an example, there is provided a flow control apparatus for controlling fluid flows associated with carrying particles. The flow control apparatus comprises a delivery tube having a lumen for carrying a particle flow of liquid containing particles from a delivery tube inlet to a delivery tube outlet, the delivery tube extending along a longitudinal axis within a housing to a focussing chamber; one or more channels for carrying a sheath flow of liquid towards the focussing chamber, the one or more channels defined between an external surface of the delivery tube and an internal surface of the housing, the channels extending in a direction substantially aligned with the longitudinal axis of the delivery tube within the housing; the focussing chamber configured to combine the particle flow and the sheath flow to generate and issue a microfluidic stream from an aperture in the flow control apparatus, the microfluidic stream comprising a laminar flow of liquid from the sheath flow surrounding liquid from the particle flow.
In an example, the housing comprises a cavity for receiving the delivery tube, the cavity terminating in the focussing chamber, wherein the geometry of the cavity is different than the geometry of the focussing chamber at the termination; the geometries comprising one or more of the following: dimensions; cross-sectional shape; longitudinal tapering angle.
In an example, an end of the delivery tube is positioned within the cavity a predetermined distance from the termination.
In an example, an end of the delivery tube is positioned within the focussing chamber a predetermined distance from the termination.
In an example, the one or more channels extend in a direction parallel to the extension of the delivery tube within the housing, or parallel to a direction of flow of the particle flow.
In an example, the one or more channels extend along a screw axis wherein the longitudinal axis of the delivery tube within the housing or the direction of flow of the particle flow is the axis of rotation of the screw axis.
In an example, the flow control apparatus comprises a plurality of channels for carrying a sheath flow of liquid between the delivery tube and the housing towards the focussing chamber, wherein a downstream outlet of each said channel is regularly spaced around the delivery tube with respect to a flow path of the particle flow.
In an example, the one or more channels are defined by corresponding projections which extend along the delivery tube and/or the housing.
In an example, the projections extend from the delivery tube towards an internal surface of the housing or from the internal surface of the housing towards the delivery tube in a direction substantially perpendicular to the direction in which the channels defined by said projections extend.
In an example, the projections extend from the delivery tube to engage with the inner surface of the housing or from the inner surface of the housing to engage with the delivery tube.
In an example, an interior lateral cross-sectional shape of the housing within which the delivery tube is positioned is different to an exterior lateral cross-sectional shape of the delivery tube at a corresponding longitudinal position, and wherein the one or more channels are defined by said difference in lateral cross-sectional shape at different longitudinal positions.
In an example, the one or more channels: a. comprise a substantially semi-circular or triangular cross-section; and/or b. extend from a sheath fluid inlet to the focussing chamber; and/or c. are configured to provide the only flow path or paths for the sheath fluid between a sheath flow inlet and the focussing chamber over at least a portion of the longitudinal extension of the delivery tube within the housing; and/or d. comprise a larger cross-sectional area at a downstream position compared with a cross-sectional area at an upstream position of said one or more channels.
In an example, the one or more channels are defined by two or more ridges defining the cross-sectional shape of the channels and wherein said ridges are dimensioned to smoothly engage with the housing such that there is substantially zero volume between the ridge and an interior surface of the housing.
In an example, the one or more channels are angled with respect to the longitudinal axis of the delivery tube.
In an example, the one or more channels are arranged at regular intervals around the entirety of the external sectional circumference of the delivery tube and merge into the focussing chamber which is at least partially defined by a tapered delivery tube and an inversely tapered housing wall.
In an example, a longitudinal section of the exterior of the delivery tube adjacent the delivery tube outlet comprises one or more of the following: a conical shape; a hemispherical shape; a cylindrical shape; bevelling in one lateral axis; bevelling at two angles in one lateral axis; partial bevelling in a second perpendicular lateral axis; a notch in a lateral axis.
In an example, the interior shape of a longitudinal section of the housing corresponding to the longitudinal section of the exterior of the delivery tube comprises a constant or a tapering lateral cross-sectional shape and/or dimensions.
In an example, the delivery tube and the housing define multiple longitudinal cavity sections between them for carrying the sheath flow, the longitudinal cavity sections having different cross-sectional shapes and/or dimensions from each other.
In an example, the interior shape of the housing or the exterior shape of the delivery tube of any one of the longitudinal sections comprises one or more of the following: longitudinal tapering; a cross-sectional shape that is circular or elliptical or rectangular.
In an example, the interior shape of the housing has a first portion with a longitudinal cross-sectional shape arranged to engage at multiple longitudinal locations with a first portion of the delivery tube in order to position the delivery tube outlet at a predetermined lateral location within the focussing chamber.
In an example, the first portion of the interior shape of the housing has a rectangular longitudinal cross-sectional shape, and the first portion of the delivery tube has a rectangular longitudinal cross-sectional shape, and wherein the interior shape of the housing has a second cross-section shape with a reducing transverse width arranged to position the delivery tube outlet at a predetermined longitudinal location within the focussing chamber.
In an example, the delivery tube has an delivery tube inlet portion having delivery tube inlet cross-sectional dimensions, a delivery tube tapering portion and a delivery tube distal portion having delivery tube distal cross-sectional dimensions which are smaller than the delivery tube inlet cross-sectional dimensions, and wherein the interior shape of the housing has a cavity inlet portion having cavity inlet cross-sectional dimensions, a cavity tapering portion and a cavity distal portion having cavity distal portion dimensions.
In an example, the delivery tube distal cross-sectional dimensions and the cavity distal cross-sectional dimensions are both uniform in the longitudinal direction.
In an example, the cross-sectional shape of any one of the delivery tube inlet portion, the delivery tube tapering portion, the delivery tube distal portion is: circular; noncircular; elliptical; rectangular.
In an example, the cross-sectional shape of any one of the cavity inlet portion, the cavity tapering portion, the cavity distal portion is: circular; non-circular; elliptical; rectangular.
In an example, the cross-sectional shape of any one of the delivery tube inlet portion, the delivery tube tapering portion, the delivery tube distal portion is different to the cross-sectional shape of the corresponding cavity inlet portion, cavity tapering portion, cavity distal portion.
In an example, a delivery tube tapering angle of the delivery tube tapering portion is the same as a cavity tapering angle of the cavity tapering portion.
In an example, a delivery tube tapering angle of the delivery tube tapering portion is less than a cavity tapering angle of the cavity tapering portion.
In an example, the delivery tube at the delivery tube outlet has a conical shape.
In an example, the delivery tube at the delivery tube outlet is bevelled in one axis.
In an example, the delivery tube at the delivery tube outlet is partially bevelled in a second perpendicular axes.
In an example, the delivery tube at the delivery tube outlet has a notch in a notch axis. In an example, the lumen comprises one or more of the following: a non-circular cross- sectional shape along at least part of its length; a plurality of portions having different cross-sectional dimensions.
In an example, the lumen comprises a plurality of portions having different cross- sectional dimensions.
In an example, the focussing chamber comprises a substantially constant lateral cross- sectional shape along a longitudinal axis. The lateral cross-sectional shape may have an aspect ratio of substantially 1 : 1.
In an example, the downstream second focussing chamber comprises a longitudinally extending projection.
In an example, the height of the longitudinally extending projection tapers along its length.
In an example, the longitudinally extending projection forms a V-shaped inner surface of the focussing chamber.
In an example, the V-shaped inner surface forms at least one side of a lateral rectangular cross-sectional shape of the focussing chamber.
In an example, a second V-shaped inner surface forms a second side of the lateral rectangular cross-sectional shape of the focussing chamber.
In an example, the focussing chamber tapers longitudinally such that the height of the focussing chamber in at least one axis perpendicular to the longitudinal axis varies.
In an example, the focussing chamber tapers longitudinally such that the height and width of the focussing chamber in both axes perpendicular to the longitudinal axis both vary.
In an example, the flow control apparatus comprises a second focussing chamber fluidically coupled between the first focussing chamber and the aperture, wherein the geometry of the first focussing chamber is different than the geometry of the second focussing chamber; the geometries comprising one or more of the following: dimensions; cross-sectional shape; longitudinal tapering angle; a longitudinally extending projection.
In an example, the second focussing chamber comprising a lateral cross-sectional shape having a higher aspect ratio than a lateral cross-sectional shape of the first focussing chamber. In an example, the second focussing chamber comprises a longitudinally extending projection.
In an example, the height of the longitudinally extending projection tapers along its length.
In an example, the longitudinally extending projection forms a V-shaped inner surface of the second focussing chamber.
In an example, the V-shaped inner surface forms one side of a lateral cross-sectional shape of the second focussing chamber.
In an example, a second V-shaped inner surface forms a second side of the lateral rectangular cross-sectional shape of the second focussing chamber.
In an example, the second focussing chamber extends longitudinally with a substantially constant cross-sectional dimension in a first lateral axis.
In an example, the second focussing chamber extends longitudinally with a substantially constant cross-sectional dimension in a second lateral axis perpendicular to the first lateral axis.
In an example, the focussing chamber tapers longitudinally such that the height of the focussing chamber in at least one axis perpendicular to the longitudinal axis varies.
In an example, the focussing chamber tapers longitudinally such that the height and width of the focussing chamber in both axes perpendicular to the longitudinal axis both vary.
In an example, the second focussing chamber is fluidically coupled to the aperture by a delivery microchannel.
In an example, the delivery microchannel has a cross sectional shape with an aspect ratio higher than the first focussing chamber and with a dimension in one lateral axis smaller than the second focussing chamber.
In an example, the delivery microchannel has one of the following cross-sectional shapes: circular; elliptical; triangular; square; rectangular.
In an example, the delivery microchannel increases cross-sectional area towards the aperture.
In an example, the delivery microchannel forms an inverse conical shape towards the aperture. In an example, the flow control apparatus comprises a confinement chamber fluidically coupled to the second focussing chamber, the confinement chamber tapering longitudinally in at least one lateral axis from the second focussing chamber.
In an example, the lateral axis comprises the same axis that the second focusing chamber has as it's longest lateral axis dimension.
In an example, the flow control apparatus comprises an engagement structure configured to secure the delivery tube during use with the housing to form at least one of the one or more channel between the housing and the delivery tube, and position the delivery tube outlet at a predetermined longitudinal, lateral, and/or rotational location within the focusing chamber.
In an example, the housing has a first portion with a longitudinal cross-sectional shape arranged to engage at multiple longitudinal locations with a first portion of the delivery tube in order to position the delivery tube outlet at a predetermined lateral location within the focussing chamber.
In an example, the engagement structure comprises longitudinally extending ridges within the cavity of the housing, the ridges configured to position the delivery tube outlet according to the predetermined lateral relationship with the housing when received therein.
In an example, the ridges are formed with the housing and/or the delivery tube.
In an example, the engagement structure comprises a rotational alignment feature arranged to secure the delivery tube outlet at a predetermined rotational angle with respect to the housing.
In an example, the rotational alignment feature is a projection from one of the delivery tube or housing and a corresponding recess in one of the housing or delivery tube.
In an example, a flow control apparatus a delivery tube having a lumen for carrying a particle flow of liquid containing particles from a delivery tube inlet to a delivery tube outlet; the delivery tube extending longitudinally within a housing to a focussing chamber adjacent the delivery tube outlet; the flow control apparatus configured to carry a sheath flow of liquid towards the focussing chamber; the focussing chamber configured to join the particle flow and the sheath flow in order to issue a microfluidic stream from an aperture in the flow control apparatus, the microfluidic stream comprising a laminar flow of liquid from the sheath flow surrounding liquid from the particle flow; wherein the focussing chamber comprises a substantially constant lateral cross-sectional shape along a longitudinal axis and has an aspect ratio of substantially 1: 1. In an example, the delivery tube and the housing together define one or more longitudinally extending channels for carrying the sheath flow.
In an example, the one or more longitudinally extending channels are defined by corresponding longitudinally extending projections extending laterally from the delivery tube portion and/or the housing portion.
In an example, an interior lateral cross-sectional shape of the housing portion within which the delivery tube portion is positioned is different to an exterior lateral cross- sectional shape of the delivery tube portion at a corresponding longitudinal position, and wherein the one or more longitudinally extending channels are defined by said difference in lateral cross-sectional shape at different longitudinal positions.
In an example, the longitudinally extending channels have a semi-circular or triangular cross-section.
In an example, the longitudinally extending channels comprise at least one of: a. a semi-circular or triangular cross-section; b. extend from a sheath fluid inlet to the focussing chamber; c. configuration to provide the only flow path for the sheath fluid between a sheath flow inlet and the focussing chamber over at least a portion of the longitudinal extension of the delivery tube within the housing.
In an example, the longitudinally extending channels are angled with respect to the longitudinal axis of the delivery tube.
In an example, a longitudinal section of the exterior of the delivery tube adjacent the delivery tube outlet comprises one or more of the following: a conical shape; a hemispherical shape; a cylindrical shape; bevelling in one lateral axis; bevelling at two angles in one lateral axis; partial bevelling in a second perpendicular lateral axis; a notch in a lateral axis.
In an example, the interior shape of a longitudinal section of the housing corresponding to the longitudinal section of the exterior of the delivery tube comprises a constant or a tapering lateral cross-sectional shape and/or dimensions.
In an example, the delivery tube and the housing define multiple longitudinal cavity sections between them for carrying the sheath flow, the longitudinal cavity sections having different cross-sectional shapes and/or dimensions from each other.
In an example, the interior shape of the housing or the exterior shape of the delivery tube of any one of the longitudinal sections comprises one or more of the following: longitudinal tapering; a cross-sectional shape that is circular or elliptical or rectangular. In an example, the lumen comprises one or more of the following: a non-circular shape along at least part of its length; a plurality of portions having different cross-sectional dimensions.
In an example, the focussing chamber tapers longitudinally.
In an example, the flow control apparatus comprises a second focussing chamber fluidically coupled between the first focussing chamber and the aperture, the second focussing chamber comprising a lateral cross-sectional shape having a higher aspect ratio than the first focussing chamber.
In an example, the second focussing chamber extends longitudinally with a substantially constant cross-sectional dimension in a first lateral axis.
In an example, the second focussing chamber extends longitudinally with a substantially constant cross-sectional dimension in a second lateral axis perpendicular to the first lateral axis.
In an example, the second focussing chamber is fluidically coupled to the aperture by a delivery microchannel having a cross sectional shape with an aspect ratio higher than the first focussing chamber and with a dimension in one lateral axis smaller than the second focussing chamber.
In an example, the flow control apparatus comprises a confinement chamber fluidically coupled to the second focussing chamber, the confinement chamber tapering longitudinally in one lateral axis from the second focussing chamber.
In an example, a particle processing system comprises a flow control apparatus as defined above, an interrogation apparatus and/or a sorting apparatus arranged to direct electromagnetic radiation at particles in a microfluidic stream issued by the flow control apparatus. The flow control apparatus is arranged to orient the asymmetric particles such that the electromagnetic radiation is directed at a predetermined facet of the asymmetric particles.
In an example, the delivery tube and the housing form are formed together as a single unified material unit or separately as respective material units.
In an example, the flow control apparatus comprises an engagement structure configured to: secure the delivery tube during use with the housing to form at least one longitudinally extending channel between the housing and the delivery tube, and position the delivery tube outlet at a predetermined longitudinal, lateral, and/or rotational location within the focusing chamber. In an example, a cavity of the housing has a first portion with a longitudinal cross- sectional shape arranged to engage at multiple longitudinal locations with a first portion of the delivery tube in order to position the delivery tube outlet at a predetermined lateral location within the focussing chamber.
In an example, the engagement structure comprises longitudinally extending ridges within the cavity of the housing, the ridges configured to position the delivery tube outlet according to the predetermined lateral relationship with the housing when received therein.
In an example, the engagement structure comprises a rotational alignment feature arranged to secure the delivery tube outlet at a predetermined rotational angle with respect to the housing.
In an example, a method of processing a stream of particles emitted from a delivery tube comprising receiving a first fluid stream comprising one or more particles and passing the stream through at least one of: a flow control apparatus as defined above or herein; a particle processing system as defined above or herein; a housing as defined above or herein; and a delivery tube as defined above or herein.
In an example, the method of processing comprises one or more of the following: inspecting the particles; displacing the particles into a different flow path within the processing channel; orienting the particles.
In an example, the method comprises exposing particles to one or more radiation sources.
In an example, the method comprises directing electromagnetic radiation at the particles to expose the particles to electromagnetic radiation to displace the particles by a predetermined amount and/or to orient the particles to a predetermined orientation and/or to ablate the particles.
In an example, the method comprises inspecting the particles and further comprises the steps of determining one or more characteristics of the particle within the stream to yield a particle characteristic; and selecting a subpopulation of the particles based on the particle characteristic.
In an example, the particle comprises a cell or a sperm cell.
In an example, the step of determining one or more characteristics comprises detecting fluorescence emitted from a particle.
In an example, the step of selecting a subpopulation of the particles comprises sorting the particles using an electromagnetic sorting method. In an example, the electromagnetic sorting method comprises a pulse or continuous flow of energy to displace a charged or uncharged particle.
In an example, the sorting method comprises a radiation source configured to direct radiation on to the particle to effect at least one of a force and torque on each particle so as to induce at least one of displacing and orienting each particle relative to an axis defined by the direction of the fluid flow along the microfluidic channel.
In an example, a flow control apparatus comprises a housing having a particle flow outlet in fluid communication with a cavity for receiving a delivery tube, the delivery tube having a lumen for carrying a source fluid containing particles from a delivery tube inlet to a delivery tube outlet; an engagement structure arranged to secure the delivery tube in the cavity to form a channel between the housing and the delivery tube, and a focusing chamber in fluid communication with the particle flow outlet, the channel for carrying a sheath fluid to the focussing chamber; the engagement structure arranged to position the delivery tube outlet at a predetermined longitudinal, lateral or rotational location within the focusing chamber.
In an example, the cavity of the housing has a first portion with a longitudinal cross- sectional shape arranged to engage at multiple longitudinal locations with a first portion of the delivery tube in order to position the delivery tube outlet at a predetermined lateral location within the focussing chamber.
In an example, the first portion of the cavity has a rectangular longitudinal cross- sectional shape, and the first portion of the delivery tube has a rectangular longitudinal cross-sectional shape, and wherein the cavity has a second cross-section shape with a reducing transverse width arranged to position the delivery tube outlet at a predetermined longitudinal location within the focussing chamber.
In an example, the engagement structure comprises longitudinally extending ridges on at least one of the delivery tube or within the cavity of the housing, the ridges configured to position the delivery tube outlet according to the predetermined lateral relationship with the housing when received therein.
In an example, the ridges are configured to form a plurality of channels between the housing and delivery tube for carrying the sheath fluid to the focussing chamber.
In an example, the channels have a semi-circular or triangular cross-section.
In an example, the ridges are angled with respect to the longitudinal axis of the delivery tube. In an example, the engagement structure comprises a rotational alignment feature arranged to secure the delivery tube outlet at a predetermined rotational angle with respect to the housing.
In an example, the rotational alignment feature is a projection from one of the delivery tube or housing and a corresponding recess in one of the housing or delivery tube.
In an example, the delivery tube at the delivery tube outlet has a conical shape.
In an example, the delivery tube at the delivery tube outlet is bevelled in one axis.
In an example, the delivery tube at the delivery tube outlet is partially bevelled in a second perpendicular axes.
In an example, the delivery tube at the delivery tube outlet has a notch in a notch axis.
In an example, the lumen has a non-circular shape along at least part of its length.
In an example, the lumen comprises a plurality of portions having different cross- sectional dimensions.
In an example, the delivery tube has an delivery tube inlet portion having delivery tube inlet cross-sectional dimensions, a delivery tube tapering portion and a delivery tube distal portion having delivery tube distal cross-sectional dimensions which are smaller than the delivery tube inlet cross-sectional dimensions, and wherein the cavity of the housing has a cavity inlet portion having cavity inlet cross-sectional dimensions, a cavity tapering portion and a cavity distal portion having cavity distal portion dimensions.
In an example, the delivery tube distal cross-sectional dimensions and the cavity distal cross-sectional dimensions are both uniform in the longitudinal direction.
In an example, a delivery tube tapering angle of the delivery tube tapering portion is the same as a cavity tapering angle of the cavity tapering portion.
In an example, a delivery tube tapering angle of the delivery tube tapering portion is less than a cavity tapering angle of the cavity tapering portion.
In an example, the cross-sectional shape of any one of the delivery tube inlet portion, the delivery tube tapering portion, the delivery tube distal portion is: circular; noncircular; elliptical; rectangular.
In an example, the cross-sectional shape of any one of the cavity inlet portion, the cavity tapering portion, the cavity distal portion is: circular; non-circular; elliptical; rectangular.
In an example, the cross-sectional shape of any one of the delivery tube inlet portion, the delivery tube tapering portion, the delivery tube distal portion is different to the cross-sectional shape of the corresponding cavity inlet portion, cavity tapering portion, cavity distal portion.
In an example, a flow control apparatus as defined above or herein, a combined flow microchannel connected to the particle flow outlet and a downstream module are arranged to direct electromagnetic radiation at particles carried by the combined flow microchannel.
In an example, the flow control apparatus is arranged to orient the asymmetric particles such that the electromagnetic radiation is directed at a predetermined facet of the asymmetric particles.
In an example, the delivery tube and the direction of the electromagnetic radiation are rotationally aligned about the longitudinal axis of the particle flow.
In an example, the downstream module comprises one or more of the following: a particle inspection module; a particle displacement module arranged to displace particles into a different flow path within the combined flow microchannel; a particle orientation module arranged to expose particles to one or more radiation sources to orient particles.
In an example, a housing for a flow control apparatus comprises a cavity in fluid communication with a particle flow outlet in fluid communication and configured to receive a delivery tube for carrying a source fluid containing particles to a delivery tube outlet; an engagement structure arranged to secure the delivery tube in use in the cavity to form a channel between the housing and the delivery tube, and to form a focusing chamber in fluid communication with the particle flow outlet, the channel for carrying a sheath fluid to the focussing chamber; the engagement structure arranged to position the delivery tube outlet at a predetermined longitudinal, lateral or rotational location within the focusing chamber.
In an example, the cavity has a first portion with a longitudinal cross-sectional shape arranged to engage at multiple longitudinal locations with a first portion of the delivery tube in order to position the delivery tube outlet at a predetermined lateral location within the focussing chamber.
In an example, the first portion of the cavity has a rectangular longitudinal cross- sectional shape, configured to engage with the first portion of the delivery tube with a rectangular longitudinal cross-sectional shape, and wherein the cavity has a second cross-section shape with a reducing transverse width arranged to position the delivery tube outlet at a predetermined longitudinal location within the focussing chamber. In an example, the engagement structure comprises longitudinally extending ridges within the cavity of the housing, the ridges configured to position the delivery tube outlet according to the predetermined lateral relationship with the housing when received therein.
In an example, the ridges are configured to form a plurality of channels between the housing and delivery tube for carrying the sheath fluid to the focussing chamber.
In an example, the engagement structure comprises a rotational alignment feature arranged to secure the delivery tube outlet at a predetermined rotational angle with respect to the housing.
In an example, a delivery tube for a flow control apparatus for controlling fluid flows associated with carrying particles is provided. The delivery tube comprises a lumen for carrying a source fluid containing particles from a delivery tube inlet to a delivery tube outlet; an engagement structure arranged to secure the delivery tube in a cavity of a housing in use to form a channel between the housing and the delivery tube, and a focusing chamber in fluid communication with a particle flow outlet, the channel for carrying a sheath fluid to the focussing chamber; the engagement structure arranged to position the delivery tube outlet at a predetermined longitudinal, lateral or rotational location within the focusing chamber.
In an example, the delivery tube comprises a first portion with a longitudinal cross- sectional shape arranged to engage at multiple longitudinal locations with a first portion of the cavity of the housing in order to position the delivery tube outlet at a predetermined lateral location within the focussing chamber.
In an example, the first portion of the delivery tube has a rectangular longitudinal cross- sectional shape which is configured to engage with a second portion of the cavity with a reducing transverse width in order to position the delivery tube outlet at a predetermined longitudinal location within the focussing chamber.
In an example, the engagement structure comprises longitudinally extending ridges on the delivery tube, the ridges configured to position the delivery tube outlet according to the predetermined lateral relationship with the housing when received therein.
In an example, the ridges are configured to form a plurality of channels between the housing and delivery tube for carrying the sheath fluid to the focussing chamber.
In an example, there is provided a delivery tube for a flow control apparatus for controlling fluid flows associated with carrying particles. The delivery tube comprises a lumen for carrying a source fluid containing particles from a delivery tube inlet to a delivery tube outlet; wherein the delivery tube at the delivery tube outlet is bevelled in one axis and is partially bevelled in a second perpendicular axis and/or has a notch in a notch axis.
In an example, the delivery tube at the delivery tube outlet is both partially bevelled in a second perpendicular axis and has a notch in a notch axis, wherein the notch axis is the same as the second perpendicular axis.
In an example, a flow control apparatus for controlling fluid flows associated with carrying particles is provided. The flow control apparatus comprises a housing having a particle flow outlet in fluid communication with a cavity for receiving a delivery tube, the delivery tube having a lumen for carrying a source fluid containing particles from a delivery tube inlet to a delivery tube outlet; wherein the delivery tube has an delivery tube inlet portion having delivery tube inlet cross-sectional dimensions, a delivery tube tapering portion and a delivery tube distal portion having delivery tube distal cross- sectional dimensions which are smaller than the delivery tube inlet cross-sectional dimensions, and wherein the cavity of the housing has a cavity inlet portion having cavity inlet cross-sectional dimensions, a cavity tapering portion and a cavity distal portion having cavity distal portion dimensions.
In an example, the delivery tube distal cross-sectional dimensions and the cavity distal cross-sectional dimensions are both uniform in the longitudinal direction.
In an example, a delivery tube tapering angle of the delivery tube tapering portion is the same as a cavity tapering angle of the cavity tapering portion.
In an example, a delivery tube tapering angle of the delivery tube tapering portion is less than a cavity tapering angle of the cavity tapering portion.
In an example, the cross-sectional shape of any one of the delivery tube inlet portion, the delivery tube tapering portion, the delivery tube distal portion is: circular; noncircular; elliptical; rectangular.
In an example, the cross-sectional shape of any one of the cavity inlet portion, the cavity tapering portion, the cavity distal portion is: circular; non-circular; elliptical; rectangular.
In an example, the cross-sectional shape of any one of the delivery tube inlet portion, the delivery tube tapering portion, the delivery tube distal portion is different to the cross-sectional shape of the corresponding cavity inlet portion, cavity tapering portion, cavity distal portion.
In an example, the lumen has a non-circular shape along at least part of its length. In an example, the lumen comprises a plurality of portions having different cross- sectional dimensions.
Aspects of the invention may also be said broadly to consist in the parts, elements and features referred to or indicated in the specification of application, individually or collectively, in any or all combinations of two or more of said parts, elements or features, and where specific integers are mentioned 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. BRIEF DESCRIPTION OF THE DRAWINGS
Examples will now be described by way of example only and with reference to the drawings in which:
Figure 1 shows a perspective view of a flow control apparatus according to an example;
Figure 2 shows a side and end view of a system having the flow control apparatus of Figure 1;
Figure 3 shows a longitudinal section of a flow control apparatus according to an example;
Figure 4A shows a cross-section view through section line AA of the flow control apparatus of Figure 3;
Figures 4B - 41 show alternative cross-sections through section line AA of the flow control apparatus of Figure 3;
Figure 5A and 5B show detailed longitudinal sections of the control chamber area of the flow control apparatus of Figure 3;
Figure 6A shows side and end views of a delivery tube according to an example;
Figure 6B shows a longitudinal section of a flow control apparatus according to an example;
Figure 6BA and 6BB show lateral section views of the flow control apparatus of Figure 6B according to examples;
Figure 6C shows a longitudinal section of a flow control apparatus according to an example;
Figure 6D and 6E show longitudinal sections of a flow control apparatus according to examples; Figure 6F shows orientation efficiency and discrimination measurements at differing flow velocities for the example of Figure 6B;
Figure 7A, 7B and 7C show side views of a delivery tube according to other examples;
Figure 8 shows end views and longitudinal sections through different axes and a perspective view of a conical tip of a delivery tube according to an example;
Figure 9 shows end views and longitudinal sections through different axes and a perspective view of a tip of a delivery tube which is bevelled in one axis, and according to an example;
Figure 10 shows end views and longitudinal sections through different of a tip of a delivery tube which is bevelled in two axes , and according to an example;
Figure 11 shows end views and longitudinal sections through different axes and a perspective view of a tip of a delivery tube which is bevelled in one axis, partially bevelled in a second perpendicular axis and includes a notch through the second axis, and according to an example;
Figure 12 shows end views and longitudinal sections through different axes and a perspective view of a tip of a delivery tube which includes a notch through one axis, and according to an example;
Figure 12A shows longitudinal sections through one axis for a number of other tapered tips of a delivery tube according to some examples;
Figure 13 shows section views through the flow control apparatus having different combinations of housing cavity and delivery tube geometries according to some examples;
Figure 14 illustrates experimental results of particle flow confinement at a particle velocity of 200mm/s according to one example;
Figure 15 illustrates experimental results of particle flow confinement at a particle velocity of lOOmm/s according to one example;
Figure 16 illustrates experimental results of particle flow confinement at a particle velocity of 50mm/s according to one example;
Figure 17 illustrates experimental results of particle flow confinement at a particle velocity of 500mm/s according to one example;
Figure 18 illustrates experimental results of particle flow position under different particle velocity conditions according to one example; Figure 19 illustrates experimental results of particle flow confinement results using different delivery tube tips according to one example;
Figure 20 illustrates experimental results of particle flow position in a minor axis under different particle velocity conditions according to one example;
Figure 21 illustrates experimental results of particle flow position for an elliptical delivery tube outlet cross-section under different particle velocity conditions according to one example.
Figure 22 illustrates a method of controlling flows associated with particles and according to an example.
Figures 23A and 23B show respectively a summary plot of experimental orientation efficiencies (y axis) vs PODT tip position in z (bottom x axis, [mm]) and max cell speed (top x axis, [m/s]),
Figure 24 illustrates how velocity components induce drag forces and can lead to a preferred orientation for aspheric particles in the focusing apparatus geometry described herein.
Figure 25A shows simulation vx/vy (y axis) vs max cell speed (top x axis, [m/s]) and PODT tip position in z (bottom x axis, [m]).
Figure 25B shows simulation vxx/vyy (y axis) vs max cell speed (top x axis, [m/s]) and PODT tip position in z (bottom x axis, [m]).
Figure 26A, 26B and 26C illustrate the effect of a mis-aligned or tilted tip;
Figure 27a and 27b illustrate example longitudinal positions of the delivery tube in the second focussing chamber;
Figure 28a-e illustrate example configurations of a second focussing chamber; and
Figure 29 illustrates a cross-section of an aperture according to an example.
5. DESCRIPTION OF EXAMPLES
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 that or those prefaced by the term may also be present. Related terms such as "comprise" and "comprises" are to be interpreted in the same manner.
The term "about" as used herein 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 construed as including a deviation of+/- 5% of the value.
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.
The indefinite articles "a" and "an," as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean "at least one."
The terms "can" and "may" are used interchangeably in the present disclosure, and indicate that the referred to element, component, structure, function, functionality, objective, advantage, operation, step, process, apparatus, system, device, result, or clarification, has the ability to be used, included, or produced, or otherwise stand for the proposition indicated in the statement for which the term is used (or referred to) for a particular example(s).
The phrase "and/or," as used herein in the specification and in the claims, should be understood to mean "either or both" of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases.
Multiple elements listed with "and/or" should be construed in the same fashion, i.e., "one or more" of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the "and/or" clause, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, a reference to "A and/or B", when used in conjunction with open-ended language such as "comprising" can refer, in one example, to A only (optionally including elements other than B); in another example, to B only (optionally including elements other than A); in yet another example, to both A and B (optionally including other elements); etc.
As used herein in the specification and in the claims, the phrase "at least one," in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present 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 elements specifically identified. Thus, as a non-limiting example, "at least one of A and B" (or, equivalently, "at least one of A or B," or, equivalently "at least one of A and/or B") can refer, in one example, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another example, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another example, 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); etc.
It is intended that reference to a range of numbers disclosed herein (for example, 1 to 10) also incorporates reference to all rational numbers within that range (for example, 1, 1.1, 2, 3, 3.9, 4, 5, 6, 6.5, 7, 8, 9 and 10) and also any range of rational numbers within that range (for example, 2 to 8, 1.5 to 5.5 and 3.1 to 4.7) and, therefore, all sub-ranges of all ranges expressly disclosed herein are hereby expressly disclosed. These are only examples of what is specifically intended and all possible combinations of numerical values between the lowest value and the highest value enumerated are to be considered to be expressly stated in this application in a similar manner.
Whenever a range is given in the specification, for example, a dimensional range, a time range, or a composition range, all intermediate ranges and subranges, as well as all individual values included in the ranges given are intended to be included in the disclosure. In the disclosure and the claims, "and/or" means additionally or alternatively. Moreover, any use of a term in the singular also encompasses plural forms.
The following sets forth specific details, such as particular examples or examples for purposes of explanation and not limitation. It will be appreciated by one skilled in the art that other examples may be employed apart from these specific details. In some instances, detailed descriptions of well-known methods, nodes, interfaces, circuits, and devices are omitted so as not obscure the description with unnecessary detail. Those skilled in the art will appreciate that the functions described may be implemented in one or more nodes using hardware circuitry (e.g., analog and/or discrete logic gates interconnected to perform a specialized function, ASICs, PLAs, etc.) and/or using software programs and data in conjunction with one or more digital microprocessors or general purpose computers. Nodes that communicate using the air interface also have suitable radio communications circuitry. Moreover, where appropriate the technology can additionally be considered to be embodied entirely within any form of computer- readable memory, such as solid-state memory, magnetic disk, or optical disk containing an appropriate set of computer instructions that would cause a processor to carry out the techniques described herein.
Hardware implementation may include or encompass, without limitation, digital signal processor (DSP) hardware, a reduced instruction set processor, hardware (e.g., digital or analogue) circuitry including but not limited to application specific integrated circuit(s) (ASIC) and/or field programmable gate array(s) (FPGA(s)), and (where appropriate) state machines capable of performing such functions. Memory may be employed to storing temporary variables, holding and transfer of data between processes, nonvolatile configuration settings, standard messaging formats and the like. Any suitable form of volatile memory and non-volatile storage may be employed including Random Access Memory (RAM) implemented as Metal Oxide Semiconductors (MOS) or Integrated Circuits (IC), and storage implemented as hard disk drives and flash memory.
Some or all of the described apparatus or functionality may be instantiated in cloud environments such as Docker, Kubenetes or Spark. This cloud functionality may be instantiated in the network edge, apparatus edge, in the local premises or on a remote server coupled via a network such as 4G or 5G. Alternatively, this functionality may be implemented in dedicated hardware.
The term "confinement" as referred to herein refers to the restriction of the cross- sectional shape and size of a flow of particles in a sample stream. For example, the diameter of a circular section of the flow may be restricted or the dimensions of the major and minor axes of an elliptical section flow may be restricted which may result in a single narrow trajectory with minimal deviation in any polar axis of particles from a defined central longitudinal axis of the flow. This constriction of flow simultaneously has the effect of reducing the average distance of individual particles from a nominal central flow vector along the axis of flow. It is generally desirable to increase confinement to result in an accurate focussing of an interrogation beam and a sorting beam for downstream interrogation and sorting. If the particles are not accurately confined, measurements may be less accurate which can negatively affect the yield of selected cells and the speed with which cells can be accurately detected.
The term "orientation" of asymmetric particles (including cells) means the predominant angle of a face of a representative sample of said particles with respect to an axis substantially perpendicular to the axis of flow of the particles. Without any features imparting an orienting torque on the particles, it is expected that the orientation of said face will be randomly distributed and facing any angle around 360°. A sample of cells that have had an orienting torque applied via an orienting feature will have a nonrandom angular orientation that preferentially directs the face of the particle in a particular angle so that a predominant angle can be determined or observed.
"Orientation efficiency" of a method or apparatus for orienting asymmetric particles corresponds to the percentage or proportion of particles in a sample of particles which are oriented with predominant angle, within a predetermined tolerance.
"Cells" and "X-cells" are referred to herein as examples of particular types of particles that may be desirable to retain within a microfluidic sorting arrangement. Where the term cell is used herein, it may be substituted with the term "particle" and there is no requirement for the cell/pa rticle to be a living cell. Those of skill in the art will readily appreciate that the mention of X-cells is intended to be indicative of any other cells or particles that have characteristics suitable for interrogation and sorting according to the present invention. In particular, X-cells may be substituted herein for any type of particle or cell, including substantially symmetric and asymmetric cells, neurons, red blood cells, tagged cells, viruses, or microbiota as will be known to those of skill in the art.
The term "microfluidic stream" as referred to herein refers to a flow of liquid having at least one geometrically constrained dimension at which surface forces dominate volumetric forces. In an example this may include a liquid stream having a submillimetre diameter or other cross-sectional dimension. In an example the microfluidic stream may be a continuous phase flow of liquid such as an unbroken stream of one or more aqueous solutions. This could be a laminar flow having a particle flow comprising particles and a sheath flow surrounding the particle flow. The microfluidic stream may alternatively or additionally comprise a dispersed flow of liquid drops. The microfluidic stream may be associated with one or more performance metrics such as flow rate, cross-sectional diameter and/or dimensions, distance to droplet formation.
The term "flow environment" as referred to herein refers to an environment in which the microfluidic stream may flow through. An example includes a microchannel which may comprise a material such as glass forming an elongate lumen or pathway through which the microfluidic stream flows. The pathway may be fully encompassed by the material between each end of the pathway; or the pathway may have at least one boundary exposing the microfluidic stream to a fluid environment with the material forming a substrate interfacing with the other boundary(s) of the microfluidic stream. In another example, the flow environment may be a fluid environment or volume which may be substantially static or which itself may be flowing. In this example, the microfluidic stream may not interface with a material substrate but be fully encompassed by the fluid environment. The fluid environment may be a liquid such as an aqueous solution or a gas such as air.
Figure 1 is a perspective view of a flow control apparatus according to an example. The flow control apparatus 100 comprises a delivery tube 130 which in use is installed within a housing 105. The figure shows these two components 105, 130 coupled together for use as well as separated. The two components 105, 130 may be replaceable separately of each other and may be consumable parts of a larger system. Alternatively, the components may be formed as part of a single monolithic structure. The housing 105 comprises a cavity 110 into which the delivery tube 130 is located, and in use may be coupled to a processing microchannel 180 at an aperture from the flow control apparatus. The delivery tube 130 comprises a lumen 145 extending from an inlet end as shown to a distal end within the cavity 110.
The housing 105 and delivery tube 130 may be constructed of various materials including: plastics, polymers, metal, glass, ceramic and composites. In one example, the flow control apparatus comprises both the delivery microchannel and focusing and/or confinement chambers. At least one of the flow control apparatus and the microfluidic channel may be formed from any one or more of a polymer, glass, ceramic, or other solid substrate, or may be pre-formed components such as PTFE tubing or glass capillaries. In some examples, a variety of materials and methods can be used to form any of the above-described components of the present disclosure. For example, the flow focusing apparatus, tube, channels and chambers of the present disclosure can be formed from solid materials, in which the channels can be formed via micromachining, film deposition processes such as spin coating and chemical vapor deposition, laser fabrication, photolithographic techniques, 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 apparatus is formed of silicon by etching features in a silicon chip. Technologies for precise and efficient fabrication of various fluidic systems and devices of the present disclosure from silicon are known. In another example, various components of the systems and devices of the present disclosure can be formed of a polymer, for example, an elastomeric polymer such as polydimethylsiloxane (PDMS), polytetrafluoroethylene (PTFE), or the like. In another example, the channels of the present disclosure can be formed from a polymer, glass, ceramic or other solid substrate, or may be pre-formed components such as PTFE tubing or glass capillaries. Different components can be fabricated of different materials. For example, at least one of the flow control apparatus and the microfluidic channel can be fabricated from an opaque material such as silicon, or from a transparent or at least partially transparent material, such as glass or a transparent polymer, for observation and/or control of the flow process. Components can be coated so as to expose a desired chemical functionality to fluids that contact interior channel walls. For example, components can be fabricated with interior channel walls coated with another material. Material used to fabricate various components of the systems and devices of the present disclosure (e.g., materials used to coat interior walls of fluid channels) may desirably be selected from among those materials that will not adversely affect or be affected by fluid flowing through the fluidic system (e.g., material(s) that are chemically inert in the presence of fluids to be used within the device). In one embodiment, various components of the present disclosure are fabricated from polymeric and/or flexible and/or elastomeric materials, and can be conveniently formed of a hardenable fluid, facilitating fabrication via moulding (e.g., replica moulding, injection moulding, cast moulding, etc.). The hardenable fluid can be essentially any fluid that can be induced to solidify, or that spontaneously solidifies, into a solid capable of containing and/or transporting fluids contemplated for use in and with the described microfluidic system. In one embodiment, the hardenable fluid comprises a polymeric liquid or a liquid polymeric precursor (i.e. a "prepolymer"). Suitable polymeric liquids can include, for example, thermoplastic polymers, thermoset polymers, or a mixture of such polymers heated above their melting point. As another example, a suitable polymeric liquid may include a solution of one or more polymers in a suitable solvent, which solution forms a solid polymeric material upon removal of the solvent, for example, by evaporation. Such polymeric materials, which can be solidified from, for example, a melt state or by solvent evaporation, are well known to those of ordinary skill in the art. A non-limiting list of examples of such polymers includes polymers of the general classes of silicone polymers, epoxy polymers, and acrylate polymers. Silicone polymers including PDMS have several beneficial properties simplifying fabrication of the microfluidic structures of the present disclosure. For instance, such materials are inexpensive, readily available, and can be solidified from a prepolymeric liquid via curing with heat. For example, PDMSs are typically curable by exposure of the prepolymeric liquid to temperatures of about, for example, about 65 °C to about 75 °C for exposure times of, for example, about an hour. Also, silicone polymers, such as PDMS, can be elastomeric and thus may be useful for forming very small features with relatively high aspect ratios, necessary in certain embodiments of the present disclosure. Flexible (e.g., elastomeric) moulds or masters can be advantageous in this regard. In further examples, the components of the present invention may be formed from recycled polymers or biodegradable polymers such as polylactic acid (PLA) and polyhydroxyalkanoates (PHAs).
In some examples the material is pre- or post- processed to increase smoothness. This example has surprisingly been found to substantially improve some of the flow properties of the material, for example reducing turbulence or drag. For example, stainless steel may be electropolished for improved smoothness and 3D printed plastics may be sanded, surface melted and/or coated with a smooth surface material.
The delivery tube 130 comprises a plurality of ridges or projections which extend longitudinally and in use engage with the inside wall of the cavity. In practice this secures the delivery tube within the housing such that the lumen 145 at the distal end of delivery tube is securely and accurately positioned within the housing. In some examples this feature also influences the fluid flow characteristics.
In an example, accurate positioning enables a flow of particles from the delivery tube to be carefully controlled within a microfluidic stream issued into the processing microchannel 180 for downstream processing. For example, the flow of particles in the microfluidic stream may benefit from improved characteristics selected from the group consisting of: enhanced positional stability, reduced mechanical stresses on the particles, reduced clumping or more uniform distribution of particles, increased or more accurate confinement, and/or more effective particle orientation. The microfluidic stream comprises a laminar combination of a particle flow from inside the delivery tube and a sheath flow from around the outside of the delivery tube, with the sheath flow surrounding the particle flow in the microfluidic stream. The generation of a stable laminar flow from the flow control apparatus in which particles are desirably arranged enables more effective downstream processing as described in more detail below. The microfluidic stream may be issued into a processing microchannel 180 as shown or into another flow environment such as a gas or liquid. The microfluidic stream issued from an aperture of a delivery microchannel within the flow control apparatus. The other end of the delivery microchannel couples to one or more focussing chambers within the flow control apparatus which contribute to the desired arrangement of the particles within the stable laminar flow of the microfluidic stream. As described herein, the focussing chamber may comprise the upstream focusing chamber, the downstream focusing chamber or the downstream confinement chamber. In some examples, downstream processing may include detection of particles (e.g. sperm) characteristics, particle orientation, particle displacement and/or particle sorting according to said characteristics.
An advantage of some examples is the controlled and stable confinement of particles within a narrow stream emitted from the delivery tube. In certain examples this confined stream passes into a processing microchannel 180 and the uninterrupted fluid stream is subjected to a sorting step to sort particles according to certain detected particle characteristics. The controlled confinement achieved enables enhanced sorting efficiency.
In particular examples, the processing microchannel 180 may have a width in the range 10 to 500 microns, or 100 to 400 microns, and a depth in the range 5 to 250 microns, for example. In some examples, the width and depth are identical, and the cross-section of the processing microchannel may be of any shape. Particularly preferred shapes of the cross-sectional shape are circular or square. The dimensions of the processing microchannel supports laminar flow, with minimal turbulence. In some examples, the processing microchannel has a planar form with in-plane length and width greater than depth transverse to the plane. In alternative examples, the depth may be greater than the length and/or width of the microchannel. In alternative examples, the processing microchannels may comprise one or more capillaries, and may feature curved segments as well as angles.
In some alternative examples, the microfluidic stream from the flow control apparatus 100 is ejected into free space or another flow environment rather than the processing microchannel 180. Particles within the microfluidic stream may then be subjected to interrogation and sorting, absent processing microchannel 180. This may provide some advantages including faster flow rate and reduced diffraction of interrogation and sorting beams. This may in turn lead to greater accuracy in the focussing of these beams and a reduction in their power, reducing their impact on wanted particles.
In use, a sheath flow of liquid is passed through channels formed between the ridges 135 and the inside surface of the cavity 110. Alternatively or additionally, the ridges may be formed on the inside surface of the cavity to create channels when engaged with the delivery tube. These channels stabilise the sheath flow, for example by reducing turbulence which may be present in the introduced fluid and/or by homogenising the flow. In particular, turbulence is introduced by having one or more sheath flow channels which enter the flow control apparatus orthogonally with respect to the longitudinal or Z- axis of flow of the particle flow or sample stream/fluid.
These channels provide a 360 degree confinement and focussing of the particle flow or sample stream which obviates the need for multiple sheath flow intersections at different directions. This simplifies the chip architecture used for the flow control apparatus.
A particle flow of liquid containing particles, preferably asymmetric particles, is passed through the lumen 145. The flow control apparatus 100 is configured such that a combined particle and sheath flow having the desired characteristics noted above emerges as a microfluidic stream into the processing microchannel, with a central particle flow and a surrounding sheath flow in a coaxial arrangement stabilised into a single laminar flow. The particle and sheath flows may be independently formed to control a number of particle flow properties, and/or combined flow properties.
In one example, asymmetric or non-spherical particles are substantially oriented in a common axis or plane within the microfluidic stream and/or the particles are focussed or confined such that the particles flow more evenly. This enables the particles to be more easily investigated and/or manipulated downstream, for example in the processing microchannel or a free space flow environment. Some examples also allow maintaining orientation and confinement properties of particles across many flow rate ranges. Other particle and/or flow properties may additionally or alternatively be adjusted using the flow control apparatus. As will be described in more detail below, examples provide more accurate lateral and longitudinal positioning of the delivery tube 130 within the housing 105 resulting in more effective focussing of the particles within a microfluidic stream. This also results in enhanced positional stability of the particles within the microfluidic stream which improves downstream interrogation and sorting performance.
Examples also reduce particle direction changes or shearing forces as well as flow turbulence thereby reducing the mechanical stresses on the particles. This in turn can improve the viability of biological cells such as sperm cells.
Examples also more evenly distribute particles within the microfluidic stream, resulting in less clumping and therefore better downstream interrogation and sorting performance.
Examples provide improved and more accurate confinement by reducing the displacement of particles from an axis of the microfluidic stream. This also results in better downstream interrogation and sorting performance.
Examples provide improved orientation efficiency, increasing the proportion of particles that are optimally oriented for downstream interrogation and sorting processes.
Figure 2 illustrates a system 200 according to an example and including a flow control apparatus 205, 230 which has an aperture 235 coupled to a processing microchannel 280. A side view of the system 200 is shown in an XY plane on the upper left. The flow control apparatus 205 controls separate sheath and particle flows which are combined to generate a microfluidic stream with desired properties. The microfluidic stream is emitted into the processing microchannel 280 as described in more detail below. In one example, the processing microchannel terminates prior to downstream interrogation or sorting. In another example, the microfluidic stream is emitted from an aperture 380 of the flow control apparatus 300 into a different flow environment which may comprise a free space volume with a static or moving gaseous atmosphere. In these examples, the processing microchannel terminates at an aperture 380 of a delivery microchannel 375 within the flow control apparatus.
Once in the processing microchannel 280 or issued into another flow environment from the aperture 235, 380, the microfluidic stream is stabilised into a combined single laminar flow 260. A detail on the lower left of Figure 2 illustrates the flow of particles 277 within a particle flow path 275 within a sheath flow path 265 in the XY plane. An end view of the system 200 is shown in a YZ plane on the upper right of Figure 2 with a detail on the lower right illustrating the flow of particles 277 within the particle flow path 275, within the sheath flow path 265 of the combined flow 260 in the YZ plane. In one example the external surface of the processing microchannel 280 is transparent along at least part of its length and may be comprised of plastic or glass or other suitable substrates for example. A particle interrogation apparatus 285 is positioned to direct a beam of electromagnetic radiation 285L at particles 277 passing within the particle flow path 275. The asymmetric particles may be oriented to present a desired face to the incident beam 285L. The lower left figure shows the particles side-on and having a narrow ellipse shape which in some examples may be a less optimal surface shape onto which the beam may impinge due to its reduced presentation area. Alternative orientations of ellipsoid particles and angles of incident beam may be preferred. The beam 285L may be a laser of predetermined wavelength and intensity and which interacts with a particle to generate a number of secondary fluorescent wavelengths 285S which may be detected by a detector 290. The intensity and/or spread of the detected fluorescent wavelengths may be used to infer the type of particle under interrogation. For example, the particles may be identified as sperm cells having X or Y chromosomes.
In one example the interrogation beam 285L is directed to interact with particles within a flow environment so that the interrogation beam intersects the microfluidic stream 260 directly and not through an processing microchannel 280. The processing microchannel 280 may be omitted, with the microfluidic stream 260 emerging from the aperture 380. Alternatively, a shortened processing microchannel 280 may be employed which is coupled to the aperture 380 of the delivery microchannel 375 but terminates prior to the interrogation beam 285L. The interrogation beam 285L may be located close to the aperture 235, 375 or shortened processing microchannel termination which enables the interrogation event to occur close to the focussing chamber where asymmetric particles are oriented. This may ensure maximal orientation and reduces possible turbulence.
Downstream of the particle interrogation apparatus 285 is a sorting apparatus 295 which directs a second beam ("sorting beam") of electromagnetic radiation 295L at some particles depending on their type as identified by the particle interrogation module 285 and detector 290. The second beam 295L may be a laser of predetermined wavelength and intensity and which is directed at one type of cell but not another. For example, the second beam 295L may be directed at sperm cells having Y chromosomes but not sperm cells having X chromosomes. Alternatively, the second beam 295L may be directed at sperm cells having X chromosomes but not sperm cells having Y chromosomes. The second beam 295L may cause particles upon which it impinges to be displaced along the optical axis of the incoming beam. This is configured such that these particles are displaced out of a flow path 275 corresponding to an original particle flow and into one or more different flow paths with positions which may correspond to an original sheath streams and/or into one or more different particle trajectories. The two or more types of particles may then be separated by separating the two or more particle flow paths. The flows of differently selected particles may be collected in different vessels to create discrete cell populations.
Details of operation of the interrogation or first beam 285L, the displacement or second beam 295L, their associated equipment and downstream separation of the particles is described in published PCT patent document W02020/013903 according to one example, and which is incorporated herein by reference. However other particle interrogation and/or particle selection/displacement technologies may alternatively be employed with the flow control apparatus described herein and 205, 230 of the examples.
In order to optimise operation of downstream particle interrogation and/or particle sorting apparatus, the particles 277 may be controlled to orient themselves at a preferred angle with respect to the beams 285L, 295L. The particles may also, or alternatively, be laterally aligned such that they intersect the beams.
The particles 277 are also controlled to be largely within the same plane or distance from the interrogation module 285 or displacement/selection module 295. In order to achieve this, in some examples the delivery tube 230 is located accurately within the housing 205 of the flow control apparatus. Accurate longitudinal, lateral and/or rotational location of the delivery tube with respect to the housing, and accurate rotational location of the housing with respect to the direction of the beams may enhance the accuracy and efficiency of these downstream processes. In an example, the delivery tube and/or housing comprise an engagement structure arranged to improve the longitudinal, lateral and/or rotational positioning of the delivery tube within the housing, and by extension the delivery tube's longitudinal, lateral and/or rotational positioning with the rest of the system 200.
In an alternative example, the particle flow control apparatus may be employed in a system which does not use an extended microchannel, and instead may be used with alternative cell sorting approaches that employ any method of displacing or selecting a particle in space, whether in fluid, aerosol droplet or in the gas phase. Such displacement/selection methods include, but are not limited to, forces derived from electric fields, electrostatics, electromagnetics, acoustic pulses or pneumatic pulses. The flow control apparatus also may be used with selection/sorting approaches that employ damaging or destroying the unwanted population of cells rather than physical displacement. This is referred to herein as "ablation". For example, such selection/sorting methods may include a radiation source (RS) configured to direct radiation on to the particle to effect at least one of a force and torque on each particle so as to induce ablation or at least one of displacing and orienting each particle relative to an axis defined by the direction of the fluid flow. Ablation may be considered the process of transferring energy to the particle sufficient to permanently inactivate the particle. In the context of biological cells, this may include rendering the cell unviable for its normal function or purpose. For example, sperm cells may be ablated to rapidly induce permanent immotility, or they may be ablated to "prime" them to be incapable of surviving downstream processes such as freezing and thawing. In the former, the ablation may involve rupturing the cell surface membrane which destroys cell integrity. During "priming", the cell surface membrane remains substantially intact, even though motility may be reduced or cease.
The methods and apparatus of some examples may have utility in achieving 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 with respect to one or more radiation beams that may be used in downstream processing of the particles. In one particular example the particles are oriented to optimise downstream processes interacting with beams of radiation where the absorption and emission of radiation may be highly orientation dependent in asymmetric particles such as sperm cells. Therefore it may be desired to orient a preferred facet of an asymmetric particle towards an incoming radiation beam, the facet having a maximum or minimum surface area or some other property.
In an alternative example, downstream processing may be implemented using an orienting module or stage employed downstream of the flow control apparatus 230, 250 in order to orient particles. For example, an orienting module may be located to direct electromagnetic radiation at particles in the combined flow 260. This may expose the particles to radiation pressure causing asymmetric particles to adopt a preferred orientation. Details of operation of an orientation stage are described in published PCT patent document WO2014/017929 which is incorporated herein by reference. However other particle orientation technologies may alternatively or additionally be employed with the flow control apparatus 205, 230 of the examples.
In some examples, various combinations of one or more downstream processes may be performed by installing corresponding modules or stages downstream of the flow control apparatus 205, 230, for example as described above. These downstream processes may include particle orienting and/or particle interrogation and/or particle displacement/selection; and may be performed separately or in any combination.
Figures 3 and 4 illustrate longitudinal section and transverse cross-section views respectively of a flow control apparatus according to an example. The transverse cross- section of Figure 4 is through section line AA in the longitudinal cross-section of Figure 3. The flow control apparatus 300 comprises a delivery tube 330 fitting within a housing 305. In particular the delivery tube 330 is securely and accurately received within a cavity 310 of the housing 305. The delivery tube 330 comprises a lumen 340 for carrying a particle flow 345 which is a moving liquid such as an aqueous solution containing particles such as sperm cells. The lumen 340 is open at an input end of the delivery tube 330 to a delivery tube inlet 332 and is open at a distal end of the delivery tube to a delivery tube outlet 333.
The delivery tube 330 also comprises ridges, fins, or projections 335 which extend longitudinally along the delivery tube or the inside surface of the housing. The ridges 335 engage with the internal surface of the cavity 310 of the housing 305 in order to secure the delivery tube within the housing. In an alternative example the housing 305 comprises ridges, fins or projections which extend longitudinally along the housing and also engage with the external surface of the delivery tube. The ridges 335 may be dimensioned to ensure a friction fit with the featureless walls of the cavity 310 or the walls of the cavity may comprise corresponding grooves into which the ridges locate. Various other mechanical fixing mechanisms may alternatively be used. By extending longitudinally, the ridges 335 improve the lateral positioning of the distal end of the delivery tube 330 so that the delivery tube outlet 333 is securely and accurately located within the housing 305. Further, the channels formed result in the sheath flow being stabilised and aligned with the Z-axis of flow prior to intersecting with the sample fluid containing particles.
Various alternative engagement structures are possible. Whilst the ridges 335 are longitudinally extending, they may also be angled with respect to the longitudinal axis to form a spiral shape along the outside of the delivery tube. Furthermore, whilst the ridges have been shown as continuous, they may be discontinuous with parts engaging with the cavity walls at different longitudinal locations. In a further alternative arrangement, longitudinally extending ridges may extend from the cavity to engage with the delivery tube. In this alternative, the delivery tube 330 may or may not also have ridges 335 extending to the cavity wall. In a yet further alternative, the outer circumference of the delivery tube 330 may be dimensioned to mate directly with the inner wall of the cavity to ensure a friction fit. The outer surface of the delivery tube and/or the inner surface of the cavity 310 may include recesses to form channels between the housing 305 and delivery tube 330.
In some examples, the ridges on the housing and/or delivery tube are arranged so as to provide channels through which sheath fluid flows. In some examples, the sheath flow is homogenous. Homogenous sheath flow means that the flow distance through each channel is substantially the same. This ensures that when the ridges end and the sheath fluid from one channel combines with sheath flow from a different channel, there is minimal turbulence caused. In this example, the sheath flow from each channel flows at the same speed and along the same distance as sheath flows from the other channels. This homogenous sheath flow is achieved by having the cross-sectional arrangement of channels have at least one line of symmetry.
This configuration improves 360° confinement and focusing of the fluid flows and obviates the need for multiple sheath intersections. If multiple sheath intersections interact with each other or the sample flow, this can cause turbulence and different directions of sheath flow.
Figures 4B-4I show non-limiting examples of channels having at least one line of symmetry where the hatched area comprises solid material and the open areas comprise flow channels.
In the examples described above, the channels may have a scalloped shape where the channel is formed at the delivery tube or housing. A scalloped shape means that one or more edges of the channel has a concave or convex profile as shown in figure 4F-H and 6A. In some examples, the scallop forms channels between one or more vertices of the shape of the delivery tube when viewed in cross-section. In other examples, one or more vertices of the shape of the delivery tube is rounded or otherwise shaped to smoothly engage with the housing. Figure 4H and 6A show the smooth engagement of rounded ridges or vertices 635. Smooth engagement means that there is substantially zero volume between the smoothed ridge/vertex and the housing wall. The engagement of at least one vertex, preferably all vertices, with the housing is preferably for a portion of at least 1% of the internal circumference of the housing. In other examples, the smooth engagement of the smoother vertex with the housing is for a portion of at least 5%, at least 10%, at least 20%, or at least 50%. In general, the higher the portion engaged, the more secure the delivery tube is within the housing. Conversely the higher the proportion of engagement, the lower volume for flow of sheath fluid through the channels.
In other examples, the housing comprises channels formed in the internal surface to enable sheath fluid to flow over and past the delivery tube. In this example, the delivery tube may be of a circular cross-section or may have channels as described above. Figure 41 shows a housing with channels and a circular cross section delivery tube. In other examples, both the housing and delivery tube may have channels, for example opposing channels where vertices engage to form elliptical or circular channels, or staggered channels formed in the housing and the delivery tube. The inventors have found that this scalloped profile minimises hydrodynamic turbulence when the sheath flows combine in the focussing chamber or prior to the focussing region, for example in region S2, S3 or S4 in figure 3. The scalloped profile also assists with maintaining a hydrodynamically smooth and non-turbulent flow when the sheath fluid combines with the sample fluid (particle fluid) in the focussing and/or confinement chamber(s)). The scalloped profile also enables easier manufacturing and facilitates cleaning of the delivery tube and housing without removal and the use of special tools to clean corners.
In the above examples, the number of channels may vary. However, in some examples at least 3 channels, preferably 4, 5, 6, 7, 8, 9, 10, 11, or 12 channels provides enhanced throughput and secure location of the delivery tube in the housing.
The engagement structure may extend for a portion of the length of the delivery tube. For example, Figure 7C indicates an example where the engagement structure only extends for a portion of the length of the delivery tube.
In alternative examples, the flow through the channels may be asymmetric, for example with more flow on one side of the delivery tube 330 than the other. This may be used to displace the particle flow within the sheath flow to one side of a central axis. Having a non-concentric particle stream with the sheath flow may be useful in some downstream interrogation and/or sorting processes. In some examples, the channels may be angled with respect to a longitudinal direction such that the flows within the channels circle around the delivery tube, forming helical or partially helical flows. This may result in a swirling sheath flow encompassing the particle flow which may provide some additional confinement.
In the example of Figures 3 and 4A, one or more sheath flow channels 360 are formed between the delivery tube 330 and housing 305 to carry a sheath flow 365 such as an aqueous solution. The sheath flow channel(s) 360 may extend from a sheath flow inlet 362 at the input end of the delivery tube and which includes the channels formed between the ridges 335. The sheath flow channel(s) extends along the outside of the delivery tube 330 to a focussing chamber 370 defined by a volume formed within the housing 305 at the end of the delivery tube 330 and into which the particle flow 345 is discharged from the delivery tube outlet 333. The focussing chamber 370 is also fluidly coupled to a delivery microchannel 375 from which a microfluidic stream is output via an aperture 380 into a processing microchannel or into a flow environment for downstream processing.
The sheath flow inlet 362 is configured to receive pumped sheath fluid from a sheath reservoir. In some examples, the inlet may feed sheath fluid at substantially a 90° angle to the direction of flow Z-axis. The inventors have found that receiving the sheath flow at this angle reduces bubbles in the system and enables the sheath channel to fill completely before the fluid flows down the channels. In alternative examples, the sheath inlet is positioned to inject sheath fluid in a downstream direction at an angle of between 90° to 0° (as illustrated) with respect to the Z-axis direction of sample flow. In one example the sheath fluid is received via the sheath inlet into a sheath chamber upstream of the sheath flow channel(s). This sheath chamber receives the sheath fluid and allows the flow to stabilise prior to the fluid moving down the channels. Preferably a purge outlet is positioned in fluid communication with the sheath chamber to receive air and excess sheath fluid and extract it from the system. Preferably the purge outlet is positioned upstream of the sheath inlet.
A central particle flow 345 is surrounded by one or more sheath flows 365 in a coaxial arrangement. The shape and size of the focussing chamber 370, the geometry and dimensions of the sheath flow channel(s) 360 and the lumen 340, together with the flow rates of the particle flow 345 and sheath flow 365 all contribute to the control of the combined fluid flows from the delivery microchannel 375. Example use cases include controlling orientation and confinement of particles within the microfluidic stream 385.
In one example, the invention provides a flow control apparatus for controlling fluid flows associated with carrying particles, the flow control apparatus comprising : a. a delivery tube comprising a lumen for carrying a flow of liquid, the delivery tube extending longitudinally within a housing which together define a focussing chamber adjacent the delivery tube outlet; and b. two, three, four or more longitudinally extending channels for carrying a sheath flow of liquid between the delivery tube and the housing towards the focussing chamber, wherein a downstream outlet of each channel is regularly spaced around the delivery tube at a single longitudinal location with respect to a flow path.
The longitudinal axis described in this example is substantially aligned with the direction of flow of the sample fluid through the delivery tube. The focusing chamber is an area where the sheath fluid flow meets the sample fluid flow. As described herein, the regular spacing of the channel exits around the delivery tube exit results in a non- turbulent flow which compresses and focuses the sample fluid flow from multiple directions simultaneously. This reduces turbulence and enhances confinement. This multi-directional flow focusing arrangement also avoids the need for multiple sheath flow intersections along the chip. The delivery microchannel 375 fluidically couples the focussing chamber 370 with an external surface of the flow control apparatus 300. The delivery microchannel 375 is a conduit which has an inlet from the focussing chamber 370 and an aperture 380 which may be in the form of an external aperture or may be connected to a processing microchannel 280. The cross-sectional shape of the delivery 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 delivery microchannel 375, its inlet and aperture 380 comprise the same rectangular cross-section with an aspect ratio of greater than 1: 1.
As noted above, the longitudinally extending ridges or other engagement structure ensures accurate and stable lateral positioning of the delivery tube outlet 333 within the focussing chamber 370. In more conventional arrangements, a delivery needle is introduced into a tapering volume of sheath fluid however the distal end of the needle is buffeted by the fluid flows and moves laterally causing the resulting particle flow to move within the surrounding sheath fluid flow or even to partially mix with the sheath fluid flow resulting in poorly oriented and poorly confined sample fluid (particle fluid) flows. This may make downstream processing difficult, inaccurate and inefficient.
In some examples, accurate longitudinal positioning of the delivery tube outlet 333 within the focussing chamber 370 helps to optimise the control and stability of orientation and/or confinement of particles or other flow properties of the microfluidic stream 385 delivered through the delivery microchannel 375 and from the aperture 380. In the example of Figure 3 and 4, this is achieved by dimensioning the ridges 335 of the delivery tube 330 to complement the dimensions of the cavity 310 of the housing 305 to prevent the delivery tube 330 from being inserted into the cavity beyond a predetermined longitudinal position.
The inventors have found that the distance of the delivery tube tip from the opening of the aperture (labelled LI in figure 6B) contributes to the orientation efficiency of asymmetric particles. Example 2 indicates that the PODT and housing configurations described herein provide consistently high orientation efficiency across a range of PODT tip distances and flow velocities.
The cavity 310 can be divided into several longitudinal sections or portions including a first portion 310-S1 which has a longitudinal cross-sectional shape, such as rectangular, which is arranged to engage at multiple longitudinal locations with a corresponding first portion of the delivery tube 330-S1. The first portion of the cavity may be substantially uniform transverse cross section along the longitudinal direction, for example of circular shape having a fixed diameter. In other examples, the first portion of the cavity may include channels or scallops formed in the housing while the delivery tube itself is of substantially circular cross-section. In other examples, the first portion of the cavity may include channels or scallops formed in the housing and channels or scallops formed in the delivery tube where each channel facilitates flow of sheath fluid through the channel. Preferably, the channels in the housing and delivery tube in this example are aligned to provide a single larger channel volume versus the volume of the channel formed in the housing or delivery tube alone. This first portion SI of the cavity 310 is used to receive the ridges 335 of the delivery tube 330. A second portion of the cavity 310-S2 tapers, having reducing dimensions when extending towards the distal end of the delivery tube. The ends of the ridges 335, having larger dimensions, prevent the delivery tube extending beyond this point, thereby ensuring accurate and stable longitudinal positioning of the delivery tube outlet 333 within the focussing chamber 370. The ends of the ridges may be shaped as shown to complement the internal shape of the cavity 310 to further improve this positioning. In other arrangements, grooves or channels in the wall of the cavity 310 may be used to receive the ridges 335 and the length of the grooves controlled to control the longitudinal position of the delivery tube outlet 333 within the focussing chamber 370. In this example, the engagement of the delivery tube ridges in the housing provide rotational engagement to ensure angular alignment, the advantages of which are described further herein. By contrast, in more conventional arrangements a delivery needle may be placed into a tapering volume of sheath fluid however if the distal end of the needle is not correctly positioned the sheath flows may turbulently interact with the particle flow causing unwanted mixing, chaotic misalignment of the particle flow and poor particle orientation and confinement.
The tapering second portion 310-S2 of the cavity 310 of the housing 305 may comprise a tapering angle a to the longitudinal axis of the housing, and the tapering second portion 330-S2 of the delivery tube 330 may comprise a tapering angle P to the longitudinal axis of the delivery tube 330. The sheath flow channel(s) 360 can be defined by the volume between the inner walls of the housing and the outer walls of the delivery tube. The housing and its inner walls in turn can be defined by the cavity shape and dimensions and the delivery tube can be defined by the shape and dimensions of its outer walls. The tapering angles can be modulated to achieve control of the acceleration of the sheath flow in portions of the sheath flow channels. In preferred examples, the tapering angle a is from 5-90°. In other examples is from 10 to 45°. In preferred examples, the tapering angle 3 is from 5 to 90°. In other examples q>/2 is from 10 to 45°.
The third portion 310-S3 of the cavity of the housing 305 may comprise uniform dimensions extending over a longitudinal length. Similarly, the third portion 330-S3 of the delivery tube 330 may comprise uniform, although smaller, dimensions extending over 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 stabilise to ensure laminar flow and reduce turbulence.
A fourth portion 330-S4 of the delivery tube includes a distal tip containing the delivery tube outlet 333. The tip may be shaped to enhance orientation and/or confinement of particles as described in more detail below. This tip area may be complemented by a further tapering fourth portion 310-S4 of the cavity 310 of the housing. The focussing chamber 370 is formed in a fifth portion 310-S5 of the cavity 310 of the housing when the delivery tube outlet 333 is positioned to discharge the particle flow 345 into the sheath flow 365 entering the focussing chamber 370 formed by the housing. The focussing chamber and other components of the flow control apparatus 300 are configured to cause a combined laminar flow of the particle and sheath flows through the delivery microchannel 375 and out of the aperture 380, in which the particles are largely oriented in one axis and largely confined to a plane containing that axis.
Additional portions of the delivery tube and/or sheath flow channel 360 may be included, or some described portions may be removed from some examples, such as the third portion 330-S3 from delivery tube as needed to impart targeted characteristics, such as but not limited to confinement, to the particle flow. Different geometries of the sheath flow channel to those illustrated may alternatively be employed.
The sheath flow 365 through the sheath flow channel 360 may be symmetric or asymmetric. For example, a larger volume in the upper half of the sheath flow channel 360 may cause the particle flow 345 to be displaced downwards. The sheath flow may also be caused to rotate about the delivery tube to generate a vortex flow which may assist with particle flow confinement. For example this is achieved using angled ridges to define a spiral or "corkscrew" flow as shown in figure 7. The different cross-sectional volumes of the sheath flow channel 360 along its length enables fine control of the sheath flow, including acceleration and stabilisation of the flows. The volumes of the sheath flow channels 360 also control the flow rates of the sheath flow through the channel. The ridges 335 and the channels formed between them act to stabilise the sheath flow as this may be introduced as a turbulent flow from outside the flow control apparatus.
Figure 4A shows a cross-section through section line AA of Figure 3, where the input area of the housing 305 and delivery tube 330 can be seen. This shows four evenly spaced ridges 335 extending from the delivery tube 330, although any number of ridges could alternatively be used. Various other parts of the delivery tube and housing are illustrated with the same reference numerals as used for those part in Figure 3. One of the ridges 335L is longer than the others and corresponds with a groove 315 in the outer wall of the cavity 310 of the housing. This arrangement comprises an indexing arrangement comprising an indexing means which ensures that the delivery tube can only be received into the housing in a single orientation illustrated generally by R. In a further example, the delivery tube may have multiple longer ridges for example 2,3,4,5,6,7,8,9,10,11 or 12 longer ridges where the ridges engage in a groove in the housing wall. Optionally, the delivery tube may comprise one or more grooves and the housing comprises one or more extending ridges to enable alignment in an inverse manner to that previously described. Where a single extending ridge aligns with a groove, this arrangement enables 360° rotational alignment so that the delivery tube can only be inserted in a single orientation. In an alternative example, the indexing means may comprise two extended ridges that are opposite each other, the delivery tube may be inserted in one of two orientations at 180° to each other to enable alignment of the ridges with the corresponding grooves. Similar angular arrangements may be employed, for example three extended ridges which align with corresponding grooves to enable three orientations at 120° to each other, or four extended ridges which align with corresponding grooves to enable four orientations at 120° to each other. These examples are particularly useful to enable correct alignment of the delivery tube outlet 333 when it comprises a non-circular cross-section i.e. it is bevelled or otherwise asymmetric, or has a non-circular lumen, e.g. an elliptical or rectangular lumen.
In examples having multiple grooves in the cavity wall 310 each for receiving a single ridge 335, the groove 315 for the longer ridge 335L is deeper such that the longer ridge 335L will still only fit within that one groove in order to ensure a predetermined rotational alignment of the delivery tube 330 within the housing 305. In an alternative arrangement using grooves for all ridges, one of these grooves may be wider than the others to receive a wider, though not necessarily longer, ridge. In a further alternative, a pin and corresponding hole arrangement may be used to correctly index the delivery tube within the housing. For example, the pin may extend through the housing into a ridge of the delivery tube, or the delivery tube or a ridge may include a pin which extends through a hole in the housing. In another arrangement, a magnet in one of the delivery tube or housing may be used with another magnet (or metallic feature) within the corresponding housing or delivery tube. Various other mechanical rotational alignment features may alternatively or additionally be used.
The external visually accessible surfaces of the housing and delivery tube may be marked to assist a user to align the delivery tube when inserting this into the housing to ensure rotational alignment. In the examples described above in which an indexing arrangement is used to determine the rotational alignment of the delivery tube in the housing, the ridges or other indexing means may extend substantially the length of the delivery tube or housing to the position of the termination of the lumen 333, or may extend along a portion of the delivery tube or housing. This portion may be defined by the beginning or end of a taper angle of the delivery tube or housing, or may be a portion of a linear region of the delivery tube or housing. For example the indexing means may extend for a portion (e.g. a quarter or half) of the distance SI, S2, or S3.
Various other examples of internal housing or cavity cross-sectional shapes and external delivery tube cross-sectional shapes are shown in Figures 4B to 41. Figures 4B - 4E illustrate longitudinal channels formed by differences in cross-sectional shape between the inner housing and the outer delivery tube. Figures 4F - 41 illustrate longitudinal channels formed by laterally extending projections from the exterior of the delivery tube and/or the interior of the housing.
Figure 5A and 5B show a detailed section of a flow control apparatus around the focussing chamber 370 according to an example. The rest of the apparatus may be the same as the example of Figures 3 and 4 or may vary from this. The lumen 540 of the delivery tube 530 comprises multiple portions 540-U, 540-T, 540-N of different cross- sectional dimensions. A first wide section 540-W carries an unfocussed particle flow 545- U which carries particles in various orientations and through a relatively large volume. A tapering portion 540-T of the lumen reduces the volume through which the particles flow, accelerating the particle flow and improving some particle flow properties. The cross-sectional shape of the lumen may also change along its length, for example going from circular in the first portion 540-W to elliptical in a final portion 540-N which causes the particles to orient along the longer axis of the ellipse. This is illustrated in the detail below the apparatus in which a particle 577-C in the circular section of the lumen 540-W is oriented into a different axis 577-E in the elliptical section of the lumen 540-N. It can be seen that the unfocussed particle flow 545-U is narrowed in at least one axis with the particle oriented and discharged from the delivery tube outlet into the focussing chamber 370. The lumen may change cross-sectional shape and/or dimensions along its length within the delivery tube. In some examples, the lumen may comprise an upper portion and a lower portion which have different cross-sectional shape and/or dimensions.
Example dimensional ranges include 50um - 1mm for the lower portion and 0.5 - 3mm for the upper portion.
In one example applicable to any of the flow control apparatus described herein, the delivery tube immediately adjacent the lumen comprises a taper from a smaller-cross- sectional area upstream in the microchannel, to a larger cross-sectional area downstream at the exit lumen of the delivery tube. This trumpet-shaped delivery tube exit transition has been found to minimise turbulence and assist laminar flow as the sample flow meets the sheath flow.
The delivery tube exit 645 is designed to be of a size appropriate to deliver cells and sample fluid. In one example, the internal diameter of the exit of the delivery tube lumen is 50pm-lmm. In another example where a more tightly confined stream is required, the delivery tube exit is from 50-500pm.
In one example shown in Figure 5B, the transition from a first wide section of the lumen 540-U to the narrowed portion 540-N comprises a step of substantially 90° to the axis of flow or a steeply tapered portion 541. The steeply tapered portion 541 is preferably at an angle of 45° or more to the axis of flow. The lumen may be adapted to receive an input tube 542 which delivers the fluid containing the particles. In this example the difference in width of the lumen at point 540-U compared to point 540-N substantially corresponds to the thickness of the input tube walls. The input tube internal diameter substantially corresponds to the lumen internal diameter beyond the end of the input tube. This enables a smooth transition between the input tube and the delivery tube lumen and reduces turbulent flow which may affect the particles or interfere with downstream orientation or confinement. The step or steeply tapered portion is an input tube arresting means at point 540-T that stops the input tube from travelling any further down the delivery tube and provides a seal between the input tube and delivery tube. In one example an o-ring or gasket is included at point 540-T to improve the seal and prevent leakage of the fluid. The input tube arresting means is also labelled 640 in figure 6.
The input tube arresting means 541 may be used in conjunction with the tapered portion 540-T a different points along the longitudinal axis of the delivery tube lumen to achieve both a) enhancements in flow confinement and/or orientation (for example using non circular cross-sectional lumens) and b) a smooth transition from the input tube to the delivery tube lumen.
Laminar sheath flows 365 interact with the partly focussed particle flow to further optimise properties of the particle flow. For example, this may be adapted to further confine and/or further orient the particles. A combined laminar fluid is output from the apparatus which comprises an inner focussed particle flow 545-F within a surrounding sheath flow 365. An orifice or aperture 580 is formed in the housing which issues this microfluidic stream from the delivery microchannel. In one example the aperture 380 may be coupled to a processing microchannel or other conduit for onward transport and downstream processing. In another example, the microfluidic stream is delivered into a fluid environment such as a liquid or gaseous environment.
In this example, the microfluidic stream issues from the aperture into a flow environment. The microfluidic stream may issue in a downwards or gravity-based direction or in a direction at an angle with respect to gravity, for example perpendicular or angled generally upwards. In one example, the flow environment is bounded by an extended microchannel or conduit which allows the microfluidic stream to travel in a controlled way from the aperture and confines the flow of particles as they pass the particle interrogation apparatus 285 and sorting apparatus 295. In an example, the flow environment may comprise a liquid of substantially the same viscosity as the liquid in the microfluidic stream. In some examples, the flow environment is a liquid of higher viscosity than the microfluidic stream. In some examples a fluid in the flow environment may be moving in the direction of movement of the microfluidic stream. The velocity of this movement may be the same or different from the velocity of the microfluidic stream. In an alternative example, the flow environment comprises a gaseous environment. In this example, the microfluidic stream is not required to be bounded by a processing microchannel or conduit. This approach has a number of advantages and results in reduced friction with the conduit resulting in improved laminar flow and enabling higher flow rates. The inventors have found that where the aperture engages with a processing microchannel or conduit, the aperture size must be aligned with the conduit internal dimensions to ensure that the microfluidic stream maintains laminar flow and has minimal turbulence. Conduit dimensions do not always align with the aperture dimensions which limits the scope of aperture dimensions. By employing a fluid flow environment, this enables flexibility in the size and shape of the aperture. In particular, using a smaller aperture than standardised conduits allow, a reduced proportion of sheath flow compared with particle flow can be achieved. This reduction in sheath volume thereby increases selected cell concentration.
Figure 6A illustrates side and end views of a delivery tube according to an example. The delivery tube 630 comprises a lumen 640 for carrying a particle flow, a plurality of ridges 635 which comprise mechanical engagement structures which extend longitudinally. Between the ridges 635 are recesses or scallops 637 which also run longitudinally and which form channels with the cavity walls of a housing when received therein. These channels may form part of a larger sheath fluid channel which transports sheath fluid from a sheath fluid inlet adjacent the larger end of the delivery tube and a focussing chamber in an assembled flow control apparatus containing the delivery tube.
The ridges 635 and recesses 637 may be the same length as illustrated, or they may be of different lengths. The ridges 635 and recesses 637 may be the same width and/or cross-sectional profile as illustrated, or they may be of different widths and/or profiles. The ridges 635 may form hemispherical recesses 637 as illustrated, although alternative arrangements are possible including polygon or complex curvature shapes, triangular or rectangular shaped cross-sections, or shapes with straight and curved components. The ridges and recesses may be regularly or irregularly spaced.
In one example, the sheath channels formed between the ridges are tapered such that a cross-sectional area of a channel is increased at a downstream position in the housing compared to an upstream position in the housing. Figures 4BA - upstream and 4BB downstream illustrate an example of this tapered channel architecture although alternative channel architectures such as those shown in figure 4A or described elsewhere herein may also be used. This tapered arrangement allows sheath fluid flow hydrodynamically combine with a sample fluid steam at the end of the sheath channels and reduce "dead space" that may increase turbulence of flow or bubble formation during priming.
The delivery tube 630 may be formed in different portions 630-S1, 630-S2, 630-S3, 630-S4 having different geometries as described herein. Similarly, the lumen 640 may be formed into different portions having different geometries as described herein. The combination of these geometries and the corresponding geometries of the cavity of the housing into which the delivery tube is assembled enable the sheath flow and particle flow to be controlled to optimise different characteristics of the flow of particles such as particle orientation and/or confinement.
The delivery tube and housing of the present invention may be combined with controlling the flow rates of the particle flow and the sheath flow. In particular examples, the flow rate is selected from the group comprising Imm/s - 20m/s, greater than Imm/s, greater than lOmm/s, greater than lOOmm/s, greater than Im/s, greater than 2m/s, greater than lOm/s or greater than 20m/s and ranges therebetween.
The channels merge into the conical cavity defined by the tapered delivery tube and the inversely tapered housing wall. The inventors have found that the smooth transition from the channels to the conical cavity provides a directional sheath flow with minimal turbulence. The sheath flow in this example enters from each channel in a symmetrical flow. The multi-directional flow is enhanced compared to a flow from, for example a sheath inlet on one side of the delivery tube, or on opposing sides of the delivery tube. The multi-directional flow preferably flows from channels arranged at regular intervals around the entirety of the 360° delivery tube. The simultaneous sheath flow from multiple directions minimises turbulent flow prior to and after combination with the sample fluid flowing through the lumen. The individual sheath flows from the various channels therefore converge gently with the particle flow at an angle with minimal displacement of the particle or sample flow and minimal turbulent flow ensuring the particle and sheath flow components remain laminar and there is little or no mixing between them.
In one example, the focusing chamber comprises an upstream part and a downstream part. Figure 6B, 6BA, 6C, 6D and 6E provide examples of a double focussing chamber comprising a delivery tube and housing which engage and synergise with each other. The upstream first focussing chamber (Pl) may be defined by a truncated conical cavity of the housing. More generally, the upstream first focussing chamber (Pl) may be defined by an internal longitudinal taper of the inner walls of the housing in which the transverse cross-section of a cavity defined by those tapering walls has substantially an aspect ratio of 1 : 1. For example, the upstream cross-section may comprise a circle, a rounded square, or a square. In this example, the substantially equal aspect ratio (1 : 1) is intended to mean a shape in which the length of a first axis of said shape is not more than 20% longer than a width of a second axis of said shape perpendicular to the first axis.
The downstream second focussing chamber P2 may comprise a cavity defined by the inner walls of the housing and having a transverse cross-sectional shape with an unequal aspect ratio, that is substantially greater than 1 : 1 with the length of one axis being longer than the length of an orthogonal axis of the cavity. For example, the downstream cross-section may comprise a rectangle, an ellipse, a rounded rectangle, or a stadium (a geometric term for a rectangle with a pair of semi-circles positioned at either end). In particular examples, the downstream cross-section aspect ratio is greater than (i.e. more unequal than) 1 : 10, for example 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.
Referring initially to Figures 6B and 6BA, one example of a double focussing chamber is illustrated. Figure 6B shows a side view of a flow focussing apparatus 600 with a double focussing chamber Pl and P2, and Figure 6BA shows an end view of the double focussing chamber looking from the right of Figure 6B to the left. The first focussing chamber Pl has a square cross section which tapers from a largest side length Pl-Ll to a smallest side length P1-L2. The second focussing chamber P2 has a rectangular cross-section with a largest side length P2-L1 and a smallest side length of P2-L2. In this example the longest side length P2-L1 of the second focussing chamber P2 is substantially the same as the smallest side length P1-L2 of the first focussing chamber Pl. This results in a slit at the left most or tapered end of the first focussing chamber, this slit extending a length L between P2-a and P2-b to form a linear section of the second focussing chamber P2. In this example, the second focussing chamber also comprises a tapering section P3 which tapers from the largest side length P2-L2 towards the entry point 374 of a delivery microchannel 375. This tapering section P3 is also referred to herein as a confinement chamber. In this example, the width of the delivery microchannel 375 is substantially the same as the smallest length P2-L2 of the second focussing chamber P2. The interactions of the particle flow issued from the particle delivery tube into the first focussing chamber, the sheath flow through the sheath flow channels into the first focussing chamber, the combined flows moving through the first and second focussing chambers into the delivery microchannel 375 result in a laminar microfluidic stream 385 which exits the delivery microchannel 375 at an aperture 380 in the fluid focussing apparatus 300. This configuration provides for improved orientation and confinement of particles within the microfluidic stream 385.
In an alternative example shown in Figure 6BC and 6BD, at least an upper or lower surface 692, 693 of the second focussing chamber comprises a reverse gable shape (i.e. the internal shape of a v- or u-shaped roof or a chined recess). The reverse gable may taper along its length (i.e. to form the internal shape of a boat hull) such that the height (Y-axis) of the chamber along a centre-line is greater at an upstream position compared to a downstream position. The reverse gable may comprise a rounded gable 693 or a sharp gable 692. This tapered reverse gable provides a hydrodynamically streamlined shape to orient and confine the cells as they pass along the second focussing chamber. In one example, the reverse gable shape extends across the longer axis of a crosssection of the chamber defined by W in figure 6B, 6BC and 6BD. Accordingly, the crosssection of the second flow focusing chamber may comprise a diamond, a pentagon or a hexagon.
The cross-sectional shape of the second focussing chamber may be said to comprise two opposing straight lines and one or more of: a curved line between the two straight lines; a a V-shaped or chined line between the two straight lines.
Figure 6BB illustrates an alternative double focussing chamber arrangement for the flow focussing apparatus 600' in which the first focussing chamber Pl' has a circular cross- sectional shape, tapering from a large diameter Pl-Dl to a small diameter P1-D2 from right to left in Figure 6B. This terminates in a second focussing chamber P2' having a rounded rectangular cross section with a large side length P2-L1' and a small side length P2-L2'.
Various alternative cross-sectional shapes could be employed for the first and second focussing chambers. More generally, a first focussing chamber comprises a constant lateral cross-sectional shape having an aspect ratio of substantially 1: 1 and which tapers longitudinally from a larger size to a smaller size adjacent a second focussing chamber. In other words, the lateral dimensions of the shape reduce in at least one axis, and in some examples in both perpendicular axes. The second focussing chamber comprises a constant lateral cross-sectional shape having a higher aspect ratio than the first focussing chamber and which extends longitudinally with a substantially constant dimension in one lateral axis, and in some examples both perpendicular lateral axes (ie without tapering). The second focussing chamber is fluidically coupled to a delivery microchannel having a smaller cross-sectional area. The second focussing chamber may include a tapering section which tapers longitudinally towards the delivery microchannel in one or more lateral axes. The delivery microchannel 375 may comprise an aspect ratio which is higher than the first focussing chamber.
Accordingly, in one example, the flow focusing arrangement comprises: a. a first focussing chamber comprising a substantially constant lateral cross- sectional shape forming a truncated cone having an aspect ratio of substantially 1 : 1 and which tapers longitudinally from a larger diameter at an upstream position, to a smaller diameter at a downstream position; b. a second focussing chamber comprising a substantially planar chamber.
In this example, the substantially planar chamber may comprise a substantially constant lateral cross-sectional shape having a higher aspect ratio than the first focussing chamber and which extends longitudinally with a substantially constant dimension in one lateral axis. In one example, the second focussing chamber comprises a substantially constant lateral cross-sectional shape forming a rectangle that tapers longitudinally in at least one axis across at least a portion of the longitudinal length of the chamber. In other words, at least one perpendicular lateral axis of the rectangle reduces longitudinally from a larger size at an upstream position, to a smaller size at a downstream position. In some examples both perpendicular lateral axes reduce longitudinally from a larger size at an upstream position, to a smaller size at a downstream position.
The flow focusing arrangement comprising a flow focusing portion comprising at least a first and second and focussing chambers as described herein may be fluidically connected to any sheath channel architecture including the sheath channel arrangements described herein comprising a plurality of longitudinally extending channels. Similarly, the sheath channel architecture comprising a plurality of longitudinally extending channels may be fluidically connected to other flow focusing apparatus. However, the inventors have found that the combination of the flow focusing portion and the sheath channel architecture provides benefits including minimisation of bubble production during priming, minimisation of turbulence, and enhanced confinement and orientation of cells.
In some examples, a downstream confinement chamber depth tapers from a depth of the second focussing chamber to a shallower depth. In one example, the shallower depth aligns with the height of the entry point to the delivery microchannel 375. In some examples, the fluid sample stream passes through the flow control apparatus 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 and sample fluids are substantially co-planar. The fluid stream travels along a substantially flat trajectory through the centre of the apparatus without major deviations of flow direction at any of the focusing chambers, or prior to the ejection from the delivery tube. The inventors have found that this aspect of minimal deviation from the emitted flow direction is important to minimise turbulence and reduce g-forces experienced by particles within the fluid. These disruptions to flow and particles can result in stress to cells which may decrease their biological efficacy or viability in vivo.
In some examples, the cone angle q> is the internal angle between the tapering walls of the cone. Figure 6B and 6C show half of this angle 671. In some examples this angle 671 is from 5-90°. In other examples 671 is from 10 to 45°.
Figure 6C shows an example in which the delivery tube comprises an elongated substantially constant transverse cross-sectional area portion 672. In this example, the delivery tube comprises two angles of taper. The first being the angle with respect to the longitudinal Z axis of flow of the delivery tube tip 673. The second being the angle with respect to the Z axis of flow of the delivery tube body 674. In some examples, the angle 673 is between 0-90°, in other examples angle 673 is between 5 and 30°. In some examples, the angle 674 is between 0-90°, in other examples, angle 674 is between 5 and 30°. The angles, dimensions and features described in relation to figures 6B to 6E are intended to be read in conjunction with other examples described herein such as the apparatus described above in relation to figures, 2, 3, 5A and 5B.
In particular examples, the size of the downstream second focussing chamber (P2) cross-section width "W" the "orientation width" (i.e. the greater of the two cross- sectional dimensions, P2-L2) comprises 0.5mm to 10mm. In preferred examples, the width comprises 2mm to 8mm, or 3mm to 6mm. Figure 6F shows orientation efficiency and discrimination measurements at differing flow velocities (V) and with different orientation width measurements using apparatus as shown in figure 6B. It can be observed that there is substantially consistent orientation efficiency with a high level of orientation of the bovine sperm cells flowed through the flow focusing apparatus.
In particular examples, the size of the downstream second focussing chamber (P2) cross-section depth "D" (the "orientation depth" (i.e. the lesser of the two cross- sectional dimensions) comprises 0.01mm to 4mm. In further examples, the width may be from 0.01mm to 10mm. In preferred examples, the depth comprises 0.025mm to 0.5mm, or 0.05mm to 0.25mm. Figure 28a shows one example of a second focussing chamber comprising a width of 2mm. Figure 28b shows one example of a second focussing chamber comprising a width of 6mm.
In one preferred example, the depth D is equal to the diameter or major dimension of the delivery microchannel 375. The inventors have found that aligning the depth of the cross-section of the downstream second focussing chamber P2 to a major dimension of the delivery microchannel 375 and the aperture 380 enables the microfluidic stream to flow with minimal turbulence from into the delivery microchannel and from the aperture. The width of the downstream second focussing chamber P2 then tapers to the minor dimension 670 of the delivery microchannel 375 as described below. In other examples, the depth D decreases from the point P2-a to point P2-bto provide a further focusing and orientation vector over the length of the downstream second focussing chamber P2.
The aspect ratio of width to height ranges from 1: 1.25 to 1 :800 w:h. In preferred examples, the aspect ratio ranges from The preferred width and height described above may be combined in the aspect ratios previously described to define a downstream second focussing chamber with the exemplary dimensions shown in Table 1 : 5 to 1 : 160 w:h.
Figure imgf000053_0001
Figure imgf000054_0001
Table 1- Exemplary dimensions for downstream cross-section and accompanying aspect ratios
Example 1 shows experimental data which indicates that an equal aspect ratio for the upstream first focussing chamber Pl cross-sectional area and an unequal aspect ratio for the downstream second focussing chamber P2 cross-sectional area provides high orientation efficiency and discrimination. In one particular example, the inventors observed that a width of 4mm and depth of 0.1mm for P2 gives consistently high orientation and discrimination between X and Y-bearing sperm cells at a range of flow rates.
The orientation length "L" of the downstream second focussing chamber P2 may also be important when considering the orientation efficiency of asymmetric particles that have passed through the apparatus of some examples. In an example the length L of region P2 from point P2-a defined as where a wall of the upstream first focussing chamber joins a wall of the downstream second focussing chamber, to point P2-b defined as where the downstream flow focusing chamber joins the downstream confinement chamber P3, is at least 3mm. In other examples, length L is between 3 to 10mm. In some examples, the orientation width W is constant for a distance L. The inventors have found that this constant width channel has flow stabilising effects and reduces downstream turbulence. This beneficial effect is observed in the increased confinement and orientation efficiency of asymmetric particles measured downstream at an interrogation region. In a further example, the downstream second focussing chamber P2 tapers directly to the start of the delivery microchannel. In this example the region P2 and P3 are combined to achieve orientation and confinement in a single region.
A third region P3 shown in figure 6B and 6C is referred to herein as a downstream confinement chamber. In this region, the two walls of the housing taper from the width W defined in P2 to the width of the delivery microchannel 375.
Figures 6D and 6E show an alternative example in which the second focussing chamber P2 tapers longitudinally in one lateral axis. As with previous examples, the first focussing chamber Pl tapers longitudinally (z-axis) in both lateral axes (x and y axes) so that, for example, a large square cross-section on the right reduces to a smaller square crosssection as the first focussing chamber extends longitudinally to the left. In this example, the second focussing chamber P2 tapers longitudinally in one lateral axis (x axis) whilst maintaining its cross-sectional dimension in the perpendicular lateral axis (y axis). The second focussing chamber P2 tapers to the cross-sectional shape and dimensions of the delivery microchannel 375.
Figure 28e shows an example in which the second flow focussing chamber tapers from a first width Wi to a narrower width downstream of W2. This tapered configuration assists in confining and orienting cells and provides a smooth flow-route to the delivery microchannel.
In one example, the second focussing chamber is adjoined by a downstream confinement region which tapers from a first depth (y axis) defined by the depth of the second focussing chamber, to a shallower second depth. In one example the second depth is defined by the depth of the entry point 374 of the delivery microchannel 375.
The upstream first focussing chamber is employed in use with the delivery tube described above. In particular, the delivery tube described with reference to the examples shown in Figures 3, 4A-I, 6B, 6C, 6E and 7. The channelled sheath fluid flows with minimal turbulence around the ridges and through the channels/scallops 656. It then cascades over and combines around the tapered section of the delivery tube 657 which forms a cavity with the tapered housing. For example as shown in figure 3 S1-S4. This open cavity and the upstream channels minimise turbulence effects caused by unequal flow. For example this unequal flow may be caused if sheath fluid is introduced to the focussing chamber from opposing sides of the housing rather than the circumferential flow achieved in the present invention. It may also be caused by sheath fluid not being appropriately channelled using multiple channels around the circumference of the housing to obtain laminar and non-turbulent flow in the direction of flow. If the sheath fluid is not channelled it can move in a radial direction around the delivery tube which negatively affects confinement and orientation efficiency, for example by turbulent flow or unequal flow velocity around the circumference.
In one example the tip of the delivery tube projects into the downstream second focussing chamber. In another example, the tip of the delivery tube is positioned within the upstream first focussing chamber.
Without wishing to be bound by theory, it is believed that the consistently high orientation efficiency achieved using the combination of channelled sheath flow, a circular cross-section upstream first focussing chamber and an unequal aspect ratio downstream second focussing chamber results from the following effect. Firstly, the channelled sheath fluid 656 enters the upstream first focussing chamber (650 in the example shown in Figure 6B) from 360°. This compresses the particle flow 655 from all directions which has the effect of increasing confinement, i.e. reducing the distance variance of individual particles from a nominal central flow vector along the axis of flow - Z. This has the effect of increasing the speed of the flow. Secondly, the particle flow passes from a substantially circular cross section to a cross-section of unequal aspect ratio as described above and labelled P2 in fig. 6B. In this region, the confined stream is compressed in 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 "flattened" face (which is defined by a "major dimension" of the particle) with an axis defined by the major dimension of the unequal aspect ratio - the X-axis. This region is believed to "prime" the particles to attain their eventual orientation post-aperture.
The particles are then subjected to a third compressive force as the fluid flows into the third region P3 - the downstream confinement chamber - in which at least one of the width (X-axis) and the height (Y-axis) of the downstream second focussing chamber reduces. In this region the fluid flow rate increases and confinement of the particles increases. The orientation of the particles is believed to flip from the downstream orientation region so that the flattened face of the particles is aligned with the Y axis. The combined sheath and particle flow or microfluidic stream then flows into the delivery microchannel 375. An alternative arrangement is shown in Figure 6E in which the downstream second focussing chamber P2 tapers, at different angles, from the upstream first focussing chamber Pl to the delivery microchannel 375. Angle of downstream second focussing chamber confinement region
In the downstream confinement chamber, the angle 01 and 02 of the two tapering walls 660 and 661 with respect to the axis of flow Z may vary from approximately 20° to approximately 80°. In a further example, the angle 01 and 02 may vary from approximately 10° to approximately 80°. In preferred examples, these angles are in the range of from 30° to 55°. In one example shown in figures 29a and 29b, the downstream second focussing chamber P3 has angles of 01 = 02 = 45°.
Figure 28c shows an example of the invention in which 01 = 02 = 67.5°. Figure 28d shows an example of the invention in which 01 = 02 = 22.5°. Figure 28e shows an example of the invention in which 01 = 02 = 11.5°. Corner radii
In some examples the angles between the three regions Pl, P2 and P3 are curved instead of sharp angles. This concept of having a curved transition angle can be described in terms of the corner radius of the P1-P2 transition, the P2-P3 transition, and the P3-outlet transition. In one example, the radius of the angles is at least 20pm. In preferred examples the corner radius is from 20pm to 1000pm. Where distances between transitions are referred to herein, it will be understood by those of skill in the art that the distance measurement is taken from the centre point of the corner radius, i.e. the half angle of the complete angle defined by the corner radius.
Orientation efficiency may be measured using a processing unit which implements an orientation efficiency estimation function which analyses output from the interrogation beam 285L via one or more detectors 290. The orientation efficiency may be used to provide sorting system monitoring for an operator and may also be used to control upstream processes such as shutting down the system if the orientation efficiency falls too low.
The orientation efficiency is a measure of the proportion of cells which are adequately oriented with respect to a reference direction. Sperm cells and other cells such as red blood cells are asymmetric having a flat oval shape, with perpendicular dimensions defining a face or large surface plane and a short dimension defining a thickness of an edge (as well as partially defining an orthogonal short surface plane). In some examples the particle selection/displacement technologies may be arranged to achieve optimal performance when its direction of propagation 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 with the orientation of the interrogation beam 285L and/or detector(s) 290. Asymmetric cells oriented within a certain range of the reference direction may still be sufficiently well oriented for laser-based sorting or other processes. Other cells which fall outside this range, for example asymmetric cells presenting their edge to the lasers, may result in measurement datapoints that cannot be classified and/or sub-optimal sorting.
A low orientation efficiency metric is an indication that the sorting system is configured sub-optimally resulting in the wastage of cells; for example because their fluorescence emissions cannot be accurately measured due to not being well oriented with respect to the illuminators or detectors. A low orientation metric may also indicate that even if accurately classified, many cells may not be properly sorted due to poor orientation with respect to a laser when this is used for sorting.
Cell orientation efficiency can be determined by comparing a number of datapoints within a focussed region of interest with the number of datapoints overall corresponding to all cell measurements, although different definitions could alternatively be used. This may be calculated by:
Figure imgf000058_0001
wherein ZA is the number of cells in a focussed region of interest, and ZB is the number of cells in another region which may correspond to all cell datapoints, or all viable cell datapoints. Whichever populations of cell datapoints are used, this metric represents the orientation efficiency over a period of time. Over different time periods, the orientation efficiency may vary. If the orientation efficiency falls below a threshold, for example 30%, this may trigger an alarm or other measure to prompt reconfiguration of the sorting system to be undertaken in order to improve the measured orientation efficiency.
In some examples, the flow focusing apparatus is modified in response to a sub-optimal orientation efficiency. In one example, the distance dZ in figure 6B is adjusted to enhance orientation efficiency. dZ is defined as the distance between the tip of the delivery tube, and the intersection point of the first focussing chamber and the substantially planar second focussing chamber. This is shown in figure 27a and b. In one example, the tip of the delivery tube is positioned downstream of the intersection point - i.e. dZ is negative as shown in figure 27a. In another example, the tip of the delivery tube is positioned upstream of the intersection point - i.e. dZ is positive as shown in figure 27b. A negative dZ has been found by the inventors to provide enhanced orientation efficiency compared to a positive dZ. In one example, the delivery tube exit is positioned within the housing downstream of a point in lateral alignment with the start of the tapered portion of the housing at 2807 in Figure 27a and 27b. Positioning of the exit at this point provides significant advantages in flow hydrodynamics due to the constricting and accelerating effect of the taper on sheath fluid passing around the delivery tube which also results in beneficial confinement of the sample stream. Alternatively, the delivery tube exit may be positioned at a negative dZ at a point greater than or equal to 10pm upstream of the end of the tapered portion where the substantially planar second focussing chamber starts. Positioning this close to the second focussing chamber requires there to be sufficient flow space for sheath fluid to pass around the tip of the delivery tube and beneficially confine the sample fluid from multiple directions. It will be appreciated by those of skill in the art that this distance will be determined based on the outer diameter of the delivery tube tip, and the cone angle of the tapered portion. A further beneficial effect of the arrangements described herein is in providing enhanced cell health by minimising 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 throughput.
In another example, the rotational alignment between the sheath channels and the focussing chambers may be adjusted. In another example, the flow rates of the particle flow and/or the sheath flow may be adjusted. In another example, the shape and/or size of the aperture 380 may be adjusted. These adjustments may be combined in any suitable manner and may be controlled depending on estimated orientation efficiency.
Figures 7A and 7B illustrate delivery tubes according to two alternative examples in which the longitudinally extending ridges 735 can also be angled with respect to the longitudinal axis. As shown in the first example 7A, the delivery tube 730 comprises a number of angled ridges or ribs 735. These may be configured to cause a spiralling sheath flow which may be used to enhance orientation and/or confinement. The angle 9 of the ridges may be adjusted to optimise this effect.
As shown in the example of 7B, the delivery tube 730-2 comprises angled ridges or ribs 735-2 grouped together in sets which define channels 736 between the sets. In some examples, the ridges may protrude from the inside of the housing to define channels therein.
The example in 7C shows the ridges 740 extending in two directions from the delivery tube to engage with the housing. The ridges are optionally tapered in the Z-axis (axis of flow) to present a thin leading edge to the flowing liquid. The thin leading edge may expand into a wider support structure then taper again to thin trailing edge. This optimised version of the ridges is akin to a wing which reduces the turbulence caused by liquid flowing over it. In this example, the ridges may define an ellipse in their cross- section with the longer dimension of the ellipse being generally aligned with the Z-axis direction of flow. This configuration of ridges may be applied to any of the other examples described herein, in particular the examples described in relation to figures 3, 6B, 6C and 6E.
In the example shown in figure 7C, the delivery tube, ridges, upstream focusing chamber, downstream second focussing chamber, downstream confinement chamber and delivery microchannel are a monolithic flow control apparatus comprising a single piece of material. The inventors have found that fabricating this monolith enables accurate engagement of the delivery tube with the focussing chambers to enhance orientation and confinement.
Figures 8 - 12A illustrate various delivery tip geometries which may be used to optimise certain characteristics of the particle flow, such as features of the particle orientation and/or confinement and/or particle integrity. All of these tips may be used in combination with the other features of the invention described above including the ridges on said delivery tube or housing, the flow focussing chambers and confinement chamber. Figure 8 illustrates a conical tip 830C at the delivery tube outlet 840 of the delivery tube 830 and which may be used with any of the previous described example or with variations of these. The lower figure shows a perspective view of the conical tip, with the middle and upper figures show end and longitudinal cross-section views in perpendicular planes - XZ and XY. As can be seen, the conical tip tapers towards the distal end of the delivery tube in both planes.
Figure 9 illustrates a bevelled tip 930B at the delivery tube outlet of the delivery tube 930 which may be used with any of the previous described example or with variations of these. The lower figure shows a perspective view of the bevelled tip, with the middle and upper figures showing end and longitudinal cross-section views in perpendicular planes - XZ and XY. As can be seen, the bevelled tip tapers towards the distal end of the delivery tube in one plane (XY) but not the other plane (XZ). The bevel angle 6 may be changed to alter the orientation characteristics of the particle flow.
Figure 10 illustrates a double bevelled tip 1030B1, 1030B2, at the delivery tube outlet of the delivery tube 1030 and which may be used with any of the previous described examples or with variations of these. The lower figure shows a perspective view of the double bevelled tip, with the middle and upper figures show end and longitudinal crosssection views in perpendicular planes - XZ and XY. As can be seen, the double bevelled tip has one bevel 1030B2 which tapers towards the distal end of the delivery tube in one plane (XZ) and also another bevel 1030B1 which tapers towards the distal end of the delivery tube 1030 in the other plane (XY). The tapering angles of the two planes may be different or the same. In the illustrated example, the tapering in the XZ plane is shorter and shallower and retains more width of the distal end of the delivery tube than the tapering in the XY plane.
Figure 11 illustrates a double bevelled 1130B1, 1130B2 with notch 1130U tip which may be used with any of the previous described example or with variations of these. The lower figure shows a perspective view of the double bevelled and notched tip, with the middle and upper figures showing end and longitudinal cross-section views in perpendicular planes - XZ and XY. This example is similar to the double bevelled tip of Figure 10 and additionally comprises a notch 1130U which comprises material delivery tube material removed from around the delivery tube outlet 1140. This notch may be achieved in any plane, although in the example this is in the XZ plane which is the same plane as the smaller bevel 1130B2 and in which some width 1130E of the distal end of the delivery tube 1130. The notch may also be any suitable shape, for example rectangular as shown in the two upper figures or semi-circular as shown in the lower figure.
Figure 12 illustrates a tip with notch 1230U which may be used with any of the previous described example or with variations of these. The right hand figure shows a perspective view of the tip with notch, with the left hand figures showing end and longitudinal crosssection views in perpendicular planes - XZ and XY. This example is comprises a notch 1230U similar to the example of Figure 11 but does not comprise any bevelling of the tip. This notch may be achieved in any plane.
Figure 12A illustrates four further tip shape examples in longitudinal cross-section views. The two upper tips comprise a convex cross-sectional shape 1230CV (left) and a concave cross-sectional shape 1230CC (right). The lower two tips each include two tapering sections in cross-section. The left lower tip 1230T1 has a first tapering section with a more acute angle than a second distal tapering section. The right lower tip 1230T2 has a first tapering section with a less acute angle that a second distal tapering section. In a further alternative, the convex or concave profiles may not be along the entire length of the tip, but for example only at the end of the tip. Various other alternatives are possible in which the profile of the tip is not uniform along its length.
Tip profiles may be used to control both the fluid velocity and the angle at which the sheath approaches the sample fluid.
Figure 13 illustrates four examples of the sheath flow channel and lumen of a flow control apparatus and which may be used with any of the previous described example or with variations of these. The three figures are cross-sections through section line BB of Figure 3 and illustrate a cross-section through a third portion S3 of the housing and delivery tube of the flow control apparatus. In the left hand figure, the housing 1305-1 has a circular cross-section cavity 1310-S3-C, the delivery tube portion 1330-S3-E has an elliptical cross-section, and the lumen 1345 has a circular cross section.
In the second figure from the left, the housing 1305-2 has an elliptical cross-section cavity 1310-S3-E, the delivery tube portion 1330-S3-E has an (smaller) elliptical crosssection, and the lumen 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 delivery tube portion 1330-S3-D has a diamond shaped cross-section, and the lumen 1345 1345- E has an elliptical cross section.
In the right most figure, the housing 1305-4 has a circular cross-section cavity 1310-S3- C, the delivery tube portion 1330-S3-A has an asymmetric curved shaped cross-section, and the lumen 1345 1345-E has an elliptical cross-section and which is offset from the centre of the delivery tube. The cross-section shape can change down the length of the tube.
By varying the combination of cavity, delivery tube and lumen geometries, the particle flow and sheath flow through the flow control apparatus can be controlled to optimise various properties including, but not limited to, particle orientation and confinement with the flow stream, particle integrity, particle distribution within the combined fluid flow, particle transport time.
Various experimental results associated with some examples are illustrated in Figures 14 - 21. The axes of the graphs are micron (x-axis) and number of pixels (y-axis) which representative of the number of particles. Figure 14 illustrates particle confinement using a conical tip and showing particle flow images at a flow rate of 200mm/s in perpendicular axes (left) together with graphs illustrating the distribution of 80% of particles (right). It can be seen that 80% of the particles are well confined in both axes. Figures 15 - 17 illustrate similar plots and graphs for different particle flow rates, respectively lOOmm/s, 50mm/s, and 500mm/s. It can be seen that a similar tight confinement is maintained for different flow rates.
Figure 18 illustrates the position of the particles at different particle flow rates. The particles are well confined at all flow rates, though are displaced slightly to one side as the flow rate increases. However, this displacement is well within the tolerance of the downstream investigation, orientation and/or displacement/selection processes. This also compares favourably with traditional needle delivery approaches in which the particle flow changes position significantly with different flow rates and typically needs to be realigned. Figure 19 illustrates on the left for perpendicular axes the core stream distribution for 80% of particles using a delivery tube tip with a 40 degrees bevel together with the delivery tube having a circular outer section paired with a housing cavity also having a circular section. This may correspond for example to the sheath flow chamber arrangement of Figure 4 and the tip of Figure 8. On the right, core stream distributions for 80% of particles using a tip with a double bevel together with the delivery tube having an elliptical outer section paired with a housing cavity having a circular section. This may correspond for example to the sheath flow chamber arrangement of Figure 13 (left-most) and the tip of Figure 10. The graphs show these distributions for both arrangements (left and right) at a flow rate of 200mm/s. The upper and lower images show the particle spread in two orthogonally aligned sensors. It can be seen that the double bevel tip with elliptical delivery tube provides improved and stable confinement in both axes at this flow rate.
Figure 20 corresponds to a delivery tube having a 40-degree single bevelled tip together with circular section outer surface paired with a circular section housing cavity inner surface. Figure 20 illustrates the distribution and position of the particles at different particle flow rates.
Figure 21 corresponds to a delivery tube having a 40-degree single bevelled tip together with an elliptical section outer surface paired with a circular section housing cavity inner surface. Figure 21 illustrates the position of the particles at different particle flow rates. It can be seen that using the elliptical section outer surface of the delivery tube reduces particle distribution and provides a more uniform flow position across different flow rates.
Using some of these examples, it can be seen that 80% of the particles can be confined within a series of ranges, including 100, 80, 60, 50, 30 and 20 microns using different combinations of the above-described features of the flow control devices. Different ranges may be useful for different downstream particle processing.
Figure 22 illustrates a flow diagram of a method 2200 of using a flow control apparatus according to an example. At 2205, the method 2200 optionally selects a delivery tube and housing combination and engages these to form a flow control apparatus to use in the rest of the method. This may be performed manually and may involve selecting different combinations of housing and delivery tube in order to implement different controls on a stream of particles. For example, these may be selected to create a focussing chamber and/or sheath flow channels having certain dimensional properties in order to control the various properties of sheath and particle flows through the apparatus as previously described. The selected housing and delivery tube may be engaged with each other using a simple push fit with engagement of the delivery tube within the housing being controlled by an engagement structure, such as one of those previously described. In another example, the delivery tube and housing may be a monolithic component.
At 2210, the flow control apparatus receives a stream of particles in a fluid flow as well as a sheath fluid flow. The particle flow may be received into a lumen of the delivery tube and the sheath flow into channels between the delivery tube and housing as previously described.
At 2215, one or more properties of the particle flow and sheath flow may be controlled to produce a microfluidic stream having a desired property. For example, the speed of the particle and sheath flows, as well as the geometries of the selected delivery tube and housing may 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.
At 2220, the microfluidic stream of particles and sheath flow may be emitted into a flow environment comprising a processing microchannel. Such a processing microchannel may direct the microfluidic stream to a particle processing apparatus. In an alternative arrangement the flow environment may be a liquid or gaseous environment through which the microfluidic stream flows.
At 2225, the particles may be inspected to determine a particle characteristic. For example, a beam of radiation may be directed at the particles in order to determine their type, such as X or Y sperm cells.
At 2230, a sub-population of the particles may be selected based on the determined particle characteristics. For example, Y sperm cells may be displaced into a different flow path compared with X sperm cells. This could be achieved using a laser to nudge the Y cells out of their current flow path. The particle flow containing the X sperm cells may then be directed to a collection vessel and the particle flow containing the Y sperm cells discarded.
The aperture 380 emits the microfluidic stream generated using the or each focussing chamber(s) into the flow environment, and confinement chambers if used, for downstream processing. The aperture may have a cross-sectional shape having a dimension which is longer in one axis than in a perpendicular axis. The microfluidic stream issues from the aperture into a flow environment such as a processing microchannel or a gaseous atmosphere. In some examples the width 670 (X-axis) of the delivery microchannel is 10pm to 200pm. Preferably the width is between 50pm and 150pm. In some examples the width is equal to the depth (Y-axis).
In one example applicable to any of the flow control apparatus described herein, the delivery microchannel immediately adjacent the aperture comprises a taper from a smaller delivery microchannel cross-sectional area upstream to a larger delivery microchannel cross-sectional area downstream, for example at the aperture itself, where the cross-sectional area is defined perpendicular to the axis of flow. One example is shown in Figure 29 3001. This trumpet-shaped delivery microchannel-to-aperture transition has been found to produce a more stable microfluidic stream 385 within the downstream flow environment which provides enhanced confinement, interrogation and sorting.
In some examples, the length of the delivery microchannel 675 (Z-axis) is at least 10 microns to 10mm from an exit of a downstream confinement chamber to the aperture 380, wherein the aperture is defined as a point on a plane perpendicular to the z-axis of flow aligned with the terminal end of the flow focusing apparatus. In one example the channel length 675 is in the range of 50 microns to 1mm. This length is important to allow laminar flow to re-establish following orientation and confinement of particles in the orientation and confinement chambers upstream.
In one example shown in figure 6D, the width 670 is reduced at the downstream aperture 380 from a width W2 compared to the width 670 upstream at the entry point 374 of the delivery microchannel 375 at Wi. This reduction in width from Wi to W2 shown has the beneficial effect of further increasing confinement of the particles. The reduction in width may be a taper along the length of the delivery microchannel as shown in Figure 6D or along a portion of it. In one example, W2 is between 50% to 95% of Wi. In particular examples, the width reduces from between 100-150 pm to 50- 99pm. In one particular example, the width reduces from approximately 125pm to approximately 75pm. Approximately in this instance is intended to mean (+/-20%).
The cross-sectional shape of the delivery 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 delivery microchannel 375, its inlet and aperture 380 comprise the same rectangular crosssection with an aspect ratio of greater than 1: 1. In a further example, the aperture cross-sectional shape comprises a square or a rectangle. Without wishing to be bound by theory, the inventors believe that having a cross-sectional shape with right angle corners provides benefits in terms of providing a substantially flat surface for the interrogation beam to enter the stream, and any light emissions to exit from the stream. This is particularly the case where the stream is emitted into a rectangular microfluidic channel but also applies to embodiments where the microfluidic stream is emitted into a gaseous free space fluid flow environment.
Whilst it will be appreciated that various features of the examples may be combined, they may also be used independently of each other. For example, the tip geometries may be used with or without the described engagement structures or the sheath flow channel and lumen geometric features. Similarly, the sheath flow and lumen geometries may or may not be used with the engagement structures or the tip geometries. The housings and delivery tubes of the examples may also be used independently of each other, for example being assembled with different delivery tubes and housings. The housings and delivery tubes may be produced as consumable parts for a larger particle processing system and may be sold and installed separately.
The various channels, chambers, surfaces and flows are configured as microfluidic, that is having dimensions that are geometrically constrained to a small scale such as submillimetre, at which surface forces dominate. However, some examples may be configured with larger dimensions in which microfluidic phenomena are partly or wholly absent.
It will be appreciated that the examples provide many advantages including improved control of particles to facilitate downstream processing. The accurate and secure positioning control of some examples ensures that the particle flow is positionally stable to improve the accuracy and efficiency of downstream processing such as particle interrogation, particle orientation, particle displacement/selection and particle sorting. Accurate control of the sheath and particle flows also help to improve various downstream processing related properties of the particles such as orientation and confinement. This in turn enables the ability to increase the particle flow rate whilst minimising damage to the particles, thereby speeding up particle processing and the efficiency of the processing. In an example, this may allow for a faster rate of separating X and Y chromosome sperm cells whilst improving separation efficiency such as retrieving a sample with 80% or more sperm cells with X-chromosomes.
Examples also provide the ability to repeatably and accurately position a delivery tube within a housing of a flow control apparatus which reduces setup time and avoids the need for a highly trained operator. Further, the improved engagement of fitting between the delivery tube and housing provides mechanical rigidity and isolation from external forces, this ensures that the delivery tube is not only accurately positioned initially but also remains so over an extended period of time, reducing need for realignment and downtime of processing.
It should be noted that the above-mentioned examples illustrate rather than limit the 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, "a" or "an" does not exclude a plurality, and a single processor or other unit may fulfil the functions of several units recited in the claims. Any reference signs in the claims shall not be construed so as to limit their scope.
6. Examples
Example 1 - Particle orientation and discrimination characteristics using two- part focussing chamber
Methodology
Staining of bovine sperm cells was carried out 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).
The flow focussing apparatus shown in figure 6C was mounted and primed with fluid to remove any bubbles. The emitted microfluidic stream was aligned to the interrogation beam. Stained cells were injected through a sample channel and combined within the flow focusing apparatus with sheath fluid. The combined sample sheath fluid is passed through the orientation and confinement chambers and through the aperture.
Fluorescence intensity data was detected using orthogonal detectors perpendicular to the Z axis of flow and a fluorescence integral was determined. A scatterplot of integrals corresponding to independent particles was created. Orientation efficiency was measured as described in WO2022139597A1.
Two Gaussian peaks were formulated which corresponded to the two populations of sperm cells bearing X and Y chromosomes respectively. These peaks map the integrals of multiple fluorescence pulses. Discrimination is calculated as the difference between peak integral values divided by the sum of the peak standard deviations. This is a standardised method to determine the separation and width of the peaks which in turn provides a model for determining the probability of a new fluorescence pulse belonging to a first or second population. Results
Figure 6F shows results from the downstream flow focusing chamber width adjustment in a flow focusing apparatus of the invention. Flow velocity was adjusted between 5 and 9m/s and width of the downstream flow focusing chamber was adjusted from 3 to 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 best for orientation efficiency and discrimination.
The width 3 mm apparatus suffered from a PODT alignment issue at speed 5m/s. but still provided good orientation efficiency.
Conclusion
The flow focussing apparatus of the invention provides excellent orientation efficiency and discrimination across a range of widths and flow velocities.
Example 2 - Enhancement of orientation efficiency by modification of PODT tip position
Methodology
The position of the particle orientation and delivery tube (PODT) tip along the flow direction (Z axis) was modified to analyse its effect on orientation efficiency. The flow control apparatus is shown in Figure 6C and the Z-axis distance being adjusted is labelled dZ. The sheath fluid is received from an inlet above the delivery tube. The sample fluid including sperm cells was delivered through the tubing inside the delivery tube from the arresting point. The cavity which holds the delivery tube is 8mm in diameter with a cone angle of 30° to the Z-axis into a downstream second focussing chamber with 130pm depth (D) by 4 mm (L) constant width cross-section. The delivery tube itself had 4 channels/scallops to allow sheath fluid to flow and establish into a stable flow around its 20° to Z-axis conical tip. The positional variation of the PODT tip in the z direction was referenced to a nominal point where the tip of the cone would meet the downstream second focussing chamber (dZ in figure 6D). The final exit of the fluid into the delivery microchannel has the downstream confinement chamber tapering down from 4 mm wide to a 100 pm diameter outlet and aperture.
A range of PODTs with tip position in z axis from 1 mm to 6 mm were manufactured. At each position a range of flow velocities were used ranging from 3.3 m/s up to 7 m/s.
The orientation efficiency can be seen in Figures 2A and 23B.
Results
Orientation efficiencies of greater than 50% were obtained using this PODT arrangement over a range of flow speeds and delivery tube tip distances. There was a slight trend in which orientation efficiency (OE) decreases as flow velocity increases for each of the PODT tip positions. In addition, OE decreases as PODT moves forward in Z axis getting closer to where the tip of the cone joins with the downstream second focussing chamber.
Conclusion
The results show that the PODT and housing configurations provide consistently high orientation efficiency across a range of PODT tip distances and flow velocities. This indicates that the PODT configurations described herein as part of the invention are adaptable to varying positions and parameters without losing the high orientation efficiencies required for effective downstream particle discrimination and sorting.
Example 3 - Modelling of orientation characteristics of flow focussing apparatus
Finite Element Modelling (FEM) simulations have been performed with the same geometry as described in example 2. The mechanism for orientation is believed to arise from asymmetry. As such, the following parameters were calculated and compared to understand the flow dynamics of the proposed apparatus: asymmetric compression factor (ACF), ratios of velocity components ( -), ratios of velocity gradient Vgr , and vorticity (curl of the velocity field).
In examples provided herein, the particles analysed are sperm cells. Bovine sperm cells have a flattened head. For maximal fluorescence detection it is desirable to have the sperm flat side facing to the interrogation beam. Therefore it is desirable to tune the flow focussing apparatus to orient cells in a particular orientation relative to the interrogation beam (285L). The simulation data reported in this example is focused on the downstream confinement chamber (P3 in figure 6B and 6C).
Methodology - Ratio of velocity gradients
The asymmetry in velocity gradients is a metric for determining and optimising orientation. There are 9 velocity gradients in total, 3 (x, y, z) for each velocity component as shown in . The ratio between Vx and Vy was chosen as a key metric for assessing orientation. It describes the asymmetric velocity components of the fluid field at each point in space, which can reflect OE preference of the flow field.
The motivation of looking at velocity components arises from the fluidic drag force FD = ^pv2CDA, where FD is the drag force, p is the density of fluid, v is the relative velocity between the particle and the fluid, cD is the drag coefficient, and A is the particle cross- sectional area. It is a force acting opposite 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 direction drag force that can potentially suggest a preferred orientation within the flow as illustrated in figure 24.
Results
As can be seen in figure 25A, this metric shows a generally high orientation efficiency. There is also a trend that OE increases as PODT moves forward in z, and orientation efficiency increases as maximum velocity decreases (fig. 25B).
Conclusions from Orientation Efficiency experiments
Effect of flow velocity - A similar trend was observed between the experimental observations and modelling when considering the effect of flow speed, i.e. as the flow speed increased, there was an decrease in orientation efficiency.
Effect of dZ position of delivery tube - A mismatch was observed between the PODT tip position's effect on OE between the simulation and the experiment. This was investigated and is understood to be caused by an experimental deviation of the PODT tip with respect to the centre of the chip in the XY plane. Inspection indicated that this was caused by incorrect positioning of the PODT in the housing causing the tip of the PODT to be tilted or transversely shifted off to one side, causing OE to drop. As a result of this observed phenomenon, further stabilisation and alignment of the PODT within the housing was carried out. This illustrates the importance of the engagement structure comprising the ridges 335 to laterally and longitudinally position the PODT tip within the housing.
Example 4 - Modelling of mis-aligned tip of delivery tube
Methodology
Further simulations were carried out to exemplify the effect of a mis-aligned or tilted tip and are shown in figure 26A, B and C.
Results
Vx/Vy was selected as the most relevant metric for determining orientation efficiency an example in figure 26B and C. Figure 26D shows a trend of decreasing orientation efficiency as flow speed increases.
Conclusions
The plot in figure 26B clearly shows a similar behaviour of OE when compared to the experimental analysis in figure 23A. This indicates that a minor variation in XY position (tilt) of the PODT tip results in a noticeable decline in OE. Modelling with a transverse shifted PODT as shown in figure 26C shows the same results. The trend of decreasing orientation efficiency as flow speed increases aligns with the results from experimental analyses in figures 23B and 25B.
The simulations and experimental results performed show that the PODT tip position in z axis and the flow speed do affect OE.
These simulations show that the sample flow cannot be sustained in the centre of the downstream second focussing chamber if the tip is misaligned. This leads to disruptions of the asymmetric compression to the flow, and hence detrimentally affects OE.
Simulations have verified that the experimentally observed loss of orientation as flow speed increases (fig. 23B) was due to the misalignment of delivery tube tip. Simulations have also found that the tilt/transverse variation of the PODT negatively impacts OE as the PODT tip gets closer to the downstream second focussing chamber. This effect is believed to arise from an increased deviation of the particle flow path when the delivery tube tip is closer to the downstream second focussing chamber. These experiments illustrate the importance of tip positioning when using a PODT with particle flows. The inventions described herein enable the accurate positioning of the delivery tube outlet at a predetermined longitudinal, lateral or rotational location within the focusing chamber and the various features impart stabilising and orienting torques on the cells. As such the invention provide enhanced orientation efficiency and consequently improved downstream discrimination and sorting.

Claims

WHAT WE CLAIM:
1. A flow control apparatus comprising: a delivery tube having a lumen, the delivery tube extending along a longitudinal axis within a housing to a focussing chamber; one or more channels defined between an external surface of the delivery tube and an internal surface of the housing, the channels extending towards a lumen exit of the delivery tube within the housing and at least partially aligned with the longitudinal axis of the delivery tube; the focussing chamber flu id ica I ly coupled to an aperture in the flow control apparatus.
2. The apparatus of claim 1, wherein the one or more channels extend substantially parallel to the longitudinal axis of the delivery tube or are angled with respect to the longitudinal axis of the delivery tube.
3. The flow control apparatus of claim 1 or 2, wherein the one or more channels are defined by corresponding projections which extend along the delivery tube and/or the housing.
4. The flow control apparatus of claim 3, wherein at least some of the projections extend from the delivery tube to contact with the inner surface of the housing or from the inner surface of the housing to contact the delivery tube.
5. The flow control apparatus of any one of claims 2 to 4, wherein the projections are arranged to contact the delivery tube or inner surface of the housing at multiple longitudinal and lateral locations in order to position the delivery tube outlet at a predetermined position within the focussing chamber.
6. The flow control apparatus of any one preceding claim, wherein the one or more channels: a. comprise a substantially semi-circular or triangular cross-section; and/or b. extend from a sheath fluid inlet to the focussing chamber; and/or c. are configured to provide the only flow path or paths for the sheath fluid between a sheath flow inlet and the focussing chamber over at least a portion of the longitudinal extension of the delivery tube within the housing; and/or d. comprise a larger cross-sectional area at a downstream position compared with a cross-sectional area at an upstream position of said one or more channels.
7. The flow control apparatus of any one preceding claim, wherein the one or more channels are spaced around the entirety of the external sectional circumference of the delivery tube and merge into the focussing chamber which is at least partially defined by a tapered delivery tube and an inversely tapered housing wall.
8. The flow control apparatus of any one preceding claim, wherein the one or more channels are defined by two or more ridges defining the cross-sectional shape of the channels and wherein said ridges are dimensioned to provide a friction fit with the housing such that there is substantially zero volume between the ridge and an interior surface of the housing.
9 The flow control apparatus of any one preceding claim, comprising an engagement structure configured to: secure the delivery tube during use with the housing to form at least one of the one or more channels between the housing and the delivery tube, and position the delivery tube outlet at a predetermined longitudinal, lateral, and/or rotational location within the focusing chamber.
10. The flow control apparatus of claim 9, wherein the housing has a first portion with a longitudinal cross-sectional shape arranged to engage at multiple longitudinal locations with a first portion of the delivery tube in order to position the delivery tube outlet at a predetermined lateral location within the focussing chamber.
11. The flow control apparatus of any one preceding claim, wherein the engagement structure comprises longitudinally extending ridges within the cavity of the housing, the ridges configured to position the delivery tube outlet according to the predetermined lateral relationship with the housing when received therein.
12. The flow control apparatus of any one of claims 9 to 11, wherein the engagement structure comprises a rotational alignment feature arranged to secure the delivery tube outlet at a predetermined rotational angle with respect to the housing.
13. The flow control apparatus of any one preceding claim, wherein an interior lateral cross-sectional shape of the housing within which the delivery tube is positioned is different to an exterior lateral cross-sectional shape of the delivery tube at a corresponding longitudinal position, and wherein the one or more channels are defined by said difference in lateral cross-sectional shape at different longitudinal positions.
14. The flow control apparatus of any one preceding claim, wherein the inner surface of the housing defines a cavity for receiving the delivery tube, the cavity terminating in the focussing chamber, wherein the geometry of the cavity is different than the geometry of the focussing chamber at the termination; the geometries comprising one or more of the following: dimensions; cross-sectional shape; longitudinal tapering angle.
15 The flow control apparatus of claim 14, wherein an end of the delivery tube is positioned within the focussing chamber a predetermined distance from the termination.
16. The flow control apparatus of any one preceding claim, comprising a second focussing chamber fluidically coupled between the first focussing chamber and the aperture, wherein the geometry of the first focussing chamber is different to the geometry of the second focussing chamber.
17. The flow control apparatus of claim 16, wherein the geometries differ in one or more of the following: dimensions; cross-sectional shape; longitudinal tapering angle.
18. The flow control apparatus of claim 16 or 17, wherein the cross-sectional shape of the first focussing chamber has a lower aspect ratio that the cross-sectional shape of the second focussing chamber.
19. The flow control apparatus of claim 18, wherein the cross-sectional shape of the first focussing chamber is circular and the cross-sectional shape of the second focussing chamber is substantially rectangular.
20. The flow control apparatus of any one of claims 15 to 19, wherein the second focussing chamber is fluidically coupled to the aperture by a delivery microchannel with a dimension in one lateral axis smaller than the second focussing chamber.
21. The flow control apparatus of any one of claims 15 to 20, comprising a confinement chamber fluidically coupled to the second focussing chamber, the confinement chamber tapering longitudinally in one lateral axis from the second focussing chamber.
22. A flow control apparatus comprising: a delivery tube having a lumen, the delivery tube extending along a longitudinal axis within a housing to a first focussing chamber; the housing comprising a second focussing chamber fluidically coupled between the first focussing chamber and an aperture in the flow control apparatus; wherein the geometry of the first focussing chamber is different to the geometry of the second focussing chamber.
23. The flow control apparatus of claim 22, wherein the geometries differ in one or more of the following: dimensions; cross-sectional shape; longitudinal tapering angle.
24. The flow control apparatus of claim 22 or 23, wherein the cross-sectional shape of the first focussing chamber has a lower aspect ratio that the cross-sectional shape of the second focussing chamber.
25. The flow control apparatus of claim 24, wherein the cross-sectional shape of the first focussing chamber is circular and the cross-sectional shape of the second focussing chamber is rectangular.
26. The flow control apparatus of any one of claims 22 to 25, wherein the second focussing chamber is fluidically coupled to the aperture by a delivery microchannel with a dimension in one lateral axis smaller than the second focussing chamber.
27. The flow control apparatus of any one of claims 22 to 26, comprising a confinement chamber fluidically coupled to the second focussing chamber, the confinement chamber tapering longitudinally in one lateral axis from the second focussing chamber.
28. The flow control apparatus of any one of claims 22 to 27 , wherein the cross- sectional shape of the second focussing chamber comprises two opposing straight lines and one or more of: a curved line between the two straight lines; a a V-shaped or chined line between the two straight lines.
29. A particle processing system comprising: a flow control apparatus according to any one preceding claim, and a particle interrogation apparatus and/or a sorting apparatus.
30. A method of controlling fluid flows associated with carrying particles, the method comprising: carrying a particle flow of liquid containing particles in a lumen of a delivery tube extending along a longitudinal axis within a housing to a focussing chamber; carrying a sheath flow of liquid in one or more channels between the delivery tube and the housing towards the focussing chamber, wherein the channels extend towards a lumen exit of the delivery tube within the housing_and at least partially aligned with the longitudinal axis of the delivery tube; generating a microfluidic stream using the focussing chamber and issuing the microfluidic stream from an aperture, the microfluidic stream comprising a laminar flow of liquid from the sheath flow surrounding liquid from the particle flow.
31. The method of claim 30, wherein the fluid flows are controlled using a flow control apparatus as claimed in any one of claims 1 to 21.
32. A method of controlling fluid flows associated with carrying particles, the method comprising: carrying a particle flow of liquid containing particles in a lumen of a delivery tube to a first focussing chamber; carrying a sheath flow of liquid to the first focussing chamber; generating a microfluidic stream using the first focussing chamber and a second focussing chamber fluidically coupled to the first focussing chamber, wherein the geometry of the first focussing chamber is different to the geometry of the second focussing chamber.
33. The method of claim 32, wherein the fluid flows are controlled using a flow control apparatus as claimed in any one of claims 22 to 29.
34. A method of controlling fluid flows associated with carrying particles, the method comprising: carrying a particle flow of liquid containing particles in a lumen of a delivery tube extending along a longitudinal axis within a housing to a first focussing chamber; carrying a sheath flow of liquid in one or more channels between the delivery tube and the housing towards the focussing chamber, wherein the channels extend towards a lumen exit of the delivery tube within the housing_and at least partially aligned with the longitudinal axis of the delivery tube; generating a microfluidic stream using the first focussing chamber and a second focussing chamber fluidically coupled to the first focussing chamber, wherein the geometry of the first focussing chamber is different to the geometry of the second focussing chamber; issuing the microfluidic stream from an aperture, the microfluidic stream comprising a laminar flow of liquid from the sheath flow surrounding liquid from the particle flow.
35. The method of claim 33, wherein the fluid flows are controlled using a flow control apparatus as claimed in any one of claims 1 to 29.
PCT/NZ2023/050128 2022-11-11 2023-11-13 Flow control apparatus, parts and methods therefore WO2024102006A1 (en)

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