Method and system for detection of asymmetrically focused particles

Abstract

Methods and systems for detecting particles. In an exemplary method, a sample fluid including particles may be driven from a sample inlet channel through a convergence region and into a sample outlet channel defining a longitudinal axis. A focusing fluid may be introduced into the convergence region from the at least two focusing channels along respective introduction axes. The introduction may be rotationally asymmetric about the longitudinal axis. The introduction axis and the longitudinal axis may collectively extend in three dimensions. The particles may pass through an interrogation region of the sample outlet channel. The interrogation zone may be illuminated with light. Optical radiation may be detected from the interrogation zone.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is based upon and claims the benefit of 35 U.S.C. 119(e) U.S. Provisional Patent Application Serial No. 63 / 341,939, filed on May 13, 2022, which is incorporated herein by reference in its entirety for all purposes. introduction

[0002] Microfluidic methods are often used to examine individual particles (such as droplets or biological cells). In this method, a sample stream containing a collection of particles is combined with one or more sheath fluid streams under laminar flow conditions. The sheath fluid is configured to arrange the particles into a single row by creating a fluid sheath around the sample stream. The fluid sheath squeezes the sample stream, which converges the sample stream on an axis while spreading the particles along the axis. Once spread out, the particles can be optically interrogated serially with minimal interference with each other. For example, fluorophores associated with the particles can be excited by irradiation with excitation light to allow detection of fluorescence caused by the excitation.

[0003] The degree to which particles are aligned with each other can affect the consistency of particle irradiation and, thereby, the accuracy / repeatability of particle interrogation. If the particles are well aligned (remain centered on the same axis as the particles pass through the irradiation zone), the particles can be uniformly irradiated. Conversely, if the centers of the particles are significantly and variably spaced from the axis, the irradiation may be inconsistent and particle interrogation less reliable. Methods and systems for improving particle alignment and optical interrogation are needed. Summary of the invention

[0004] The present disclosure provides methods and systems for detecting particles. In an exemplary method, a sample fluid including particles can be driven from a sample inlet channel through a confluence region and into a sample outlet channel defining a longitudinal axis. A focusing fluid can be introduced into the confluence region from at least two focusing channels along corresponding introduction axes. The introduction can be rotationally asymmetric about the longitudinal axis. The introduction axis and the longitudinal axis can extend together in three dimensions. The particle can pass through an interrogation zone of the sample outlet channel. The interrogation zone can be illuminated with light. Optical radiation can be detected from the interrogation zone. BRIEF DESCRIPTION OF THE DRAWINGS

[0005] Figure 1 is a schematic diagram of a detection system for particles, wherein the particles are focused off-axis by asymmetric introduction of a focusing fluid.

[0006] Figure 2 is a somewhat schematic, partially isometric view of selected aspects of an illustrative "three-port" detection system in which focusing fluid is introduced asymmetrically into the confluence region via three ports.

[0007] Figure 3 yes Figure 2 The overall detection system of the illustrative Figure 2 A somewhat schematic partial cross-sectional view taken along line 3-3.

[0008] Figure 4 yes Figure 2 The overall detection system of the illustrative Figure 2 Another somewhat schematic partial cross-sectional view taken along line 4-4.

[0009] Figure 5 is a somewhat schematic, partially isometric view of selected aspects of an illustrative "two-port" detection system in which focusing fluid is introduced asymmetrically into the confluence region via two ports.

[0010] Figure 6 yes Figure 5 An illustrative detection system, roughly along the Figure 5 A somewhat schematic partial cross-sectional view taken along line 6-6.

[0011] Figure 7 yes Figure 5 The overall detection system of the illustrative Figure 5 Another somewhat schematic partial cross-sectional view taken along line 7-7.

[0012] Figure 8 is a partial schematic diagram of an illustrative detection system in which focusing fluid is introduced symmetrically (rather than asymmetrically) into the confluence region at four ports, taken downstream of the confluence region through an interrogation zone of the sample outlet channel, the interrogation zone being orthogonal to the longitudinal axis of the sample outlet channel, and indicating a particle localization zone (in dashed outline) that is relatively wide and centered on the longitudinal axis.

[0013] Fig. 9 It can be passed through serial Figure 8 A graph of the signal detected from the same particle in an interrogation zone of FIG. 1 and illustrating the variation in the signal amplitude measured from the same particle due to the width of the particle localization zone.

[0014] Fig.10 yes Figure 2 A partial schematic diagram of the detection system, such as Figure 8 , taken through the interrogation zone of the sample outlet channel, and indicating (in dashed outline) the off-axis particle localization region that is radially offset from the longitudinal axis of the sample outlet channel and larger than the sample outlet channel due to the rotationally asymmetric introduction of the focusing fluid at the three ports. Figure 8 The broad particle localization zone shown in is much narrower.

[0015] Fig.11 It is possible to pass through Fig.10 A graph of the signal detected from the same particle with a narrow interrogation zone and the graph illustrates the signal measured from the same particle (relative to Fig. 9 ) for more consistent signal amplitude.

[0016] Fig.12 yes Figure 5 A partial schematic diagram of the detection system, such as Figure 8 , taken through the interrogation zone of the sample outlet channel, and indicating (in dashed outline) the off-axis particle localization region that is radially offset from the longitudinal axis of the sample outlet channel and is larger than the sample outlet channel due to the rotationally asymmetric introduction of the focusing fluid at the two ports. Figure 8 The broad particle localization zone shown in is much narrower.

[0017] Fig.13 It is possible to pass through Fig.12 A graph of the signal detected from the same particle with a narrow interrogation zone and the graph illustrates the signal measured from the same particle (relative to Fig. 9 ) for more consistent signal amplitude.

[0018] Fig.14 is a flow chart listing exemplary steps that may be performed in a method of detecting particles.

[0019] Fig.15 is a somewhat schematic partial axonometric illustration of selected aspects of a working model of a "four-port" detection system for droplets (as particles) in which the focusing fluid is introduced into the confluence region at four ports rotationally symmetrically (rather than asymmetrically).

[0020] Fig.16 and Fig.17 It is from passing through Fig.15 The detection system interrogates a small droplet in the area ( Fig.16 ) and standard droplet ( Fig.17 ) collected location data map.

[0021] Fig.18 yes Fig.16 The are supplemented with diagrams illustrating the lubrication forces and how they can affect the distribution of small droplets.

[0022] Fig.19 yes Fig.17 The are supplemented with diagrams illustrating the lubrication forces and how they may affect the distribution of standard droplets.

[0023] Fig. 20 yes Fig.16A graph of only part of the location data, where the part is Fig.19 The figure is generally indicated at "20".

[0024] Fig.21 is a view of the working model of the "three-port" detection system for droplets (as particles), which is generally similar to Fig.15 The same as in , except that the focusing fluid is rotationally asymmetrically introduced into the confluence region at three ports instead of four ports.

[0025] Fig. 22 is to use Fig.21 A graph of the position data collected from a small droplet by the three-port detection system, generally with Fig.16 Same as in.

[0026] Fig.23 is a view of another working model of a "three-port" detection system for droplets (as particles), generally similar to Fig.21 The difference is that Fig.23 The three ports are relative to Fig.21 The three ports are collectively rotationally offset by 90 degrees about the longitudinal axis of the sample outlet channel.

[0027] Fig.24 is to use Fig.23 A graph of the position data collected from a small droplet by the three-port detection system, generally with Fig.16 Same as in.

[0028] Fig.25 is a view of yet another working model of a "three-port" detection system for droplets (as particles), generally similar to Fig.23 The difference is that Fig.25 The three ports are relative to Fig.23 The three ports are collectively rotationally offset by 180 degrees about the longitudinal axis of the sample outlet channel.

[0029] Fig.26 is to use Fig.25 A graph of the position data collected from a small droplet by the three-port detection system, generally with Fig.16 Same as in.

[0030] Fig. 27 is a view of the working model of a "two-port" detection system for droplets (as particles), which is generally similar to Fig.15 The same as in , except that the focusing fluid is introduced symmetrically into the confluence region at two ports instead of four ports.

[0031] Fig.28 is to use Fig. 27A plot of the position data collected from a small droplet by the two-port detection system, generally with Fig.16 Same as in.

[0032] Fig.29 is a view of another working model of a "two-port" detection system for droplets (as particles), generally similar to Fig. 27 The difference is that Fig.29 The two ports are relative to Fig. 27 The two ports are collectively rotationally offset by 90 degrees about the longitudinal axis of the sample outlet channel.

[0033] Fig.30 is to use Fig.29 A plot of the position data collected from a small droplet by the two-port detection system, generally with Fig.16 Same as in.

[0034] Fig.31 is a view of another working model of a "two-port" detection system for droplets (as particles), generally similar to Fig. 27 The difference is that Fig.31 The two ports are rotationally offset from each other by only 90 degrees about the longitudinal axis of the sample outlet channel.

[0035] Fig.32 is to use Fig.31 A plot of the position data collected from a small droplet by the two-port detection system, generally with Fig.16 Same as in.

[0036] Fig.33 is a view of yet another working model for a "two-port" detection system for droplets (as particles), generally similar to Fig.31 The difference is that Fig.33 The two ports are relative to Fig.31 The two ports are collectively rotationally offset by 90 degrees about the longitudinal axis of the sample outlet channel.

[0037] Fig.34 is to use Fig.33 A plot of the position data collected from a small droplet by the two-port detection system, generally with Fig.16 Same as in.

[0038] Fig.35 is a view of the working model of a "one-port" detection system for droplets (as particles), which is generally similar to Fig.15 The method is the same as in , except that the focusing fluid is asymmetrically introduced into the confluence region at one port instead of four ports.

[0039] Fig.36 is to use Fig.35A plot of the position data collected from a small droplet by a one-port detection system, generally with Fig.16 Same as in.

[0040] Fig.37 is a view of another working model of a "one-port" detection system for droplets (as particles), which is generally similar to Fig.35 The difference is that Fig.37 A port relative to Fig.35 The rotational offset is up to 90 degrees about the longitudinal axis of the sample outlet channel.

[0041] Fig.38 is to use Fig.37 A plot of the position data collected from a small droplet by a one-port detection system, generally with Fig.16 Same as in.

[0042] Fig.39 is a fluidics diagram of an exemplary asymmetric three-port detection system for droplets.

[0043] Fig.40 yes Fig.39 Isometric view of the splitter device of a three-port detection system.

[0044] Fig.41 yes Fig.40 The overall upper edge of the separator device Fig.40 A cross-sectional view taken along line 41-41.

[0045] Fig.42 yes Fig.40 The overall upper edge of the separator device Fig.40 Another cross-sectional view taken along line 42-42. DETAILED DESCRIPTION

[0046] Various aspects and examples of methods and systems for detecting particles are described below and illustrated in the associated drawings. Unless otherwise specified, methods and systems may, but are not required to, include at least one of the structures, components, functions, and / or variants described, illustrated, and / or included herein. In addition, unless specifically excluded, the process steps, structures, components, functions, and / or variants described, illustrated, and / or included herein may be included in other similar methods and systems, including being interchangeable between the disclosed examples. The following description of various examples is merely illustrative in nature and is in no way intended to limit these examples, their applications, or their uses. Additionally, the advantages provided by the examples described below are illustrative in nature, and not all examples provide the same advantages or the same degree of advantages.

[0047] The present disclosure provides methods and systems for detecting particles. In an exemplary method, a sample fluid including particles can be driven from a sample inlet channel through a confluence region and into a sample outlet channel defining a longitudinal axis. A focusing fluid can be introduced into the confluence region from at least two focusing channels along corresponding introduction axes. The introduction can be rotationally asymmetric about the longitudinal axis. The introduction axis and the longitudinal axis can extend together in three dimensions. The particle can pass through an interrogation zone of the sample outlet channel. The interrogation zone can be illuminated with light. Optical radiation can be detected from the interrogation zone.

[0048] Further aspects of the disclosure are described in the following sections: (I) Definitions, (II) Overview, (III) Examples, Components, and Alternatives, (IV) Illustrative Combinations and Additional Examples, (V) Advantages and Benefits, and (VI) Conclusion.

[0049] The features, functions, and advantages can be achieved independently in various examples of the present disclosure or may be combined in yet other examples, further details of which can be seen with reference to the following description and drawings. I. definition

[0050] The technical terms used in the present disclosure have meanings generally recognized by those skilled in the art. However, the following terms may be further defined as follows.

[0051] As used herein, the term "asymmetric" means a lack of rotational symmetry about a specified axis.

[0052] A "carrier fluid" is a bulk fluid used to encapsulate and transport particles. The bulk fluid can be a liquid or a gas. In some examples, the carrier fluid can be described as a continuous phase, and the particles therein can be described as a dispersed phase. The carrier fluid can be immiscible with each particle and can encapsulate each particle. In some examples, the carrier fluid can be an oil, such as fluorocarbon oil or silicone oil. In other examples, the carrier fluid can be aqueous.

[0053] A "channel" is a passageway for a fluid to flow through. A channel may be elongated to define a longitudinal axis along which the fluid flows. The longitudinal axis of the channel may be linear, curved, angled, or a combination thereof, etc. The channel may have any suitable cross-sectional shape, such as circular, semicircular, elliptical, polygonal, etc. The cross-sectional shape may be uniform or may vary along the longitudinal axis of the channel. The channel has a diameter measured orthogonally to the longitudinal axis. The diameter may vary along the longitudinal axis or may remain constant.

[0054] A "channel network" is a collection of three or more channels fluidly connected to one another to define two or more distinct paths for fluid flow at one or more channel intersections.

[0055] "Comprise," "include," and "have" (and their variations) may be used interchangeably to mean including but not necessarily limited to, and are open-ended terms that are not intended to exclude additional, unrecited elements or method steps.

[0056] A "junction region" is a junction where two or more fluid streams meet. The fluid streams may include a sample fluid stream and one or more focusing fluid streams, each of which may be directed to the junction through a respective channel of the channel network.

[0057] “Coupled” means a relationship in which the properties of one affect the properties of the other, and may include permanent or releasable connection, whether directly or indirectly through intermediate components, and is not necessarily limited to physical connection(s).

[0058] A "detection station" is an optical subsystem location that includes: at least one light source for illuminating an interrogation zone with light; at least one optical sensor for detecting optical radiation from the interrogation zone, and optionally includes: one or more optical elements for relaying or conditioning light / optical radiation between (one or more) light sources and the interrogation zone and / or between the interrogation zone and (one or more) optical sensors.

[0059] A "droplet" is a small volume of liquid encapsulated by an immiscible fluid (e.g., by an immiscible liquid that can form a continuous phase of an emulsion). The immiscible liquid can include oil and / or can consist essentially of oil. For example, the droplets disclosed herein can have an average volume of less than about 500 nL, 100 nL, 10 nL, or 1 nL, etc.

[0060] “Exemplary” means “illustrative” or “serving as an example.” Similarly, the term “exemplary” (or “exemplary”) means “illustrating by giving an example.” Neither term implies preference or superiority.

[0061] "First," "second," "alpha," "beta," and similar terms are used to distinguish or identify members of a group, etc., in the order in which they are introduced in a particular context, and are not intended to indicate a sequential or numerical limitation.

[0062] A "fluid sheath" is one or more fluid layers positioned on opposite sides of a fluid core (such as a fluid core formed by a sample fluid) and / or surrounding a fluid core (such as a fluid core formed by a sample fluid). Both the fluid sheath and the fluid core can flow in the same direction but do not substantially mix with each other due to laminar flow.

[0063] "Fluorescence" is the emission of optical radiation in response to the absorption of light. As used herein, fluorescence is intended to encompass any form of photoluminescence in which the absorption of one or more photons promotes an electron to an excited state and results in the subsequent emission of a new photon, whether from a singlet, triplet, or other state. The excited state resulting from the absorption may have any suitable lifetime.

[0064] A "fluorophore" is any atom, functional group, moiety or substance that is capable of fluorescing.

[0065] A "focusing fluid" is any fluid that is combined with a sample fluid stream containing particles to position the particles relative to each other, such as to align, radially position, and / or separate the particles. The focusing fluid may be interchangeably described as a sheath fluid.

[0066] An "interrogation zone" is the area in a channel or network of channels that is illuminated with light and from which optical radiation is directed.

[0067] "Microscopic" means too small to be visible to the naked eye and / or having a largest dimension of less than one millimeter.

[0068] "Optical radiation" means electromagnetic radiation in the spectrum, i.e. ultraviolet, visible and / or infrared light. Accordingly, the term "light" has the same meaning as optical radiation.

[0069] An "optical sensor" is any type of light sensing device. Exemplary optical sensors include photodiodes, avalanche photodiodes, photomultiplier tubes, phototransistors, etc. An optical sensor can be configured to convert detected light into an electrical output (e.g., voltage, current, or resistance) and / or a digital output.

[0070] A "particle" is any small (e.g., tiny) discrete object (such as a piece or volume of matter). A particle can be substantially solid (i.e., solid phase particle), liquid (i.e., liquid phase particle), or a combination thereof, etc. Exemplary particles include droplets, beads, biological cells, and viral particles. Exemplary sizes (e.g., length or diameter) of particles include less than 2000, 1000, 500, 200, or 100 microns, and / or at least 5, 10, or 20 nanometers, etc.

[0071] A "port" is an opening at and through which fluid enters or leaves a structure. A port may be located at the intersection of a first structure, such as a channel, and a second structure, such as another channel or a confluence area.

[0072] A "positive / negative pressure source" is any device that can apply positive or negative pressure (suction) to a fluid. Exemplary positive / negative pressure sources include pumps, vacuum chambers, compressed gas chambers, and the like.

[0073] A "pump" is any device that drives movement of a fluid (such as flow through a channel(s)) by mechanical action or application of an electric field. A pump can be a mechanical pump, such as a positive displacement pump (e.g., a piston pump, a syringe pump, a diaphragm pump, a gear pump, a peristaltic pump, a cam pump, etc.), a centrifugal pump, etc. Alternatively, a pump can be an electric pump that uses an electric field to drive fluid flow. Exemplary electric pumps include electroosmotic pumps.

[0074] A "sample fluid" is any fluid containing a sample and / or particles of interest.

[0075] A "separator" is a device and / or structure that combines fluid streams to arrange particles into a single file and / or to increase the average axial spacing between particles.

[0076] "Substantially" means conforming primarily to a particular size, range, shape, concept, or other aspect modified by the term, such that the feature or component need not conform exactly as long as it is suitable for the intended purpose or function of the feature or component. For example, a "substantially cylindrical" object means that the object resembles a cylinder, but may have one or more deviations from a true cylinder.

[0077] "Transverse" means perpendicular or oblique to the longitudinal axis (such as within 45 degrees of exactly perpendicular). II. Overview

[0078] This section provides an overview of the methods and systems described herein.

[0079] A method for detecting particles is provided. In the method, a sample fluid including particles can be driven from a sample inlet channel through a junction area and into a sample outlet channel defining a longitudinal axis. A focusing fluid can be introduced into the junction area from at least two focusing channels along respective introduction axes. The introduction can be rotationally asymmetric about the longitudinal axis. The introduction axis and the longitudinal axis can extend together in three dimensions. The particle can pass through an interrogation zone of the sample outlet channel. The interrogation zone can be illuminated with light. Optical radiation can be detected from the interrogation zone.

[0080] Another method of detecting particles is provided. In this method, a sample fluid including particles can be driven from a sample inlet channel through a junction area and into a sample outlet channel defining a longitudinal axis. A focusing fluid can be introduced into the junction area along two or more introduction axes that collectively have a rotational asymmetry about the longitudinal axis. The particles can pass through an interrogation zone of the sample outlet channel. The interrogation zone can be illuminated with light. Optical radiation can be detected from the interrogation zone.

[0081] A system for detecting particles is provided. The system may include a channel network including a confluence region that fluidically connects a sample inlet channel and a sample outlet channel to each other. The sample outlet channel may define a longitudinal axis. The channel network may also include at least two focusing fluid channels that define corresponding introduction axes and are fluidically connected to the confluence region at two or more ports. The introduction axis and the longitudinal axis may extend together in three dimensions. The system may also include one or more positive / negative pressure sources that are operably connected to the channel network and are configured to: drive a sample fluid including particles from the sample inlet channel through the confluence region and through an interrogation region of the sample outlet channel; and drive the focusing fluid to be introduced asymmetrically into the confluence region around the longitudinal axis. The system may include a further detection station that includes a light source configured to illuminate the interrogation region with light, and may also include an optical sensor configured to detect optical radiation from the interrogation region. III. Examples, Parts, and Alternatives

[0082] The following subsections A to D describe selected aspects of exemplary methods and systems for detecting off-axis positioned particles by the introduction of rotational asymmetry of a focusing fluid. The examples in these subsections are intended to be illustrative and should not be construed as limiting the overall scope of the present disclosure. Each subsection may include one or more different examples and / or contextual or related information, functions, structures, and / or processes. A. Detection systems for particles

[0083] This section provides an overview of exemplary detection systems for particles, each of which positions the particle off-axis upstream of a detection station; see Figure 1-Figure 13 .

[0084] Figure 1 An illustrative detection system 100 is shown for particles 102. The detection system has a fluidics subsystem 104 extending through a detection station 106. The fluidics subsystem 104 is configured to separate particles 102 from one another along a flow path upstream of the detection station 106, position the particles laterally (off-axis) relative to the flow path, and pass the separated, positioned particles serially through the detection station.

[0085] The fluidics subsystem 104 includes a sample reservoir 107, a focusing reservoir 108, and a waste container 109, which are fluidically connected to each other through a channel network 110. The sample reservoir 107 contains a sample fluid 112 including particles 102 enclosed by a carrier fluid 114 and supplies the sample fluid 112 to the channel network 110. The focusing reservoir 108 contains a focusing fluid 116 and supplies the focusing fluid 116 to the channel network 110. The waste container 109 receives the sample fluid 112 combined with the focusing fluid 116 from the channel network 110 downstream of the detection station 106.

[0086] The channel network 110 has a collection of channels that carry sample fluid 112 and focus fluid 116 to (and from) a separator 117, which provides a junction area 118. A sample inlet channel 120 delivers the sample fluid 112 to the junction area 118 of the separator. At least one focus fluid channel 122 delivers the focus fluid 116 to the junction area 118 of the separator. In some examples, two or more focus fluid channels can deliver the focus fluid 116 to the junction area 118, as indicated by the ellipsis 123 between the focus fluid channel 122 and another (dashed) focus fluid channel 122a. A sample outlet channel 124 delivers the combination of the sample fluid 112 and the focus fluid 116 from the junction area 118 through the detection station 106.

[0087] The focusing fluid 116 enters the confluence region 118 from each focusing fluid channel 122, 122a at a corresponding focusing fluid port 126, 126a. The number of focusing fluid ports utilized by the detection system to introduce the focusing fluid into the confluence region is provided in the present disclosure as a shorthand to distinguish between different system examples (e.g., a "one-port," "two-port," "three-port," or "four-port" detection system). The (one or more) focusing fluid channels and (one or more) ports of the detection system are configured (e.g., sized, oriented, and / or positioned) to rotationally asymmetrically introduce the focusing fluid 116 into the confluence region 118, which positions the particle 102 at an off-axis position in the sample outlet channel 124.

[0088] The fluidics subsystem 104 includes a pair of positive / negative pressure sources (i.e., a sample pump 128a and a focus pump 128b) for driving the flow of sample fluid 112 and focus fluid 116 from the sample reservoir 107 and the focus reservoir 108, respectively, into and through the channel network 110. In other examples, the fluidics subsystem includes a pump downstream of the separator 117 and / or the detection station 106, alternatively or additionally.

[0089] The detection station 106 includes (one or more) light sources 130, (one or more) optical sensors 132, and one or more optical elements 133. The light source 130 is configured to illuminate an interrogation zone 134 of the sample outlet channel 124 with light. The optical sensor 132 is configured to detect optical radiation from the interrogation zone 134. The optical element 133 provides an illumination optic to relay light from the light source 130 to the interrogation zone 134 and / or condition the light. The optical element 133 also provides a detection optic to collect optical radiation from the interrogation zone 134, condition the collected optical radiation, and / or relay the optical radiation to the optical sensor 132. In some examples, illumination can be performed directly by the light source 130 without a separate illumination optic, and / or detection can be performed by the optical sensor 132 without a separate detection optic.

[0090] Figure 2-Figure 4 An illustrative "three-port" detection system 200 is shown in which the focus fluid 216 is introduced into the confluence region 218 at three ports 226a-226c rotationally asymmetrically. The detection system 200 can have any combination of system components and features described above in connection with the detection system 100 (see also Figure 1 ). For example, the detection system has a channel network 210 that includes a sample inlet channel 220, three focusing fluid channels 222a-222c that terminate at respective ports 226a-226c, and a sample outlet channel 224. A junction region 218 is formed where the sample inlet channel 220 and the focusing fluid channels 222a-222c join one another at or near the upstream end of the sample outlet channel 224. The sample outlet channel extends downstream through the detection station 206 where an interrogation zone 234 of the channel is illuminated.

[0091] The sample fluid 212 including the particles 202 and the carrier fluid 214 flow along the sample inlet channel 220 toward the junction area 218, as indicated by the flow arrows 235. The sample inlet channel 220 includes a tapered portion 236 that narrows toward the junction area 218. The tapered portion 236 reduces the diameter of the sample inlet channel 220 so that the particles 202 are positioned, on average, closer to the longitudinal axis of the sample inlet channel and with increased axial alignment with each other for at least approximately single-file entry into the junction area 218.

[0092] The focusing fluid 216 flows in the respective focusing fluid channels 222a-222c toward the confluence region 218, as indicated by the flow arrows 237a-237c. More specifically, the three streams of the focusing fluid 216 enter the confluence region 218 via the three ports 226a-226c, where they are combined with the sample fluid stream. The ports 226a-226c are rotationally asymmetrically arranged about a longitudinal axis 238 defined by the sample outlet channel 224 (and / or defined jointly by the sample inlet channel 220 and the sample outlet channel 226). The longitudinal axis 238 is centered across one or both channels, and the longitudinal axis 238 may be linear or at least partially non-linear (e.g., curved at one or more locations along the axis). The asymmetric arrangement of the focusing fluid channels 222a-222c and ports 226a-226c enables off-axis focusing of the particles 202, with a lateral offset (eg, radial offset) from the longitudinal axis 238 (see Figure 3 ). More specifically, in this example, the average position of the centers 239 of the particles 202 is offset vertically below the longitudinal axis 238, but other offset directions may be suitable.

[0093] The focusing fluid channels 222a-222c define respective introduction axes 242a-242c along which the focusing fluid 216 is introduced into the confluence region 218 (see Figure 3 and Figure 4). In the depicted example, the three lead-in axes 242a-242c considered collectively lack rotational symmetry in that rotationally adjacent pairs of lead-in axes are rotationally offset from each other about the longitudinal axis 238 by different angles. More specifically, lead-in axes 242a and 242b are offset by up to 90 degrees, lead-in axes 242b and 242c are offset by up to 90 degrees, but lead-in axes 242a and 242c are offset by up to 180 degrees. In other examples using two or more focusing channels, rotationally adjacent pairs of lead-in axes defined by the two or more focusing channels (in the absence of any intervening lead-in axes) may be rotationally offset from each other by at least 120, 150, 180, or 210 degrees, etc., and / or rotationally adjacent pairs of lead-in axes may be rotationally offset from each other by less than 120, 110, 100, 90, or 80 degrees. In the depicted example, the lead-in axes 242a-242c are coplanar with each other (each lead-in axis lies in the same plane orthogonal to the longitudinal axis 238), and are each radially arranged relative to the longitudinal axis. In other examples using one or more focusing channels, at least one of the introduction axes (and / or each of the introduction axes) may not be orthogonal to the longitudinal axis 238, such as forming an angle of at least 10, 20, or 30 degrees with a plane orthogonal to the longitudinal axis. Alternatively or in addition, at least one of the introduction axes (and / or each of the introduction axes) may not be radial relative to the longitudinal axis of the sample outlet channel (i.e., at least one and / or each introduction axis does not intersect the longitudinal axis. Arranging the introduction axes to be non-orthogonal and / or non-radial to the longitudinal axis allows for more precise off-axis focusing of particles to obtain more consistent detection results.

[0094] Inspection station 206 illustrates a possible arrangement for its components (see Figure 2 ). Light source 230 illuminates interrogation zone 234 on illumination axis 240 via optical element(s) 233 that focus the light onto the interrogation zone. The light may induce fluorescence from particles 202, which is detected on detection axis 241 by optical sensor 232, which receives optical radiation from interrogation zone 234 via optical element(s) 233, which collects the optical radiation and directs it to the optical sensor. In this example, illumination axis 240 and detection axis 241 are orthogonal to each other, but other arrangements, such as epi-fluorescence detection using the same objective lens for focusing illumination light and collecting fluorescence, may also be suitable. In other examples, the detected optical radiation may be, for example, illumination light that has been scattered.

[0095] Figure 5-Figure 7An illustrative "two-port" detection system 300 is shown in which the focus fluid 316 is introduced into the confluence region 318 at two ports 326a, 326b rotationally asymmetrically. The detection system 300 can have any combination of system components and features described above in connection with the detection systems 100 and 200 (see also Figure 1-Figure 4 ). For example, the detection system has a channel network 310 that includes a sample inlet channel 320, two focusing fluid channels 322a, 322b that terminate at respective ports 326a, 326b, and a sample outlet channel 324. A junction region 318 is formed where the sample inlet channel 320 and the focusing fluid channels 322a, 322b join one another at or near the upstream end of the sample outlet channel 324. The sample outlet channel extends downstream through the detection station 306 where an interrogation zone 334 of the sample outlet channel 324 is illuminated.

[0096] The sample fluid 314 including the particles 302 and the carrier fluid 312 flows along the sample inlet channel 320 toward the confluence area 318, as indicated by the flow arrows 335. The sample inlet channel 320 includes a tapered portion 336 as described above for the detection system 200 (see also Figure 2 and Figure 3 ).

[0097] The focusing fluid 316 flows in the respective focusing fluid channels 322a, 322b toward the confluence region 218, as indicated by the flow arrows 337a, 337b. More specifically, the two streams of the focusing fluid 316 enter the confluence region 318 via the two ports 326a, 326b, where the streams mix with the sample fluid stream at the confluence region 318. The ports 326a, 326b are rotationally asymmetrically arranged about a longitudinal axis 338 defined by the sample outlet channel 324 (and / or defined jointly by the sample inlet channel 320 and the sample outlet channel 324). The asymmetric arrangement of the focusing fluid channels 322a, 322b and the ports 326a, 326b causes the particle 302 to be positioned off-axis, with a lateral offset (e.g., radial offset) from the longitudinal axis 338 (see Figure 6 ), generally as described above for detection system 200 (see also Figure 2 and Figure 3 ).

[0098] The focusing fluid channels 322a-322b define respective introduction axes 342a, 342b along which the focusing fluid 316 is introduced into the confluence region 318 (see Figure 6 and Figure 7). In this example, the lead-in axes 342a, 342b are rotationally offset from each other by 90 degrees, are coplanar with each other, and are orthogonal to the longitudinal axis 338. However, as described above, other arrangements for the lead-in axes may be suitable.

[0099] Figure 8 An illustrative "four-port" detection system 400 is shown in which the focus fluid 416 is introduced symmetrically (rather than asymmetrically). The focus fluid 416 is delivered into the confluence region by four focus fluid channels 422a-422d through their four ports along respective introduction axes 442a-442d. Due to the rotational symmetry of the focus fluid channels 422a-422d and their equal diameters, the orthogonal forces exerted on the sample fluid stream 412 entering the confluence region cancel each other out, so that the net lateral displacement force on the sample fluid is zero.

[0100] Figure 8 The view is taken downstream of the confluence region through the interrogation zone 434 of the sample outlet channel 424, the interrogation zone 434 being orthogonal to the longitudinal axis 438 defined by the sample outlet channel. Due to the lack of net displacement forces, the particles 402 are focused to a particle localization zone 443 (in dashed outline) centered about the longitudinal axis 438. The particle localization zone represents the area or volume within which substantially all (e.g., greater than 95% or 99%) of the particles pass through the interrogation zone 434, given the flow rates of the sample fluid 412 and the focusing fluid 416, a given particle size, etc. The particle localization zone can be defined relative to the center 439 of the particle, as in the present example, or relative to the periphery of the particle, etc. As explained in more detail below in Section C, "small" particles having an average diameter that is significantly smaller than the channel diameter at the interrogation zone 434 (such as less than 70%, 60%, 50%, or 40%, etc., of the channel diameter) may not be effectively aligned with each other by symmetric focusing.

[0101] Fig. 9A graph of the signal that can be detected from the interrogation zone 434 when the same small particle 402 passes through serially is shown. Due to the larger diameter of the particle positioning zone 443 relative to the size of the small particle, the efficiency of uniform illumination of the small particle and / or signal detection without light is variable. For example, the excitation light (e.g., from an LED) can be transmitted to the small particle through one or more optical lenses with a focal length. The light emitted from the small particle in response to the excitation light can be transmitted through another set of one or more optical lenses (with another focal length) to reach the optical sensor. When the position of the small particle is offset from the focal point by different distances, the intensity of the excitation light incident on the small droplet will change, and the intensity of the emitted light reaching the optical sensor will change. Therefore, the signal amplitude of the four signal peaks 444 generated by the four identical particles 402 varies significantly, which reduces the accuracy and precision of particle detection.

[0102] Fig.10 An asymmetric three-port detection system 200 is shown for use with Figure 8 symmetrical four-port detection system 400 for comparison (see also Figure 2-Figure 4 ). The focusing fluid 216 is introduced asymmetrically via the three focusing channels 222a-222c along respective introduction axes 242a-242c. Due to the lack of rotational symmetry of the focusing fluid channels 222a-222c, and their equal diameters, the orthogonal forces exerted on the sample fluid flow 212 entering the confluence region do not completely cancel each other out, so that there is a net lateral force 245 on the sample fluid and / or particles 202 therein. In this example, the net lateral force is a radial force.

[0103] Fig.10 The view and Figure 8 The view of FIG. 2 is obtained in the same manner, i.e., downstream of the junction region 218 through the interrogation zone 234 of the sample outlet channel 224, the interrogation zone 234 being in a plane orthogonal to the longitudinal axis 238 (see also FIG. 2 ). Figure 2-Figure 4 ). Due to the presence of the net lateral force 245, the particle 202 is focused to a particle localization region 243 (in dashed outline) having a lateral offset 246 from the longitudinal axis 238. The particle localization region 243 is as described above for Figure 8 245, the particle center 239 of the particle 202 is relatively close to the particle center 239 of the focusing fluid. Figure 8 The particles 402 in are focused into a much smaller area.

[0104] Fig.11 2 shows a graph of the signal that can be detected from the interrogation zone 234 when the same small particle 202 passes through in series. Figure 8The particle localization area 443 of the symmetrical detection system 400 is compared to the particle localization area 443 of the symmetrical detection system 400), so that the small particles are more uniformly illuminated with light and / or the efficiency of signal detection is more uniform. Therefore, the signal amplitudes of the four signal peaks 244 generated by the four identical particles 202 have less variation, which improves the accuracy and precision of particle detection relative to the accuracy and precision of the symmetrical detection system 400.

[0105] Fig.12 An asymmetric two-port detection system 300 is shown for use with Figure 8 The symmetrical four-port detection system 400 and Fig.10 The asymmetric three-port detection system 100 is compared with Figure 2-Figure 7 ). The focusing fluid 316 is introduced rotationally asymmetrically via the two focusing channels 322a, 322b along respective introduction axes 342a, 342b. Due to the lack of rotational symmetry of the focusing fluid channels 322a, 322b, and their equal diameters, the orthogonal forces exerted on the sample fluid flow 312 entering the confluence region do not cancel each other out, so that there is a net lateral force 345 on the sample fluid and / or particles 302 therein. In this example, the net lateral force 345 is a radial force.

[0106] Fig.12 The view and Figure 8 The view of FIG. 302 is taken in the same manner, i.e., through the interrogation zone 334 of the sample outlet channel 324 downstream of the confluence region 318, which is in a plane orthogonal to the longitudinal axis 338. Due to the presence of the net lateral force 343, the particle 302 is focused to a particle localization zone 345 (in dashed outline) having a radial offset 346 from the longitudinal axis 338. The particle localization zone 343 is described above with respect to FIG. Figure 8 345, the particle center 339 of particle 302 is relatively Figure 8 The particles 402 in are focused into a much smaller area.

[0107] Fig.13 34 shows a graph of the signal that can be detected from the interrogation zone 334 when the same small particle 302 passes through serially. Figure 8 The particle localization area 443 of the symmetrical detection system 400 is compared to the particle localization area 443 of the symmetrical detection system 400), so that the small particles are more uniformly illuminated with light and / or the efficiency of signal detection is more uniform. Therefore, the signal amplitudes of the four signal peaks 344 generated by the four identical particles 302 have less variation, which improves the accuracy and precision of particle detection relative to the accuracy and precision of the symmetrical detection system 400.

[0108] The direction and magnitude of the radial offset from the longitudinal axis can be controlled by the number, orientation, diameter, and / or relative flow rates of the focusing fluid channels. In some examples, a radial offset that is at least generally orthogonal (within 20, 10, or 5 degrees of exact orthogonality) to the illumination axis of the detection station can provide optimal consistency for particle illumination. In some examples, a radial offset that is at least approximately orthogonal (within 20, 10, or 5 degrees of exact orthogonality) to the detection axis of the detection station can provide optimal consistency for detecting optical radiation from the particles. B. Detection methods for particles

[0109] This subsection describes illustrative methods for detecting particles in which the particles are positioned off-axis by introducing a rotational asymmetry in the focusing fluid. Each method may include Fig.14 Any suitable combination of the steps listed in flowchart 550, performed in any suitable order, with any of the steps omitted or performed any suitable number of times, and using or including any of the features, aspects, configurations, additional steps, or modifications described elsewhere in the present disclosure.

[0110] In preparation step 551, a sample fluid is prepared. The sample fluid includes particles and a carrier fluid in which the particles are encapsulated. In some examples, the carrier fluid may include oil. In some examples, the carrier fluid may be aqueous. The particles may be droplets, beads, fragments, biological cells, viral particles, microparticles, granules, etc.

[0111] In driving step 552, the sample fluid is driven through the confluence area and enters into the sample outlet channel. One or more pumps and / or positive / negative pressure sources can be used to drive. The sample fluid can be driven into the confluence area from the sample inlet channel. The sample inlet channel can have a tapered area that tapers toward the confluence area. The sample inlet channel and the sample outlet channel can be coaxial with each other.

[0112] In an introduction step 553, the focusing fluid is introduced into the confluence area rotationally asymmetrically. The focusing fluid can be introduced as a single focusing stream along a single introduction axis, or can be introduced as two or more focusing streams along two or more corresponding introduction axes. The two or more focusing streams can have the same diameter and volume flow rate, or can have different diameters and / or different volume flow rates. The focusing fluid can be introduced in a rotationally asymmetrical manner about a longitudinal axis defined by the sample outlet channel. The rotational asymmetry can be a rotational asymmetry of a single introduction axis or two or more introduction axes along which the focusing fluid is introduced. Alternatively or additionally, the rotational asymmetry can be a rotational asymmetry of a single focusing stream or two or more focusing streams into which the focusing fluid is introduced.

[0113] Each introduction axis can have any suitable orientation. Each introduction axis can be orthogonal or non-orthogonal to the longitudinal axis defined by the sample outlet channel. In some examples, the introduction axes are coplanar with each other to define a plane orthogonal to the longitudinal axis.

[0114] The introduction may be performed at any suitable number of ports, such as a single port, two or more ports, three or more ports, etc. Each of the ports may be formed at the intersection of a corresponding focusing fluid channel and a confluence region.

[0115] Introducing the focusing fluid can be performed at any suitable flow rate relative to the flow rate at which the sample fluid is driven into the confluence region. In some examples, the introduced flow rate is at least 5, 10, 15, or 20 times the driven flow rate.

[0116] The introduction may produce an average position of the particle center in the interrogation zone that is radially offset from the longitudinal axis of the sample outlet channel. The introduction may exert a net force on the particles and / or sample fluid in a direction transverse to the longitudinal axis.

[0117] In a conveying step 554, the particles pass through an interrogation zone of a sample outlet channel. As described above, the particles may pass through the interrogation zone serially and / or with an average radial offset from the longitudinal axis.

[0118] The interrogation zone is illuminated with light in an illumination step 555. The light may include excitation light for fluorophore(s) associated with (eg, located on or in) the particle.

[0119] In a detection step 556, optical radiation is detected from the interrogation zone. The optical radiation may include fluorescent and / or scattered light.

[0120] In a determination step 557, a characteristic of the particle is determined based on the detected optical radiation. The characteristic may include the presence / absence or amount of an analyte (eg, nucleic acid target), particle size, and the like. C. Comparison of symmetric and asymmetric detection system configurations

[0121] This subsection describes test data obtained using a symmetric four-port detection system for droplets (as particles), as well as various asymmetric three-port, two-port, and one-port detection systems.

[0122] Fig.15Aspects of a working model of a "four-port" detection system 600 for aqueous droplets 602 are shown. The droplets are present in a sample fluid 612 that includes an immiscible carrier fluid, which is an oil that encapsulates each of the aqueous droplets and provides a continuous phase. A focusing fluid 616 has the same composition as the carrier fluid and is injected symmetrically (rather than rotationally asymmetrically) from four focusing fluid channels 622a-622d at four ports into a confluence region 618.

[0123] The detection system 600 has a channel network 610 including a sample inlet channel 620, focusing fluid channels 622a-622d, a separator 617, and a sample outlet channel 624 (defined by a capillary 658). The sample inlet channel 620 delivers a sample fluid 612 including aqueous droplets 602 to a confluence region 618, as indicated by flow arrows 635, and as described above for other detection systems 100, 200, and 300 (see subsections A and Figure 1-Figure 7 ). The sample inlet channel 620 has a tapered region 636 at its downstream end, which is formed by the separator 617. The focusing fluid 616 is delivered to the confluence region 618 through the focusing fluid channels 622a-622d, as indicated by the corresponding flow arrows 637a-637d. The focusing fluid is introduced into the confluence region orthogonal to the longitudinal axis of the sample outlet channel 624 as a focusing fluid stream traveling along a corresponding introduction axis orthogonal to the longitudinal axis. In addition, the focusing fluid streams are rotationally symmetrically arranged with the same focusing fluid flow rate to balance each other so that the sample fluid 612 is focused on the longitudinal axis of the sample outlet channel 624 (i.e., centered in the sample outlet channel). The focusing fluid streams from the focusing fluid channels 622a and 622c are coaxial with each other and flow in opposite directions, as are the focusing fluid streams from the focusing fluid channels 622b and 622d. The volume flow rate of the focusing fluid 616 is 25 times the volume flow rate of the sample fluid 612 so as to firmly focus the sample fluid flow to approximately 4% of the cross-sectional area of ​​the sample outlet channel 624 .

[0124] The sample fluid 612 and the focus fluid 616 travel downstream of the junction region 618 through the detection station 606. The detection station 606 includes a light source 630 for illuminating an interrogation zone 634 of the detection station along an illumination axis 640 of the sample outlet channel 624. As the droplets 602 pass serially through, a high-speed camera 632 captures images of the illumination zone 640 as viewed along a horizontal detection axis 641.

[0125] Separator 617 provides 4-way oil injection to create a sheathing flow designed to keep the droplets in the center of the capillary. Based on the droplet velocity within capillary 658, the Reynolds number is calculated to be 430. This means that the flow regime within capillary 658 is laminar flow including inertia. Therefore, the droplets may be affected by the following factors: (1) lubrication forces directed radially inward toward the longitudinal axis of the capillary at a position close to the periphery due to the sheathing flow; (2) buoyancy forces directed upward due to the density difference between the oil phase (carrier fluid and focusing fluid) and the droplets; (3) Saffman forces in random directions due to inertial flow conditions; and (4) instabilities caused by fluid flow pulsations created by the syringe pumps that drive the flow of sample fluid and focusing fluid.

[0126] Fig.16 and Fig.17 Shown from passing through Fig.15 The "small" droplet 602a ( Fig.16 ) and the "standard" droplet 602b ( Fig.17 ) . The small droplet 602a has a radius of 31 microns, and the standard droplet 602b has a radius of 56 microns. An exemplary captured image 660 showing one or two droplets 602a or 602b is presented in the lower right corner of each figure.

[0127] The measured vertical intra-capillary positions of a collection of approximately 1300-1500 droplets 602a and 602b are plotted in each graph. The vertical position of the center of each droplet is determined from the image of the droplet captured by camera 632 and is indicated by an asterisk in the graph. The capillary 658 forming the sample outlet channel 624 has an inner diameter of 150 microns. Accordingly, the droplets concentrated in the capillary within the interrogation zone are plotted at 75 microns in the graph.

[0128] In each of the graphs, the average vertical position of the droplets is close to or at 75 microns. However, the standard deviation of the average varies significantly depending on the size and type of the droplets. The center of the small droplet 602a is the most widely distributed, from about 50 microns to 100 microns, with a standard deviation of 15.1. The high-speed video of the small droplet 602a traveling along the capillary 658 reveals the substantial vertical motion of the small droplets. As further explained below, many of the small droplets appear to bounce up and down between invisible barriers within the capillary 658. In contrast, the center of the standard droplet 602b is tightly clustered along the horizontal line at 75 microns, with a standard deviation of 1.2.

[0129] Fig.18 and Fig.19 Use respectively Fig.16 and Fig.17658 can affect the positioning of the small droplet 602a and the standard droplet 602b within the capillary. The lubrication force creates a boundary layer 662 having a thickness of approximately 19 microns. Neither the small droplet 602a nor the standard droplet 602b enters the boundary layer 662. Fig.18 and Fig.19 As shown in FIG, the difference in size between the small droplet 602a (radius 31 microns) and the standard droplet 602b (radius 56 microns) explains the effect of the boundary layer on the degree of droplet alignment. The center of the small droplet 602a was allowed to travel 50 microns off-axis (see Fig.18 ). The travel distance is determined by the difference between: (a) the inner diameter of the capillary 658 (i.e., 150 microns) and (b) the sum of two times the diameter of the small droplet 602a (i.e., 62 microns) and the thickness of the boundary layer 662 (i.e., 38 microns). Accordingly, the small droplet 602a may be affected by the different forces described above, causing the small droplet not to travel in a straight line, but to travel laterally and bounce within the capillary. In contrast, the center of the standard droplet 602b is restricted from moving off-axis because the inner diameter of the capillary 658 is approximately the same as the sum of two times the diameter of the standard droplet 602 and the thickness of the boundary layer 662. In other words, because the standard droplet is squeezed by the wall lubrication force boundary layer and thereby moves in a straight line through the center of the capillary, the standard droplet does not have room for lateral migration and / or rebound within the capillary.

[0130] Fig. 20 Shows Fig.18 Graph of the measured positions within the capillary for a sequence of about 40 droplets, which are Fig.18 For unknown reasons, the droplet positions have a somewhat periodic pattern.

[0131] Fig.21 A working model of a "three-port" detection system 700 for droplets is shown. The detection system 700 is similar to Fig.15 The detection system 600 is the same as that of the embodiment of the present invention: the focusing fluid is rotationally asymmetrically introduced into the confluence area of ​​the separator 717 from the three focusing fluid channels 722a, 722b and 722d at three ports instead of four ports.

[0132] Fig. 22 Shows the use of Fig.21 The graph of the position data in the capillary collected from about 1300 droplets by the three-port detection system 700 is generally similar to Fig.16 The droplets have vertical positions distributed across about 50 microns with a standard deviation of 9.5. Accordingly, the droplet alignment provided by the asymmetric three-port configuration of the detection system 700 does not appear to be any better than Fig.15The symmetrical configuration of the detection system 600 aligns the droplets much better. The net lateral force exerted by the three streams of focusing fluid on the sample fluid stream is horizontal to produce a horizontal radial offset of the droplet position that has a much smaller effect on the vertical positioning. In other words, the camera of the detection station is not arranged to detect changes in the horizontal position of the droplet.

[0133] Fig.23 Another working model of a "three-port" detection system 800 for droplets is shown. The detection system 800 is similar to Fig.21 The three-port detection system 700 is the same as the one of the embodiments of the present invention: the focusing fluid is rotationally asymmetrically introduced into the confluence area of ​​the separator 817 from the three focusing fluid channels 822a, 822b and 822c at three ports, and the three ports are rotationally offset by 90 degrees from the three ports of the separator 717.

[0134] Fig.24 Shows the use of Fig.23 The graph of the position data in the capillary collected from about 1200 small droplets by the three-port detection system 800 is generally similar to Fig.16 The droplets have vertical positions that are generally concentrated at a position within the capillary of 50 microns, with moderate dispersion upward to a position of 100 microns, to produce a standard deviation of 6.4. Accordingly, the asymmetric three-port configuration of the detection system 800 provides a standard deviation of 6.4. Fig.15 The symmetrical configuration of the detection system 600 can detect better droplet alignment. The net lateral force exerted by the three streams of focusing fluid on the sample fluid stream is vertical to produce a vertical downward radial shift in the droplet position, which is efficiently detected by the camera of the detection station of the system. The majority of the droplets are positioned adjacent to the lower portion of the boundary layer. This indicates that the symmetrical configuration for the injection of the focusing fluid is relatively stable relative to other forces that encourage lateral migration and rebound of the droplets (such as Fig.15 and Fig.16 ), the net lateral force from the focused fluid pushing the droplets toward the boundary layer dominates. When viewed with high-speed video, most of the droplets appear to slide along the lower portion of the boundary layer.

[0135] Fig.25 A working model of yet another "three-port" detection system 900 for droplets is shown. The detection system 900 is similar to Fig.23 The three-port detection system 800 is the same as the three-port detection system 800: the focusing fluid is asymmetrically introduced into the confluence area of ​​the separator 917 from the three focusing fluid channels 922a, 922c and 922d at the three ports, and the three ports are Fig.23 The three ports of separator 817 are rotationally offset by 180 degrees.

[0136] Fig.26 Shows the use of Fig.25 The graph of the position data in the capillary collected from about 1400 droplets by the three-port detection system 900 is generally similar to that of Fig.16 The droplets have vertical positions that are generally clustered at a position within the capillary of 100 microns, with moderate dispersion down to 50 microns to produce a standard deviation of 5.7. Accordingly, the asymmetric three-port configuration of the detection system 900 provides a standard deviation of 5.7. Fig.15 The symmetrical configuration of the detection system 600 can detect better droplet alignment. The net lateral force exerted by the three streams of focusing fluid on the sample fluid stream is vertical to produce a vertical upward radial displacement of the droplet position, which is efficiently detected by the camera of the detection station of the system. The majority of the droplets are positioned adjacent to the upper portion of the boundary layer, indicating that other forces that encourage lateral migration and rebound of droplets (such as the force of the sample fluid) in the symmetrical configuration for focusing fluid injection are relatively stable. Fig.15 and Fig.16 ), the net lateral force from the focused fluid pushing the droplets toward the boundary layer dominates. When viewed with high-speed video, most of the droplets appear to slide along the upper part of the boundary layer.

[0137] Fig. 27 A working model of a symmetrical "two-port" detection system 1000 for droplets is shown. The detection system 1000 is similar to Fig.15 The symmetrical four-port detection system 600 is the same as: the focusing fluid is symmetrically introduced into the confluence area of ​​the separator 1017 from only two focusing fluid channels 1022b and 1022d at two ports, and the two ports are vertically aligned with each other.

[0138] Fig.28 is to use Fig. 27 A graph of the position data in the capillary collected from about 1600 droplets by the two-port detection system 1000, which is generally similar to Fig.16 The droplets have centers spread between positions within the capillary of 50 μm and 100 μm with a standard deviation of 16.2. Accordingly, the symmetric two-port detection system 1000 produces the same Fig.15 and Fig.16 The results of the symmetrical four-port detection system 600 are similar to the results.

[0139] Fig.29 A working model of a symmetrical "two-port" detection system 1100 for droplets is shown. The detection system 1100 is similar to Fig.28The symmetrical two-port detection system 1000 is the same: the focusing fluid is symmetrically introduced from the two focusing fluid channels 1122a and 1122b into the confluence area of ​​the separator 1117 at two ports, and the two ports are horizontally aligned with each other.

[0140] Fig.30 is to use Fig.29 A graph of the position data in the capillary collected from about 1400 droplets by the two-port detection system 1100 is generally similar to Fig.16 The droplets have centers spread between positions within the capillary of 50 microns and 100 microns with a standard deviation of 16.5. Accordingly, the symmetric two-port detection system 1100 produces the same Fig. 27 and Fig.28 The results of the symmetric two-port detection system 1000 are similar to the results (if not worse).

[0141] Fig.31 The working model of the asymmetric "two-port" detection system 1200 for droplets is shown. The detection system 1200 is similar to Fig.23 The three-port detection system 800 is the same as: the focusing fluid is asymmetrically introduced into the confluence area of ​​the separator 1217 from only two focusing fluid channels 1222b and 1222c at two ports, and the two ports are rotationally offset by 90 degrees from each other.

[0142] Fig.32 Shows the use of Fig.31 A graph of the position data in the capillary collected from about 1,300 droplets by the two-port detection system 1200, generally similar to Fig.16 The droplets have vertical positions that are generally clustered at about 60 microns in the capillary, with moderate dispersion upward to 90 microns to produce a standard deviation of 6.4. Accordingly, the asymmetric two-port configuration of the detection system 1200 provides a standard deviation of 6.4. Fig.15 The symmetrical configuration of the detection system 600 enables better droplet alignment to be detected. The net lateral force exerted on the sample fluid stream by the two streams of the focusing fluid is angled downward at 45 degrees, which produces a radial shift in the droplet position that is efficiently detected by the camera at the detection station of the system.

[0143] Fig.33 FIG. 1 shows a working model of another asymmetric “two-port” detection system 1300 for droplets. The detection system 1300 is similar to the Fig.31 The two-port detection system 1200 is the same as the two-port detection system 1200: the focusing fluid is asymmetrically introduced into the confluence area of ​​the separator 1317 from the two focusing fluid channels 1322c and 1322d at the two ports, and the two ports are relative to Fig.31The two ports of the detection system 1200 are rotationally offset by 90 degrees.

[0144] Fig.34 Shows the use of Fig.33 A graph of the position data in the capillary collected from about 1000 droplets by the two-port detection system 1300 is generally similar to Fig.16 The droplets have vertical positions that are generally clustered at about 90 microns in the capillary, with moderate dispersion down to 60 microns to produce a standard deviation of 6.4. Accordingly, the asymmetric two-port configuration of the detection system 1300 provides a standard deviation of 6.4. Fig.15 The symmetrical configuration of the droplet alignment of the detection system 600 enables better droplet alignment to be detected. The net lateral force exerted on the sample fluid stream by the two streams of the focusing fluid is angled upward at 45 degrees, which produces a radial shift in the droplet position that is efficiently detected by the camera at the detection station of the system.

[0145] Fig.35 A working model of an asymmetric "one-port" detection system 1400 for droplets is shown. The detection system 1400 introduces focus fluid asymmetrically from only one focus fluid channel 1422b into the confluence region of the separator 1417 in a vertically downward direction at only one port.

[0146] Fig.36 Shows the use of Fig.35 The one-port detection system 1400 collects the position data of the capillary from about 1400 small droplets, which is generally as follows: Fig.16 The droplets have vertical positions spread out by almost 50 microns across the capillary, yielding a standard deviation of 13.8. Accordingly, the asymmetric one-port configuration of the detection system 1400 provides droplet alignment no better than that from Fig.15 The symmetrical configuration of the detection system 600 allows the droplets to be aligned much better.

[0147] Fig.37 A working model of an asymmetric "one-port" detection system 1500 for droplets is shown. The detection system 1500 introduces focus fluid asymmetrically from only one focus fluid channel 1522a into the confluence region of the separator 1517 in the horizontal direction at only one port.

[0148] Fig.38 Shows the use of Fig.37 A graph of the position data in the capillary collected from about 750 droplets using the one-port detection system 1500 is generally similar to Fig.16 The droplets have vertical positions that are spread almost 50 microns across the capillary, yielding a standard deviation of 13.7. Accordingly, the asymmetric one-port configuration of detection system 1500 provides droplet alignment that is no better than Fig.15 The symmetrical configuration of the detection system 600 allows the droplets to be aligned much better.

[0149] Buoyancy and fluid dynamics can work together to focus droplets or other particles having a density less than or greater than the density of the carrier fluid (and / or focusing fluid). The focusing fluid can be introduced asymmetrically, with a net lateral force upward or downward, such as vertically (i.e., in a direction parallel to gravity), to improve particle focusing.

[0150] Using a collection of droplets loaded with FAM, CY5, ROX, HEX, CY5.5, or ATTO590 fluorescent dyes, Fig.15 Four-port symmetrical detection system 600, Fig.23 Asymmetric three-port detection system 800 and Fig.31 The three-port detection system 800 produced the smallest amplitude CV, followed by the two-port detection system 1200. The four-port detection system 600 performed the worst. D. Fluidics diagram and separators

[0151] This section describes an exemplary fluidics diagram and an asymmetric three-port separator 1617 for a droplet detection system 1600; see Figure 39-Figure 42 . .

[0152] The detection system 1600 includes a fluidics subsystem 1604 that intersects the detection station 1606, as described above in subsections A and C. The fluidics subsystem 1604 uses a sample pump 1628a and a focusing pump 1628b to move fluids into, through, and out of the channel network 1610. Each sample fluid 1612 is drawn into the channel network 1610 from a well of a multi-well microplate 1664 using a pickup head assembly 1665 in conjunction with the sample pump 1628a. The sample pump also drives each sample fluid into the separator 1617. The oil is drawn from the focusing reservoir 1608 by the focusing pump 1628b and then driven by the pump through the diverter 1666 and into the three ports of the separator 1617. The sample fluid and oil flow through the detection station 1606 in the sample outlet channel 1624 and then into the waste container 1609.

[0153] Figure 40-Figure 42An isometric view and a cross-sectional view of a separator 1617 are shown. The separator has a monolithic body defining a plurality of internally threaded openings 1667-1671 extending inwardly from a surface of the body and intersecting each other at a confluence region 1618 within the body. Each opening 1667-1671 is configured to receive an externally threaded fitting that holds an end of a tube or tubular on which a ferrule is disposed. The inner end of opening 1667 forms a tapered region 1636 upstream of a sample inlet 1672 that intersects the confluence region 1618. The inner ends of openings 1668-1670 form focusing fluid ports 1626a-1626c, respectively, that intersect the confluence region 1618. The inner end of opening 1671 forms a sample outlet 1673, which is connected to a separate capillary tube that forms a sample outlet channel including an interrogation zone, as described elsewhere herein. IV. Illustrative combinations and additional examples

[0154] This section describes additional aspects and features of the method and system of the present disclosure, which are presented without limitation in the form of a series of paragraphs, some or all of which may be indexed alphanumerically for clarity and efficiency. Each of these paragraphs may be combined with one or more other paragraphs and / or with disclosure elsewhere in the application in any suitable manner. Some of the following paragraphs explicitly mention and further limit other paragraphs, thereby providing examples of some suitable combinations without limitation.

[0155] A1. A method for detecting particles, the method comprising: (i) driving a sample fluid including particles from a sample inlet channel through a junction area and into a sample outlet channel defining a longitudinal axis; (ii) introducing a focusing fluid from at least two focusing channels into the junction area along corresponding introduction axes, wherein the introduction is rotationally asymmetric around the longitudinal axis, and wherein the introduction axis and the longitudinal axis extend together in three dimensions; (iii) passing the particles through an interrogation zone of the sample outlet channel; (iv) illuminating the interrogation zone with light; and (iv) detecting optical radiation from the interrogation zone.

[0156] A2. The method of paragraph A1, wherein each of the introduction axes is orthogonal to the longitudinal axis.

[0157] A3. The method of paragraph A1 or A2, wherein introducing comprises introducing the focusing fluid from each focusing channel into the confluence region at a respective flow rate that remains constant regardless of the detected optical radiation.

[0158] A4. The method of any of paragraphs A1 to A3, wherein introducing comprises introducing the focus fluid from each focus channel into the confluence region at the same flow rate.

[0159] A5. The method of any of paragraphs A1 to A4, further comprising: transporting each of the particles from the sample outlet channel to the same waste container regardless of the detected optical radiation.

[0160] A6. A method as described in any of paragraphs A1 to A5, wherein, under the same conditions, introducing the focusing fluid concentrates the particles into a smaller particle localization area within the sample outlet channel than would occur by introducing the focusing fluid in a manner rotationally symmetric about the longitudinal axis.

[0161] A7. A method as in any of paragraphs A1 to A6, wherein the introducing comprises creating a boundary layer adjacent to a wall of a sample fluid channel using a focusing fluid, wherein particles are excluded from the boundary layer, and wherein the introducing comprises pushing particles toward the boundary layer so that the particles slide along the boundary layer as they travel along the sample outlet channel.

[0162] A8. The method of any of paragraphs A1 to A7, wherein the interrogation zone is downstream of and spaced apart from the junction area.

[0163] A9. The method of any of paragraphs A1 to A8, wherein, in response to introducing the focusing fluid into the junction region, the average position of particles in the interrogation zone has a radial offset from the longitudinal axis.

[0164] A10. The method of paragraph A9, wherein the sample outlet channel has a radius at the interrogation zone, and wherein the radial offset is at least 10%, 15%, 20%, or 25% of the radius.

[0165] A11. The method of paragraph A9 or A10, wherein the particle has a radius, and wherein the radial offset is at least 20%, 25%, 30%, or 40% of an average value of the radius of the particle.

[0166] A12. The method of any of paragraphs A9 to A11, wherein the irradiating is performed on an irradiation axis, and wherein the radial offset is in a direction within 20 degrees orthogonal to the irradiation axis.

[0167] A13. The method of paragraph A12, wherein the radial offset is orthogonal to the irradiation axis.

[0168] A14. A method as in any of paragraphs A1 to A13, wherein introducing comprises applying a net force on the sample fluid using the focusing fluid in a direction transverse to the longitudinal axis.

[0169] A15. The method of paragraph A14, wherein applying comprises applying a net force in a direction orthogonal to the longitudinal axis.

[0170] A16. The method of paragraph A14 or A15, wherein the net force is a radially oriented force.

[0171] A17. The method of any of paragraphs A1 to A16, wherein driving comprises driving the particles through a tapered region of the sample inlet channel that tapers toward a junction region.

[0172] A18. The method of any of paragraphs A1 to A17, wherein introducing comprises introducing the focus fluid from at least two focus fluid channels into the confluence region through two or more ports.

[0173] A19. The method of paragraph A18, wherein introducing comprises introducing the focusing fluid into the confluence region through only two ports or at least three ports.

[0174] A20. The method of paragraph A19, wherein the focusing fluid is introduced into the junction region through only two ports, and wherein the respective introduction axes are rotationally offset from each other about the longitudinal axis by 120 degrees or less.

[0175] A21. The method of paragraph A18, wherein the focusing fluid is introduced into the junction region through only three ports, and wherein the respective introduction axes are rotationally offset from each other by 100 degrees or less.

[0176] A22. The method of any of paragraphs A1 to A21, wherein the sample outlet channel has a channel diameter at the interrogation zone, and wherein the particles have an average particle diameter that is less than 60% of the channel diameter.

[0177] A23. The method of any of paragraphs A1 to A22, wherein the sample outlet channel has a channel diameter at the interrogation zone, and wherein the particles have an average particle diameter greater than 20% of the channel diameter.

[0178] A24. A method as in any of paragraphs A1 to A23, wherein introducing comprises forming an asymmetric fluid sheath of a focusing fluid around the sample fluid.

[0179] A25. The method of any of paragraphs A1 to A24, wherein the particles are droplets.

[0180] A26. The method of any of paragraphs A1 to A24, wherein the particles are biological cells.

[0181] A27. The method of any of paragraphs A1 to A26, wherein the fluid flow in the sample outlet channel has a Reynolds number between 1 and 2300.

[0182] A28. A method as in any of paragraphs A1 to A27, wherein driving comprises driving the sample fluid into the confluence area at a first volume flow rate, and wherein introducing comprises introducing the focusing fluid into the confluence area at a second volume flow rate, the second volume flow rate being at least five times the first volume flow rate.

[0183] A29. The method of any of paragraphs A1 to A28, wherein introducing includes increasing alignment of the particles with respect to each other along the longitudinal axis.

[0184] A30. A method as in paragraph A29, wherein, other things being equal, the alignment caused by introducing the focusing fluid is greater than the corresponding alignment of the particles that would be produced by introducing the focusing fluid in a manner rotationally symmetric about the longitudinal axis.

[0185] A31. The method of any of paragraphs A1 to A30, wherein the sample inlet channel and the sample outlet channel are coaxial with each other.

[0186] A32. The method of any of paragraphs A1 to A31, wherein introducing comprises increasing an average spacing of particles from one another along the longitudinal axis.

[0187] A33. A method as in any of paragraphs A1 to A32, wherein driving and introducing includes creating at least one pressure differential that produces flow of the sample fluid and the focusing fluid.

[0188] A34. A method as in paragraph A33, wherein driving and introducing are performed using one or more positive / negative pressure sources.

[0189] A35. The method of paragraph A33 or A34, wherein driving and introducing includes operating one or more pumps.

[0190] A36. The method of any of paragraphs A1 to A35, wherein passing comprises passing the particles serially through the interrogation zone.

[0191] A37. The method of any of paragraphs A1 to A36, wherein the monolithic separator device defines a downstream end of the sample inlet channel, a confluence region, and an upstream end of the sample outlet channel.

[0192] A38. The method of any of paragraphs A1 to A37, wherein detecting the optical radiation includes detecting fluorescence from the particles.

[0193] A39. The method of any of paragraphs A1 to A38, wherein each of the particles is separated from a wall of the sample outlet channel surrounding the interrogation zone.

[0194] A40. The method of any of paragraphs A1 to A3 and A5 to A39, wherein introducing comprises introducing the focus fluid from at least two of the focus channels into the confluence region at different flow rates.

[0195] B1. A method for detecting particles, the method comprising: (i) driving a sample fluid including particles from a sample inlet channel through a confluence area and into a sample outlet channel defining a longitudinal axis; (ii) introducing a focusing fluid into the confluence area along two or more introduction axes, the two or more introduction axes collectively having a rotational asymmetry around the longitudinal axis; (iii) passing the particles through an interrogation zone of the sample outlet channel; (iv) illuminating the interrogation zone with light; and (iv) detecting optical radiation from the interrogation zone.

[0196] B2. The method of paragraph B1, further comprising any of the limitations of paragraphs A1 to A40.

[0197] C1. A system for detecting particles, the system comprising: (i) a channel network, the channel network comprising a confluence region fluidically connecting a sample inlet channel and a sample outlet channel to each other, the sample outlet channel defining a longitudinal axis, the channel network also comprising at least two focusing fluid channels, the at least two focusing fluid channels defining corresponding introduction axes, and being fluidically connected to the confluence region at two or more ports, wherein the introduction axis and the longitudinal axis extend together in three dimensions; (ii) one or more positive / negative pressure sources, the one or more positive / negative pressure sources being operably connected to the channel network and configured to: drive a sample fluid including particles from the sample inlet channel through the confluence region and through an interrogation region of the sample outlet channel; and to drive the focusing fluid to be introduced into the confluence region asymmetrically around the longitudinal axis; and (iii) a detection station, the detection station comprising a light source configured to illuminate the interrogation region with light, and the detection station also comprising an optical sensor configured to detect optical radiation from the interrogation region.

[0198] C2. The system of paragraph C1, wherein the channel network includes a monolithic separator device that defines a downstream end of the sample inlet channel, a confluence region, and an upstream end of the sample outlet channel.

[0199] C3. A system as described in paragraph C2, wherein the one or more positive / negative pressure sources include a first pump and a second pump, the first pump is used to drive the sample fluid through the confluence area and the interrogation area, and the second pump is used to drive the focusing fluid into the confluence area.

[0200] C4. The system of any of paragraphs C1 to C3, wherein at least two focusing fluid channels are configured such that an average position of particles in the interrogation zone has a radial offset from the longitudinal axis.

[0201] C5. A system as in any of paragraphs C1 to C4, wherein the system is configured to satisfy any of the limitations in paragraphs A1 to A40. V. Advantages and Benefits

[0202] Different examples of methods and systems for detecting particles provide several advantages over known solutions for detecting particles. For example, the illustrative examples described herein permit more accurate and efficient detection of particle collections that differ from one another in terms of particle size, particle composition (e.g., particles with different densities), particle shape, etc. using the same fluid layout.

[0203] Additionally, and among other benefits, the methods and systems disclosed herein provide for more consistent illumination of a collection of particles with light.

[0204] Additionally, and among other benefits, the methods and systems disclosed herein provide for more uniform excitation of, and thereby more informative emission from, the fluorophore(s) associated with a collection of particles.

[0205] Additionally, and among other benefits, the methods and systems disclosed herein may be used in flow cytometry.

[0206] Additionally, and among other benefits, the methods and systems disclosed herein may be used for particle tracking velocimetry.

[0207] There is no known system or method that can perform these functions.However, not all of the examples described herein provide the same advantages or the same degree of advantages. VI. in conclusion

[0208] The disclosure set forth above can encompass a plurality of different examples with independent utility. Although each of these examples is disclosed in its (one or more) preferred form, its specific examples as disclosed and illustrated herein should not be considered as restrictive, because multiple variations are possible. With regard to the section titles used in this disclosure, such titles are only used for organizational purposes. The subject matter of the present disclosure includes all novel and non-obvious combinations and sub-combinations of various elements, features, functions, and / or properties disclosed herein. The attached claims specifically point out certain combinations and sub-combinations that are considered to be novel and non-obvious. Other combinations and sub-combinations of features, functions, elements, and / or properties can be claimed in applications claiming priority to this application or related applications. Such claims, whether their scope is wider, narrower, equivalent or different than the original claims, are also considered to be included in the subject matter of the present disclosure.

Claims

1. A method for detecting particles, the method comprising: driving a sample fluid including the particles from a sample inlet channel through a junction region and into a sample outlet channel defining a longitudinal axis; introducing a focusing fluid from at least two focusing channels into the confluence region along respective introduction axes, wherein the introduction is rotationally asymmetric about the longitudinal axis, and wherein the introduction axes and the longitudinal axis together extend in three dimensions; passing the particle through an interrogation zone of the sample outlet channel; illuminating the interrogation zone with light; and Optical radiation is detected from the interrogation zone.

2. The method of claim 1, wherein: Each of the lead-in axes is orthogonal to the longitudinal axis.

3. The method of claim 1, wherein: Introducing includes introducing the focusing fluid from each focusing channel into the junction region at respective flow rates that remain constant regardless of the detected optical radiation.

4. The method of claim 1, wherein: Introducing includes introducing the focus fluid from each focusing channel into the confluence area at the same flow rate.

5. The method of claim 1, further comprising: Each of the particles is transported from the sample outlet channel to the same waste container regardless of the optical radiation detected.

6. The method of claim 1, wherein: All else being equal, introducing the focusing fluid concentrates the particles to a smaller particle localization zone within the sample outlet channel than would occur by introducing the focusing fluid in a rotationally symmetric manner about the longitudinal axis.

7. The method of claim 1, wherein: The introducing comprises creating a boundary layer adjacent to a wall of the sample outlet channel using the focusing fluid, wherein the particles are excluded from the boundary layer, and wherein the introducing comprises pushing the particles toward the boundary layer such that the particles slide along the boundary layer as they travel along the sample outlet channel.

8. The method of claim 1, wherein: The interrogation zone is downstream of and spaced apart from the merging area.

9. The method of claim 1, wherein: In response to introducing the focusing fluid into the junction region, the average position of the particles in the interrogation zone has a radial offset from the longitudinal axis.

10. The method of claim 9, wherein: The sample outlet channel has a radius at the interrogation zone, and wherein the radial offset is at least 10%, 15%, 20% or 25% of the radius.

11. The method of claim 9, wherein: The particles have a radius, and wherein the radial offset is at least 20%, 25%, 30% or 40% of an average value of the radius of the particles.

12. The method of claim 9, wherein: Irradiation is performed on an illumination axis, and wherein the radial offset is in a direction within 20 degrees orthogonal to the illumination axis.

13. The method of claim 12, wherein: The radial offset is orthogonal to the illumination axis.

14. The method of claim 1, wherein: Introducing includes exerting a net force on the sample fluid using the focusing fluid in a direction transverse to the longitudinal axis.

15. The method of claim 14, wherein: Applying includes applying the net force in a direction orthogonal to the longitudinal axis.

16. The method of claim 14, wherein: The net force is a radially oriented force.

17. The method of claim 1, wherein: Driving includes driving the particles through a tapered region of the sample inlet channel, the tapered region tapering toward the junction region.

18. The method of claim 1, wherein: Introducing includes introducing the focus fluid from the at least two focus fluid channels into the confluence region through two or more ports.

19. The method of claim 18, wherein: Introducing includes introducing the focus fluid into the confluence area through only two ports or at least three ports.

20. The method of claim 19, wherein: The focusing fluid is introduced into the junction region through only two ports, and wherein the respective introduction axes are rotationally offset from each other by 120 degrees or less about the longitudinal axis.

21. The method of claim 18, wherein: The focusing fluid is introduced into the confluence region through only three ports, and wherein the respective introduction axes are rotationally offset from each other by 100 degrees or less.

22. The method of claim 1, wherein: The sample outlet channel has a channel diameter at the interrogation zone, and wherein the particles have an average particle diameter that is less than 60% of the channel diameter.

23. The method of claim 1, wherein: The sample outlet channel has a channel diameter at the interrogation zone, and wherein the particles have an average particle diameter greater than 20% of the channel diameter.

24. The method of claim 1, wherein: Introducing includes forming an asymmetric fluid sheath of the focusing fluid around the sample fluid.

25. The method of claim 1, wherein: The particles are liquid droplets.

26. The method of claim 1, wherein: The particles are biological cells.

27. The method of claim 1, wherein: The fluid flow in the sample outlet channel has a Reynolds number between 1 and 2,300.

28. The method of claim 1, wherein: Driving includes driving the sample fluid into the confluence area at a first volume flow rate, and wherein introducing includes introducing the focus fluid into the confluence area at a second volume flow rate, the second volume flow rate being at least five times the first volume flow rate.

29. The method of claim 1, wherein: Introducing includes increasing alignment of the particles with respect to each other along the longitudinal axis.

30. The method of claim 29, wherein: All other things being equal, the alignment induced by introduction of the focusing fluid is greater than a corresponding alignment of the particles that would result from introduction of the focusing fluid in a rotationally symmetric manner about the longitudinal axis.

31. The method of claim 1, wherein: The sample inlet channel and the sample outlet channel are coaxial with each other.

32. The method of claim 1, wherein: Introducing includes increasing an average spacing of the particles from one another along the longitudinal axis.

33. The method of claim 1, wherein: Driving and introducing include creating at least one pressure differential that produces flow of the sample fluid and the focusing fluid.

34. The method of claim 33, wherein: Actuation and introduction are performed using one or more positive / negative pressure sources.

35. The method of claim 33, wherein: Actuating and introducing includes operating one or more pumps.

36. The method of claim 1, wherein: Passing includes passing the particles serially through the interrogation zone.

37. The method of claim 1, wherein: The monolithic separator device defines a downstream end of the sample inlet channel, the confluence region, and an upstream end of the sample outlet channel.

38. The method of claim 1, wherein: Detecting optical radiation includes detecting fluorescence from the particles.

39. The method of claim 1, wherein: Each of the particles is spaced apart from a wall of the sample outlet channel surrounding the interrogation zone.

40. The method of claim 1, wherein: Introducing includes introducing the focus fluid from at least two of the focus channels into the confluence region at different flow rates.

41. A method for detecting particles, the method comprising: driving a sample fluid including the particles from a sample inlet channel through a junction region and into a sample outlet channel defining a longitudinal axis; introducing a focusing fluid into the junction region along two or more introduction axes, the two or more introduction axes collectively having a rotational asymmetry about the longitudinal axis; passing the particle through an interrogation zone of the sample outlet channel; illuminating the interrogation zone with light; and Optical radiation is detected from the interrogation zone.

42. A system for detecting particles, the system comprising: a channel network comprising a junction region fluidly connecting a sample inlet channel and a sample outlet channel to each other, the sample outlet channel defining a longitudinal axis, the channel network further comprising at least two focusing fluid channels defining respective introduction axes and being fluidly connected to the junction region at two or more ports, wherein the introduction axis and the longitudinal axis collectively extend in three dimensions; one or more positive / negative pressure sources operably connected to the channel network and configured to: drive a sample fluid including the particles from the sample inlet channel through the confluence region and through an interrogation zone of the sample outlet channel; and to drive a focusing fluid into the confluence region asymmetrically about the longitudinal axis; and A detection station includes a light source configured to illuminate the interrogation zone with light and an optical sensor configured to detect optical radiation from the interrogation zone.

43. The system of claim 42, wherein: The channel network includes a monolithic separator device defining a downstream end of the sample inlet channel, the confluence region, and an upstream end of the sample outlet channel.

44. The system of claim 43, wherein: The one or more positive / negative pressure sources include a first pump for driving the sample fluid through the junction area and the interrogation zone and a second pump for driving the focusing fluid into the junction area.

45. The system of claim 42, wherein: The at least two focusing fluid channels are configured such that an average position of the particles in the interrogation zone has a radial offset from the longitudinal axis.

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