Particle sorter nozzle and use method thereof

Abstract

The invention provides a particle sorter nozzle. The target nozzle includes an elongated body and an air inlet radially disposed at a proximal end of the elongated body. The air inlet of the subject nozzle includes a radial airflow path that may supply air to the channel. The elongated body includes a proximal opening engageable with the flow chamber to establish a liquid receiving relationship, a distal opening for emitting droplets, and a channel through which liquid may be delivered from a proximal end to a distal end through the elongated body. In addition, particle sorters having the subject nozzles, methods of using and assembling the subject particle sorters, and kits are also provided.

Description

[0001] Cross-references to related patent applications

[0002] Pursuant to Title 35 of the United States Patent Act, Section 119(e), this application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 342,724, filed on May 17, 2022; the disclosure of U.S. Provisional Patent Application No. 63 / 342,724 is incorporated herein by reference in its entirety and becomes a part of the present invention. Background Art

[0003] Flow cytometry is a technique used to characterize and often sort biological material, such as cells in a blood sample, or target particles in another biological or chemical sample. A flow cytometer typically includes a sample reservoir for holding a fluid sample (e.g., a blood sample) and a sheath reservoir containing a sheath fluid. A flow cytometer delivers particles (including cells) in a fluid sample as a stream of cells to a flow chamber, while a sheath fluid is directed into the flow chamber. To characterize the components of the flowing stream, the flowing stream is irradiated with light. Changes in the material in the flowing stream, such as morphology or the presence of fluorescent markers, can result in changes in the observed light, which can be characterized and separated. To characterize the components of the flowing stream, light must be directed onto the flowing stream and collected. Light sources in a flow cytometer vary and can include one or more broad spectrum lamps, light emitting diodes, and single wavelength lasers. The light source is aligned with the flowing stream, and the optical response from the irradiated particles is collected and quantified.

[0004] Separation of biological particles is achieved by adding a sorting or collection function to a flow cytometer. Particles in the separation stream are detected to have one or more desired characteristics and can be individually separated from the sample stream by mechanical or electronic removal. A common flow sorting technique uses droplet sorting, in which a fluid stream containing linearly separated particles is split into droplets. Droplets containing the target particles are charged and deflected through an electric field into a collection tube. Typically, linearly separated particles in the fluid stream are characterized as they pass through an observation point located directly below the nozzle tip. Once a particle is determined to meet one or more desired criteria, the time when the particle will reach the droplet breakoff point and break away from the fluid stream in a droplet can be predicted. Ideally, a brief charge is applied to the fluid stream before the droplet containing the selected particle breaks away from the fluid stream, and then grounded immediately after the droplet breaks away. The droplet to be sorted retains its charge when it breaks away from the fluid stream, while all other droplets are uncharged.

[0005] Droplet formation at the nozzle tip is usually achieved by a piezoelectric oscillator (also known as a piezoelectric actuator), which produces a small perturbation in the sheath flow, causing jet instabilities and droplet formation. Figure 1 A conventional cell sorter with a piezoelectric oscillator is shown. Figure 1 As shown, a liquid sample enters at the end 101. A piezoelectric oscillator 102 generates air flow disturbances to form droplets, and a fluid flow charging wire 103 applies a charge. The sample is irradiated with light in the interrogation area 104, generating scattered light and fluorescence, which are then collected for analysis. Subsequently, the droplets break apart at the far end of the nozzle 106, and the deflector 107 attracts or repels the droplets. Uncharged droplets are transferred to waste 108, while charged droplets are received in a collection container 109. This conventional droplet formation method uses a variety of nozzle sizes to produce a variety of droplet sizes. The high pressure in the fluid flow system places stringent demands on construction materials and fluid seals. Summary of the invention

[0006] The inventors have realized that a piezoelectric oscillator (such as Figure 1 Conventional cell sorting systems (such as those shown in FIG. 1 ) generate fluid disturbances upstream of the interrogation zone, thereby reducing the quality of the data obtained thereby. In addition, such systems require the use of nozzles of different diameters to produce droplets of different sizes. Therefore, it has been determined that it is best to use a single nozzle to produce droplets of different sizes without these droplets generating upstream fluid disturbances. Embodiments of the present invention meet this need.

[0007] Various aspects of the present invention include a nozzle having an elongated body and an air inlet radially disposed at the proximal end of the elongated body. The target elongated body includes a proximal opening that can be engaged with a flow chamber to establish a liquid receiving relationship, a distal opening for emitting droplets, and a channel through which the liquid can be transported from the proximal end to the distal end through the elongated body. The subject air inlet includes a radial air flow path that can supply air to the channel. In some instances, the height of the radial air flow path ranges from 150 µm to 250 µm (e.g., 200 µm). In some cases, the radius of the radial air flow path ranges from 200 µm to 300 µm (e.g., 250 µm). In some embodiments, the air inlet may include multiple radial air flow paths, for example, the number of radial air flow paths in the multiple radial air flow paths ranges from 2 to 5 (e.g., 4). In some versions, the air inlet is operably connected to one or more gas ducts. In selected versions, the channel may have a diameter ranging from 150 µm to 250 µm (e.g., 200 µm), and the diameter may or may not be constant over the entire length of the elongated body. In some examples, the elongated body is cylindrical and has a length ranging from 4 mm to 4.5 mm (e.g., 4.3 mm).

[0008] In addition, various aspects of the present invention also include a particle sorter having a subject nozzle. The target particle sorter includes a flow chamber and a nozzle, wherein the flow chamber can convey particles in a flow stream, and the nozzle includes an elongated body and an air inlet radially arranged at the proximal end of the elongated body (for example, as described above). In an embodiment, the particle sorter of the present invention may include a compressed gas source, such as an air compressor or a gas tank. In some instances, the compressed gas source can produce a gas with a pressure range of 2 kPa-10 kPa. In some versions, the particle sorter includes one or more gas conduits, which can operably connect the compressed gas source to the air inlet. In addition, an embodiment of the present invention also includes a processor, which is operably connected to the compressed gas source and can adjust the pressure of the generated gas provided to the air inlet. In some cases, the nozzle is not operably fixed to the piezoelectric actuator. According to some versions, the particle sorter also includes one or more of the following components: a light source that can irradiate the flow stream at an interrogation point; a detector that can collect particle modulated light from the flow chamber; a plurality of containers that can receive droplets emitted from the distal opening of the elongated structure; and a deflection plate that can deflect the droplets into a given container among the plurality of containers.

[0009] Various aspects of the present invention also include methods for sorting particle samples. The subject method includes introducing a particle sample into a cell sorter, the cell sorter including a flow chamber that can deliver particles in a flow stream and a subject nozzle (e.g., as described above); sorting the particle sample by flow cytometry. In some embodiments, the method includes regulating the pressure of a gas provided to the gas inlet using a compressed gas source. In some cases, the particle sample being sorted can be a biological sample, such as a cell.

[0010] The various elements of the present disclosure further include a particle sorter assembly method. The target method includes operably connecting a nozzle, such as the nozzle described above, to a flow chamber that can transport particles in a flow stream. In the subject method, operably connecting the nozzle includes engaging the proximal opening of the elongated body with the flow chamber to establish a liquid receiving relationship. In some instances, the method includes gaseously connecting a compressed gas source (e.g., an air compressor) to an air inlet. In some instances, the method includes operably connecting one or more gas conduits to the compressed gas source and the air inlet. In selected cases, the method includes connecting a processor to a compressed gas source, wherein the processor can adjust the pressure of the generated gas provided to the air inlet. The method described in some embodiments does not include operably connecting a piezoelectric actuator to the nozzle.

[0011] Various aspects of the present invention further include kits. The subject kit includes a subject nozzle (e.g., as described above). In some cases, the kit also includes a compressed gas source (e.g., an air compressor). The kit according to some embodiments of the present invention includes one or more gas conduits that can operably connect the compressed gas source to the gas inlet. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] The present invention can be best understood from the following detailed description when read in conjunction with the accompanying drawings. The accompanying drawings include the following figures:

[0013] Figure 1 A conventional cell sorter with a piezoelectric oscillator is shown.

[0014] Figure 2A -B shows a nozzle according to some embodiments ( Figure 2A ) and catheter ( Figure 2B ).

[0015] Figure 3 A particle sorter with a nozzle according to certain embodiments is shown.

[0016] Figure 4 A functional block diagram of a flow cytometry system according to certain embodiments is shown.

[0017] Figure 5 A sorting control system according to certain embodiments is shown.

[0018] Fig. 6A -B shows a schematic diagram of a particle sorter system according to certain embodiments.

[0019] Figure 7 A block diagram of a computing system according to some embodiments is shown.

[0020] Figure 8 A schematic diagram of a nozzle according to certain embodiments is shown.

[0021] Fig. 9 Results of fluid simulation of a nozzle according to some embodiments of the present invention are shown. DETAILED DESCRIPTION

[0022] The present invention provides a particle sorter nozzle. The target nozzle includes an elongated body and an air inlet radially arranged at the proximal end of the elongated body. The air inlet of the subject nozzle includes a radial air flow path that can supply air to the channel. The elongated body includes a proximal opening that can be engaged with a flow chamber to establish a liquid receiving relationship, a distal opening for emitting droplets, and a channel that can transport liquid from the proximal end to the distal end through the elongated body. In addition, a particle sorter having a subject nozzle, a method for using and assembling the subject particle sorter, and a kit are also provided.

[0023] Before describing the present invention in more detail, it should be understood that the present invention is not limited to the specific embodiments described, which may, of course, vary. It should also be understood that since the scope of the present invention is limited only by the appended claims, the terms in the present invention are only used to describe specific embodiments and are not intended to be limiting.

[0024] If the present invention provides a range of values, it should be understood that the present invention includes each intermediate value between the upper and lower limits of this range (unless the context clearly stipulates otherwise, it is accurate to one tenth of the unit of the lower limit), as well as any other specified values ​​or intermediate values ​​within this specified range. The upper and lower limits of these smaller ranges may be independently included in the smaller ranges and are also included in the scope of the present invention, but are subject to any explicitly excluded limits in the specified range. If the specified range includes one or two of the limits, the present invention also includes a range excluding any or both of these included limits.

[0025] Certain ranges in the present invention are expressed as numerical values ​​preceded by the term "about". The term "about" as used in the present invention is used to provide literal support for the exact number that follows it, as well as numbers that are close to or approximately the number that follows it. In determining whether a number is close to or approximately a number that is explicitly recited, the close or approximate unrecited number can be a number that is substantially equivalent to the explicitly recited number in the context in which it is presented.

[0026] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those commonly understood by those of ordinary skill in the art to which the present invention belongs. Although any methods and materials similar or equivalent to the methods or materials described herein can also be used in the practice or testing of the present invention, representative illustrative methods and materials are described below.

[0027] All publications and patents cited in this specification are incorporated herein by reference as if each individual publication or patent was specifically and individually indicated to be incorporated herein by reference as a part of the present invention and is intended to disclose and describe the methods and / or materials in connection with the cited publication. Citation of any publication is for the purpose of disclosure prior to the filing date and should not be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Furthermore, the dates of publication provided may be different from the actual publication dates which need to be independently confirmed.

[0028] It is noted that, as used in the present disclosure and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. It should also be noted that any optional elements may be excluded when drafting the claims. Therefore, this statement is intended to serve as a precondition for using exclusive terminology such as "only," "only" and the like in connection with the recitation of claim elements or using a "negative" limitation.

[0029] After reading this disclosure, it will be apparent to those skilled in the art that each of the individual embodiments described and illustrated herein has discrete components and features that can be easily separated or combined with the features of any of the other multiple embodiments without departing from the scope or spirit of the invention. Any method recited may be implemented in the order of events recited or in any other order that is logically possible.

[0030] Although the systems and methods have been or will be described for the sake of grammatical fluency and functional explanation, it should be expressly understood that, except as expressly provided in 35 U.S. Patent Act § 112, the claims should not be construed as necessarily being limited in any way to “means” or “steps” but rather be given the full meaning and scope of equivalents provided by the definitions in the claims in accordance with the doctrine of judicial equivalents, and to the extent that a claim is expressly provided in 35 U.S. Patent Act § 112, it should be given its full legal equivalent pursuant to 35 U.S. Patent Act § 112.

[0031] Particle Sorter Nozzle

[0032] As described above, various aspects of the present invention include a particle sorter nozzle. The subject nozzle includes an elongated body and an air inlet radially disposed at the proximal end of the elongated body. In some cases, the nozzle of the present invention can produce droplets having any suitable diameter. In these cases, droplets having different diameters can be produced without adjusting the nozzle itself (e.g., by exchanging the nozzle with another nozzle having a different diameter). For example, in some cases, the droplets produced by the subject nozzle can have any suitable diameter in the range of 75 μm-230 μm, as desired. The droplets can have any suitable volume in the range of 0.25nL-6 nL, as desired. In addition, the subject nozzle can reduce fluid disturbances upstream of the interrogation zone, such as by 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 95% or more, 99% or more, including a 100% reduction.

[0033] In addition to the above, the subject nozzle can also be used to generate droplets without using a piezoelectric oscillator. The nozzle described in the present invention can be used to generate droplets using two immiscible fluids (dispersed phase and continuous phase). The dispersed phase is a fluid that is to be split into droplets (such as a flow stream containing particles). The continuous phase (such as air) is immiscible with the dispersed phase and can be driven independently. Without being bound by theory, it has been found that when the dispersed phase and the continuous phase meet at the junction, the interaction of the two phases at the junction causes droplets to form. For more information on the physical mechanism of using a continuous phase and a dispersed phase, see Baroud et al. "Laboratory Chip" (2010) 10, 2032-2045; the disclosure of the article is cited in the present invention and becomes a part of the present invention.

[0034] By "elongated body" is meant that the subject nozzle is longer than it is wide. In other words, the nozzle has a distinct proximal end and a distal end. The proximal end is the end at which the nozzle receives liquid from the flow chamber when the nozzle is used in a particle sorter. The distal end is the end at which the liquid splits and forms droplets when the nozzle is used in a particle sorter. The elongated body may have any suitable cross-sectional shape, wherein the target cross-sectional shape includes but is not limited to a straight cross-sectional shape (e.g., square, rectangular, trapezoidal, triangular, hexagonal, etc.), a curved cross-sectional shape (e.g., circular, elliptical), and an irregular shape (e.g., a parabolic bottom coupled to a planar top). In an embodiment, the elongated body has a substantially circular cross-sectional shape at a position along the length direction. The so-called "substantially" circular cross-section means that in an embodiment, the cross-section at one or more positions along the length direction of the nozzle may slightly deviate from the circular cross-section, while the circular cross-section characterizes the rest of the structure. For example, in some versions, the cross-section of the elongated structure at one or more positions along the length direction is a polygon (e.g., a hexagon, a pentagon, etc.). In other words, in such versions, the elongated body is not a perfect cylinder, but rather has a circular cross-section with a diameter greater than that in other regions in some areas. In other cases, the elongated body is cylindrical throughout its length. The elongated body may have any suitable length, such as a length ranging from 3 mm to 5 mm, such as 3.5 mm to 4.75 mm, such as 4 mm to 4.5 mm, including 4.2 mm to 4.4 mm. In some cases, the length of the elongated body is 4.3 mm.

[0035] The target nozzle includes a proximal opening that can be engaged with the flow chamber to establish a liquid receiving relationship. In an embodiment, the opening is located at the geometric center of the cross section of the proximal nozzle. The so-called "liquid receiving" relationship means that the subject nozzle can be connected to the flow chamber so that the liquid (e.g., analytical liquid) transported through the flow chamber is received in the nozzle at the proximal end of the elongated body. The proximal end of the elongated body can be connected to the flow chamber in any suitable manner. For example, in some cases, the proximal end of the elongated body is connected to the flow chamber in a liquid-tight and / or air-tight manner. In this case, fluid and / or air will not enter or leave the flow chamber or nozzle at the interface between the two components. In some cases, the proximal end may include a gasket or O-ring for maintaining a liquid-tight and / or airtight seal. In other cases, the proximal end includes a groove for accommodating an O-ring. The nozzle may include any suitable device that can be operably fixed to the flow chamber, including but not limited to a clamp, a magnet, a latch, a notch, a countersunk hole, a countersunk hole, a groove, a pin, a tether, a hinge, a non-permanent adhesive, or a combination thereof.

[0036] The proximal opening may have any suitable cross-sectional shape, wherein the target cross-sectional shape includes, but is not limited to, a straight cross-sectional shape (e.g., square, rectangular, trapezoidal, triangular, hexagonal, etc.), a curved cross-sectional shape (e.g., circular, elliptical), and an irregular shape (e.g., a parabolic bottom coupled with a flat top). In some cases, the opening has a circular cross-sectional shape. In this case, the opening may have any suitable diameter, such as a diameter ranging from 100 µm to 300 µm, such as 125 µm to 275 µm, such as 150 µm to 250 µm, such as 175 µm to 225 µm, such as 190 µm to 210 µm, such as 195 µm to 205 µm, including 199 µm to 201 µm. In some embodiments, the diameter of the opening is 200 µm.

[0037] The target nozzle also includes a distal opening for emitting droplets. The distal opening may have any suitable cross-sectional shape, wherein the target cross-sectional shape includes, but is not limited to, a straight cross-sectional shape (e.g., square, rectangular, trapezoidal, triangular, hexagonal, etc.), a curved cross-sectional shape (e.g., circular, elliptical), and an irregular shape (e.g., a parabolic bottom coupled to a planar top). In some cases, the opening has a circular cross-sectional shape. In this case, the distal end may have any suitable diameter, such as a diameter ranging from 100 µm to 300 µm, such as 125 µm to 275 µm, such as 150 µm to 250 µm, such as 175 µm to 225 µm, such as 190 µm to 210 µm, such as 195 µm to 205 µm, including 199 µm to 201 µm. In certain embodiments, the diameter of the opening is 200 µm.

[0038] The subject nozzle also includes a channel extending from the proximal end to the distal end in the elongated body, through which liquid can be transported. The channel can have any suitable cross-sectional shape, wherein the target cross-sectional shape includes but is not limited to a straight cross-sectional shape (e.g., square, rectangular, trapezoidal, triangular, hexagonal, etc.), a curved cross-sectional shape (e.g., circular, elliptical), and an irregular shape (e.g., a parabolic bottom coupled to a plane top). In some cases, the channel has a circular cross-sectional shape. In this case, the channel can be cylindrical. The channel can have any suitable diameter, such as a diameter range of 100 µm-300 µm, such as 125 µm-275 µm, such as 150 µm-250 µm, such as 175 µm-225 µm, such as 190 µm-210 µm, such as 195 µm-205 µm, including 199 µm-201 µm. In certain embodiments, the diameter of the channel is 200 µm. The diameter of the channel is constant or variable over the entire length. In some cases, the diameter of the passage is constant throughout the length of the elongated body. In some such cases, the proximal opening, the distal opening, and the entire length of the passage have the same diameter.

[0039] The target nozzle includes an air inlet radially disposed at the proximal end of the elongated body. The so-called "air inlet" refers to a portal including one or more paths through which gas from an external source can be delivered to the interior of the nozzle. The air inlet includes a radial air flow path that can supply air to the channel. The so-called "radial air flow path" refers to a passage disposed inside the air inlet, through which gas flows to the channel. The air inlet and its constituent radial air flow paths are arranged radially relative to an imaginary axis running through the length of the elongated body. The air inlet includes any suitable number of radial air flow paths. In some embodiments, the air inlet includes a single radial air flow path. In other embodiments, the air inlet includes multiple radial air flow paths. In such embodiments, the number of radial air flow paths can range from 2 to 6, including 2 to 5. In some cases, the air inlet includes 4 radial air flow paths. The radial air flow paths can have any suitable shape. In some instances, each radial air flow path is wedge-shaped. In this case, the radial air flow path opening facing the external environment is wider, while the radial air flow path opening facing the channel is relatively narrow. Each radial air flow path may have any suitable length. The length of each radial air flow path may be conceptualized as the radius measured from an imaginary axis running through the length of the elongated body to the external environment (i.e., the end of the path). For example, the radial air flow path may have a radius in the range of 150 µm-350 µm, such as 175 µm-325 µm, such as 200 µm-300 µm, such as 225 µm-275 µm, such as 245 µm-255 µm, including 249 µm-251 µm. In some cases, the radial air flow path includes a radius of 250 µm. Each radial air flow path may have any suitable height, i.e., the measured height from the top surface of the radial air flow path to the bottom surface of the radial air flow path, wherein the top surface is part of a plane closer to the proximal end of the elongated body, and the bottom surface is part of a plane closer to the distal end of the elongated body. In some cases, the radial air flow path has a height ranging from 100 µm to 300 µm, such as 125 µm to 275 µm, such as 150 µm to 250 µm, such as 175 µm to 225 µm, such as 190 µm to 210 µm, such as 195 µm to 205 µm, including 199 µm to 201 µm. In a selected example, the radial air flow path includes a height of 200 µm.

[0040] In some embodiments, the air inlet is operably connected to one or more gas ducts. The so-called "gas duct" refers to a channel through which gas can be provided to the air inlet or constitutes a radial air flow path. The radial air flow path may be connected to the gas duct. For example, each radial air flow path may have a shape complementary to the shape of the gas duct, so that the gas duct can be releasably fixed to the radial air flow path. In some embodiments, the gas duct is gaseously connected to the radial air flow path by a press fit. In other cases, the gas duct may be fixed to the radial air flow path by a clamp, a magnet, a latch, a notch, a countersunk hole, a countersunk hole, a groove, a pin, a tether, a hinge, a non-permanent adhesive or a combination thereof.

[0041] The subject nozzle can be made of any suitable material. In some instances, the nozzle includes one or more polymer materials. For example, in some embodiments, the nozzle includes one or more hard plastic materials, such as polycarbonate, polyvinyl chloride (PVC), polyurethane, polyether, polyamide, polyimide and other polymer plastic materials. Examples of polymer materials include acrylonitrile-butadiene-styrene (ABS), polylactic acid (PLA), acrylic-styrene-acrylonitrile (ASA), polyethylene terephthalate (PET), ethylene glycol-modified polyethylene terephthalate (PETG), polyaryletherketone (PAEK), polyetherimide (PEI), polycarbonate (PC), polypropylene (PP), aliphatic polyamide (PPA), polyoxymethylene (POM), polymethyl methacrylate (PMMA), polybutylene terephthalate (PBT), polyphenylene sulfone (PPSU), polyetheretherketone (PEEK) and nylon and its composite materials and mixed materials. In some cases, the nozzle is made of PEEK. In some embodiments, the nozzle comprises a glass-filled polymer (i.e., having glass fibers in a polymer matrix). In these embodiments, any suitable polymer (such as the above-mentioned polymers) can be combined with glass fibers to form a glass-filled polymer. In some examples, the nozzle comprises one or more metal parts, including aluminum, titanium, brass, iron, lead, nickel, steel (such as stainless steel), copper, tin, and combinations and alloys thereof.

[0042] Figure 2A 2 shows a nozzle according to some embodiments of the present invention. The nozzle 200 comprises an elongated body 201 (only a partial view is shown) having a proximal opening 202. Figure 2A In the embodiment, a portion of the elongated body 201 is absent, so that the channel 203 is visible. The nozzle 200 also includes an air inlet 204, which includes a wedge-shaped radial air flow path 205. The radial air flow path 205 can accommodate Figure 2B A gas conduit 206 for supplying gas to the channel 203 is shown.

[0043] Particle Sorter

[0044] Various aspects of the present invention further include a particle sorter. The target particle sorter includes a nozzle having an elongated body and an air inlet (e.g., as described above). A "particle sorter" is any device for flow cytometric sorting of particles (e.g., microspheres, cells, etc.). The subject particle sorter may or may not include a piezoelectric actuator. In an embodiment, the nozzle is not operably fixed to the piezoelectric actuator. By "not operably fixed" is meant that there is no piezoelectric actuator near the flow chamber or the nozzle, which would generate fluid disturbances sufficient to produce droplet breakup when in operation. In an embodiment where there is no piezoelectric actuator, droplets are formed by combining a dispersed phase including a sheath fluid (optionally containing particles) received from the flow chamber with a continuous phase including a gas. The continuous phase (e.g., air) is immiscible with the dispersed phase, and the interaction of the two phases at the nozzle air inlet is sufficient to produce droplets at the distal end of the nozzle. In addition, a version of the subject particle sorter can adjust the size / volume of the droplets produced by changing the amount of pressure applied to the nozzle at the air inlet. In selected cases, the size / volume of the droplets produced can be adjusted without changing the nozzle or otherwise altering the diameter of the nozzle.

[0045] In addition to the above-mentioned nozzle, the subject particle sorter also includes a flow chamber having a flow channel for transporting particles in a flow stream passing through the flow channel from a proximal inlet to a distal outlet. As described in the present invention, "flow chamber" in its conventional sense refers to a component for transporting particles in a sheath fluid and comprising a flow channel having a liquid flow stream. In an embodiment, the subject flow chamber includes a small glass tube. The target small glass tube includes a container having a channel running through it. The flow stream may include a liquid sample injected from a sample tube. The target flow chamber includes a light-permeable flow channel. In some cases, the flow chamber includes a transparent material (e.g., quartz) that allows light to pass. Any suitable flow chamber that propagates a fluid sample to a sample interrogation area can be used as the flow chamber described in the present invention, wherein in some embodiments, the flow chamber is a cylindrical flow chamber or a truncated conical flow chamber or a flow chamber including a proximal cylindrical portion defining a longitudinal axis and a distal truncated conical portion terminating in a flat surface (having an orifice transverse to the longitudinal axis).

[0046] In some embodiments, the particle sorter includes a sample fluid source. The sample fluid source can be any reservoir or container suitable for containing a sample fluid (e.g., having hard or flexible walls). The sample fluid container can have a volume in the range of 1 mL-100 mL; for example, the volume range of the container can be 1 mL-90 mL, 1 mL-80 mL, 1 mL-70 mL, 1 mL-60 mL, 1 mL-50 mL, 1 mL-40 mL, 1 mL-30 mL, 1 mL-20 mL, or 1 mL-10 mL.

[0047] In some embodiments, the particle sorter includes a sheath fluid source. The sheath fluid source can be any reservoir or container suitable for containing a sample fluid (e.g., having rigid or flexible walls). In some embodiments, the sheath fluid source is fluidically coupled to an input end of a flow chamber. The sheath fluid container can have a volume in the range of 1 L-100 L; for example, the volume of the container can range from 1 L-90 L, 1 L-80 L, 1 L-70 L, 1 L-60 L, 1 L-50 L, 1 L-40 L, 1 L-30 L, 1 L-20 L, or 1 L-10 L.

[0048] In some embodiments, the flow chamber includes an inlet that can provide a sample from a sample fluid source to the flow chamber. The inlet can be an orifice located on the wall of the inner cavity, or a conduit located at the proximal end of the inner cavity. If the inlet is an orifice located on the wall of the inner cavity, the inlet orifice can be any suitable shape, wherein the target cross-sectional shape includes, but is not limited to: a straight cross-sectional shape (e.g., square, rectangular, trapezoidal, triangular, hexagonal, etc.), a curved cross-sectional shape (e.g., circular, elliptical, etc.) and an irregular shape (e.g., a parabolic bottom coupled to a flat top). In some embodiments, the inlet has a circular orifice. The size of the inlet orifice can vary depending on the shape, and in some cases, its opening ranges from 0.1 mm to 5.0 mm, such as 0.2 to 3.0 mm, such as 0.5 mm to 2.5 mm, such as 0.75 mm to 2.25 mm, such as 1 mm to 2 mm, including 1.25 mm to 1.75 mm, such as 1.5 mm.

[0049] In some cases, the injection port is a conduit at the proximal end of the lumen of the flow chamber. For example, the injection port can be a conduit having an injection port orifice in line with the flow chamber orifice. If the injection port is a conduit in line with the flow chamber orifice, the cross-sectional shape of the injection tube can be any suitable shape, wherein the target cross-sectional shape includes, but is not limited to: a straight cross-sectional shape (e.g., square, rectangular, trapezoidal, triangular, hexagonal, etc.), a curved cross-sectional shape (e.g., circular, elliptical, etc.) and an irregular shape (e.g., a parabolic bottom coupled to a flat top). The conduit orifice can vary in shape, and in some cases, its opening range is 0.1 mm-5.0 mm, such as 0.2-3.0 mm, such as 0.5 mm-2.5 mm, such as 0.75 mm-2.25 mm, such as 1 mm-2 mm, including 1.25 mm-1.75 mm, such as 1.5 mm. The shape of the injection port tip can be the same or different from the cross-sectional shape of the sample injection tube. For example, the injection port orifice may include a beveled tip having a bevel angle in the range of 1°-10°, such as 2°-9°, such as 3°-8°, such as 4°-7°, including a bevel angle of 5°.

[0050] In some embodiments, the flow chamber further comprises a sheath fluid injection port that can provide sheath fluid from a sheath fluid source to the flow chamber. In embodiments, the sheath fluid injection system can provide a sheath fluid flow to the inner cavity of the flow chamber, for example, to generate a layered sheath fluid flow path around the sample flow path together with the sample. Depending on the desired flow characteristics, the rate of sheath fluid delivered to the flow chamber chamber can be 25 μL / sec-2500 μL / sec, for example 50 μL / sec-1000 μL / sec, including 75 μL / sec or more-750 μL / sec.

[0051] In some embodiments, the sheath injection port is an orifice on the inner cavity wall. The sheath injection port orifice can be in any suitable shape, wherein the target cross-sectional shape includes but is not limited to a straight cross-sectional shape (e.g., square, rectangular, trapezoidal, triangular, hexagonal, etc.), a curved cross-sectional shape (e.g., circular, elliptical), and an irregular shape (e.g., a parabolic bottom coupled with a flat top). The size of the injection port orifice can vary depending on the shape, and in some cases, its opening range is 0.1 mm-5.0 mm, such as 0.2 mm-3.0 mm, such as 0.5 mm-2.5 mm, such as 0.75 mm-2.25 mm, such as 1 mm-2 mm, including 1.25 mm-1.75 mm, such as 1.5 mm.

[0052] In some embodiments, the system further includes a pump that is connected to the flow chamber fluid for causing the flow stream to propagate through the flow chamber. Any suitable fluid pump scheme can be used to control the flow stream to flow through the flow chamber. In some cases, the system includes a peristaltic pump, such as a peristaltic pump with a pulse dampener. The pump in the subject system causes the fluid to flow through the flow chamber at a rate suitable for multi-photon counting of light from the sample in the flow stream. For example, the system may include a pump that causes the sample to flow through the flow chamber at the following rate: 1 nL / min-500 nL / min, such as 1 nL / min-250 nL / min, 1 nL / min-100 nL / min, 2nL / min-90 nL / min, 3 nL / min-80 nL / min, 4 nL / min-70 nL / min, 5 nL / min-60 nL / min, including 10 nL / min-50 nL / min. In certain embodiments, the flow rate of the flow stream is 5 nL / min-6 nL / min.

[0053] The flow stream in the flow chamber can be irradiated with light from a light source at an interrogation point. The flow stream may include a liquid sample injected from a sample tube. In certain embodiments, the flow stream may include a narrow and fast-flowing liquid stream, which is arranged in a manner that allows linearly separated particles transported in the liquid stream to be separated from each other in a single column. The "interrogation point" discussed in the present invention refers to the area in the flow chamber where particles are irradiated with light from a light source (e.g., for analysis). The size of the interrogation point can vary as needed. For example, where 0 μm represents the axis of light emitted by the light source, the interrogation point can range from -100 μm to 100 μm, such as -50 μm to 50 μm, such as -25 μm to 40 μm, including -15 μm to 30 μm.

[0054] The far end of the flow chamber is operably connected to the proximal end of the nozzle so that the nozzle and the flow chamber form a liquid receiving relationship. The proximal end of the elongated body can be connected to the flow chamber in any suitable manner. For example, in some cases, the proximal end of the elongated body is connected to the flow chamber in a liquid-tight and / or airtight manner. In this case, fluid and / or air will not enter or leave the flow chamber or nozzle at the interface between the two components. In some cases, the proximal end may include a gasket or O-ring for maintaining a liquid-tight and / or airtight seal. In other cases, the proximal end includes a groove for accommodating an O-ring. The nozzle may include any suitable device that can be operably fixed to the flow chamber, including but not limited to a clamp, a magnet, a latch, a notch, a countersunk hole, a countersunk hole, a groove, a pin, a tether, a hinge, a non-permanent adhesive or a combination thereof.

[0055] In some cases, the particle sorter includes a compressed gas source. The compressed gas source can be any device that produces and / or stores gas under pressure. In some embodiments, the compressed gas source is a gas compressor. In other cases, the compressed gas source can be a gas tank that stores gas under pressure. Any suitable gas can be used. The target gas includes, but is not limited to, O2, CO2, N2, etc. and combinations thereof. In some cases, the gas is ambient air from the surrounding environment. In this case, the gas can be purified to remove any particles that may potentially contaminate the sample being sorted. For example, the compressed gas source can be connected in a gaseous manner to a compressed air filter that can be used to remove particles. In some embodiments, the target filter can filter particles ranging in size from 1 micron to 100 microns. In some cases where the compressed gas source is a gas tank, the gas stored in the gas tank can be purified. The compressed gas source can supply gas to a nozzle having any suitable pressure. For example, in selected versions, the compressed gas source can produce gas in a pressure range of 2 kPa-10 kPa. The compressed gas source may be adjustable so that the pressure of the gas received at the gas inlet may be varied as desired (e.g., to produce droplets of different sizes). In some cases where the compressed gas source is a gas storage tank, a pressure regulator may be used to adjust the pressure. In cases where the compressed gas source is a gas compressor, the activity of the compressor may be adjusted to adjust the gas pressure produced thereby.

[0056] In some embodiments, the particle sorter includes one or more gas conduits. As described above, the gas conduit is a channel through which gas can be provided to the air inlet or the subject nozzle to form a radial air flow path. In some cases, the gas conduit can operably connect a compressed gas source to the air inlet. Any suitable number of gas conduits can be used. In some cases, the particle sorter includes a single gas conduit that connects a compressed gas source to a single radial air flow path in the air inlet in a gaseous manner. In other examples, the particle sorter includes multiple gas conduits. In some such examples, the number of gas conduits in the multiple gas conduits ranges from 2-6, including 2-5. In some cases, the particle sorter includes 4 gas conduits. The gas conduit can have any suitable cross-sectional shape, wherein the target cross-sectional shape includes but is not limited to a straight cross-sectional shape (e.g., square, rectangular, trapezoidal, triangular, hexagonal, etc.), a curved cross-sectional shape (e.g., circular, elliptical), and an irregular shape (e.g., a parabolic bottom coupled to a flat top). In some embodiments, the gas conduit has a rectangular cross-sectional shape.

[0057] The gas conduit may be made of any suitable material. In some instances, the gas conduit includes one or more polymer materials. For example, in some embodiments, the gas conduit includes one or more rigid plastic materials, such as polycarbonate, polyvinyl chloride (PVC), polyurethane, polyether, polyamide, polyimide and other polymer plastic materials. Examples of polymer materials include acrylonitrile-butadiene-styrene (ABS), polylactic acid (PLA), acrylic-styrene-acrylonitrile (ASA), polyethylene terephthalate (PET), ethylene glycol-modified polyethylene terephthalate (PETG), polyaryletherketone (PAEK), polyetherimide (PEI), polycarbonate (PC), polypropylene (PP), aliphatic polyamide (PPA), polyoxymethylene (POM), polymethyl methacrylate (PMMA), polybutylene terephthalate (PBT), polyphenylene sulfone (PPSU), polyetheretherketone (PEEK) and nylon and composite materials and mixed materials thereof. In some cases, the gas conduit is made of PEEK. In some embodiments, the gas conduit comprises a glass-filled polymer (i.e., having glass fibers in a polymer matrix). In these embodiments, any suitable polymer (such as the above-mentioned polymers) can be combined with glass fibers to form a glass-filled polymer. In some examples, the gas conduit comprises one or more metal components, including aluminum, titanium, brass, iron, lead, nickel, steel (such as stainless steel), copper, tin, and combinations and alloys thereof.

[0058] Figure 3 1 shows a view of a particle sorter 300 according to some embodiments of the present invention. Figure 3 As shown, the particle sorter 300 includes a flow chamber 301. In addition, a titanium hose barb 302 is provided, through which a fluid is provided to the flow chamber, thereby generating a laminar flow. Particles passing through the flow chamber 301 are irradiated by a laser beam at an irradiation point 303. The distal end of the flow chamber 301 is operably connected to the proximal end of a nozzle 304. The particle sorter 300 further includes a gas conduit 305, through which compressed gas is provided to the channel of the nozzle 304. Supplying gas to the channel through the gas inlet at the proximal end of the nozzle is sufficient to precipitate the generated droplets at the distal end of the nozzle.

[0059] In an embodiment, the particle sorter further includes a processor operably connected to a compressed gas source. The target processor can adjust the gas pressure provided to the inlet. For example, the subject processor can be operated in conjunction with programmable logic, and the programmable logic can be implemented in hardware, software, firmware, or any combination thereof to adjust the gas pressure provided to the inlet. For example, in the case where the programmable logic is implemented in software, the pressure regulation can be implemented at least in part by a computer-readable data storage medium including a program code, and the program code includes instructions that can adjust the gas pressure provided to the inlet when executed. For example, in the case where the compressed gas source is a gas tank, the processor can be operably connected to a regulator associated with the gas tank, so that the desired pressure level can be applied to the inlet of the nozzle. In a version where the compressed gas source is a gas compressor, the processor can be operably connected to the gas compressor so that the processor controls the activity of the compressor and reaches the desired pressure level. In some instances, the processor can increase the pressure to reduce the size of the droplets, and can reduce the pressure to increase the size of the droplets.

[0060] In an embodiment, a particle sorter includes a droplet deflector that can transfer droplets containing analyzed cells from a droplet stream generated by a flow stream originating from a flow nozzle to a receiving location. Using a droplet deflector, the transfer of target droplets to a receiving location can be achieved by electrostatically charging the droplets and deflecting the charged droplets from the flow stream by applying an external electrostatic field. Such an electrostatic field can be generated by a deflection plate disposed near the flow stream. The term "deflection" or "deflected" as used in the present invention refers to electrostatically deflecting target droplets from a flow stream of analyzed droplets, so that cells can be identified and tracked in the flow stream, and only droplets in the flow stream that include target cells will be transferred and collected by a collection container. In some instances, the particle sorter includes a deflection plate that can deflect a single droplet into each collection container.

[0061] The deflection plates in the target sorting block can be configured according to the type of cells to be sorted, the sorting rate, the applied voltage on the cells, and the number of components to be sorted in the sample. In an embodiment, the length of a suitable deflection plate can range from 5 mm to 100 mm, such as 6 mm to 90 mm, such as 7 mm to 80 mm, such as 8 mm to 70 mm, such as 9 mm to 60 mm, including 10 mm to 50 mm. The width of the deflection plate can range from 1 mm to 25 mm, such as 2 mm to 20 mm, such as 3 mm to 15 mm, including 5 mm to 10 mm. The distance between each deflection plate can vary according to the applied voltage and the size of the particles to be sorted in the flow stream. In certain embodiments, the distance between each deflection plate can be 1 mm or more, such as 2 mm or more, such as 3 mm or more, such as 4 mm or more, such as 5 mm or more, including 10 mm or more. For example, the distance between each deflector plate can range from 1 mm to 25 mm, such as 2 mm to 22.5 mm, such as 3 mm to 20 mm, such as 4 mm to 17.5 mm, including 5 mm to 15 mm. The deflector plates can also be at an angle of 15° to 75°, such as 20° to 70°, such as 25° to 65°, including 30° to 60°.

[0062] The voltage applied to the deflection plates for transferring charged particles may be 10 mV or more, such as 25 mV or more, such as 50 mV or more, such as 100 mV or more, such as 250 mV or more, such as 500 mV or more, such as 750 mV or more, such as 1000 mV or more, such as 2500 mV or more, such as 5000 mV or more, including 10000 mV or more. In some embodiments, the voltage applied to the deflection plates ranges from 0.001 V to 6000 V, including 0.001 V to 5000 V, such as 0.01 V to 4000 V, such as 0.1 V to 3000 V, such as 1 V to 2000 V, such as 5 V to 1500 V, such as 10 V to 1000 V, such as 25 V to 750 V, including 100V to 500 V.

[0063] The deflector plate can transfer particles from the flow stream to a receiving position downstream of the deflector plate. In an embodiment, the deflector plate can transfer each particle to a certain angle, and the angle can change. In certain embodiments, the deflector plate can deflect each particle from the longitudinal axis of the flow stream by an angle of 0.5° or more, such as 1° or more, such as 1.5° or more, such as 2° or more, such as 2.5° or more, such as 3° or more, such as 5° or more, such as 7.5° or more, including deflecting each particle from the longitudinal axis of the flow stream by an angle of 10° or more. For example, each particle can be deflected from the longitudinal axis of the flow stream by an angle of 0.1°-30°, such as 0.5°-25°, such as 1°-20°, such as 2°-15°, including 5°-10°.

[0064] Any suitable deflector plate scheme may be used to deflect particles in a flow stream, including but not limited to the cell sorting deflector plates described in U.S. Pat. Nos. 3,960,449; 4,347,935; 4,667,830; 5,245,318; 5,464,581; 5,483,469; 5,602,039; 5,643,796; 5,700,692; 6,372,506 and 6,809,804 and 7,880,108, the disclosures of which are incorporated herein by reference in their entirety. In certain embodiments, the deflector plates include charged plates for sorting cells in a flow stream used in a flow cytometer system, such as a BD Biosciences FACS Canto TM , BD Biosciences FACSVantage TM BD Biosciences FACSort TM , BD Biosciences FACSAria TM BD Biosciences FACSCount TM BD Biosciences FACScan TM 、BD Biosciences FACSCalibur TM System, BD Biosciences Influx TM Cell sorter, etc.

[0065] The target particle sorter may further include a light source that can irradiate particles passing through the flow chamber at the interrogation point. The light source described in the present invention can use any suitable light source. In some embodiments, the light source is a laser. In an embodiment, the laser can be any suitable laser, such as a continuous wave laser. For example, the laser can be a diode laser, such as an ultraviolet diode laser, a visible diode laser, and a near-infrared diode laser. In other embodiments, the laser can be a helium-neon (HeNe) laser. In some instances, the laser is a gas laser, such as a helium-neon laser, an argon laser, a krypton laser, a xenon laser, a nitrogen laser, a CO2 laser, a CO laser, an argon-fluorine (ArF) excimer laser, a krypton-fluorine (KrF) excimer laser, a xenon-chlorine (XeCl) excimer laser, or a xenon-fluorine (XeF) excimer laser, or a combination thereof. In other instances, the subject flow cytometer includes a dye laser, such as a stilbene, coumarin, or rhodamine laser. In other examples, the target laser includes a metal vapor laser, such as a helium cadmium (HeCd) laser, a helium mercury (HeHg) laser, a helium selenium (HeSe) laser, a helium silver (HeAg) laser, a strontium laser, a neon copper (NeCu) laser, a copper laser, or a gold laser, and combinations thereof. In other examples, the subject flow cytometer includes a solid-state laser, such as a ruby ​​laser, a Nd:YAG laser, a NdCrYAG laser, an Er:YAG laser, a Nd:YLF laser, a Nd:YVO4 laser, a Nd:YCa4O(BO3)3 laser, a Nd:YCOB laser, a titanium sapphire laser, a thulium YAG laser, an ytterbium YAG laser, a Yb2O3 laser, or a cerium-doped laser, and combinations thereof.

[0066] According to some embodiments, the laser light source may further include one or more optical adjustment elements. In some embodiments, the optical adjustment element is located between the light source and the flow chamber and may include any device capable of changing the spatial width of the irradiation or some other characteristic of the light source irradiation (such as irradiation direction, wavelength, beam width, beam intensity and focus). The optical adjustment scheme may include any suitable device for adjusting one or more characteristics of the light source, including but not limited to lenses, reflectors, filters, optical fibers, wavelength separators, pinholes, slits, collimation schemes and combinations thereof. In some embodiments, the target flow cytometer includes one or more focusing lenses. In one example, the focusing lens can be a magnifying lens. In other embodiments, the target flow cytometer includes an optical fiber.

[0067] Where the optical adjustment element is movable, the optical adjustment element may be movable continuously or in discrete intervals, for example in increments of 0.01 µm or greater, for example 0.05 µm or greater, for example 0.1 µm or greater, for example 0.5 µm or greater, for example 1 µm or greater, for example 10 µm or greater, for example 100 µm or greater, for example 500 µm or greater, for example 1 mm or greater, for example 5 mm or greater, for example 10 mm or greater, including 25 mm or greater increments.

[0068] Any displacement scheme can be used to move the optical adjustment element structure, such as coupling with a movable support table, or directly using a motor-driven translation stage, a guide rod translation assembly, a gear-type translation device, such as a stepper motor, a servo motor, a brushless motor, a brushed DC motor, a micro stepper drive motor, a high-resolution stepper motor and other types of motors.

[0069] The light source can be positioned at any suitable distance from the flow chamber, for example, the light source is separated from the flow chamber by 0.005 mm or more, for example, 0.01 mm or more, for example, 0.05 mm or more, for example, 0.1 mm or more, for example, 0.5 mm or more, for example, 1 mm or more, for example, 5 mm or more, for example, 10 mm or more, for example, 25 mm or more, including a distance of 100 mm or more. In addition, the light source can be positioned at any suitable angle relative to the flow chamber, for example, an angle in the range of 10°-90°, for example, 15°-85°, for example, 20°-80°, for example, 25°-75°, including 30°-60°, for example, 90°.

[0070] In some embodiments, the target light source includes multiple lasers to provide discrete irradiation lasers to the flow stream, such as 2 lasers or more, such as 3 lasers or more, such as 4 lasers or more, such as 5 lasers or more, such as 10 lasers or more, including 15 lasers or more. Depending on the wavelength of light required to irradiate the flow stream, the specific wavelength of each laser can range from 200 nm to 1500 nm, such as 250 nm to 1250 nm, 300 nm to 1000 nm, 350 nm to 900 nm, including 400 nm to 800 nm. In some embodiments, the target laser may include one or more of a 405 nm laser, a 488 nm laser, a 561 nm laser, and a 635 nm laser.

[0071] The target particle sorter may further include one or more particle modulated light detectors that can detect particle modulated light intensity data. In some embodiments, the particle modulated light detector includes one or more forward scattered light detectors that can detect forward scattered light. For example, the subject particle sorter may include 1 forward scattered light detector or multiple forward scattered light detectors, such as 2 or more, such as 3 or more, such as 4 or more, including 5 or more. In some embodiments, the particle sorter includes 1 forward scattered light detector. In other embodiments, the particle sorter includes 2 forward scattered light detectors.

[0072] The forward scattered light detector described in the present invention may use any suitable detector for detecting the collected light. The target detector may include, but is not limited to, an optical sensor or detector, such as an active pixel sensor (APS), an avalanche photodiode, an image sensor, a charge coupled device (CCD), an enhanced charge coupled device (ICCD), a light emitting diode, a photon counter, a bolometer, a pyroelectric detector, a photoresistor, a photovoltaic cell, a photodiode, a photomultiplier tube (PMT), a phototransistor, a quantum dot photoconductor or a photodiode and a combination thereof, and other detectors. In some embodiments, a charge coupled device (CCD), a semiconductor charge coupled device (CCD), an active pixel sensor (APS), a complementary metal oxide semiconductor (CMOS) image sensor, or an N-type metal oxide semiconductor (NMOS) image sensor is used to measure the collected light. In some embodiments, the detector is a photomultiplier tube, for example, the active detection surface area of ​​each region ranges from 0.01 cm 2 -10 cm 2 (e.g. 0.05 cm 2 -9 cm 2 , for example 0.1 cm 2 -8 cm 2 , for example 0.5 cm 2 -7 cm 2 , including 1cm 2 -5 cm 2 ) of the photomultiplier tube.

[0073] In embodiments, the forward scattered light detector may measure light continuously or at discrete intervals. In some cases, the target detector measures the collected light continuously. In other cases, the target detector measures light at discrete intervals, such as every 0.001 ms, 0.01 ms, 0.1 ms, 1 ms, 10 ms, 100 ms (including 1000 ms), or at other intervals.

[0074] In additional embodiments, the one or more particle modulated light detectors may include one or more side scattered light detectors that can detect wavelengths of side scattered light (i.e., light refracted and reflected from the surface and internal structure of the particle). In some embodiments, the particle sorter includes a single side scattered light detector. In other embodiments, the particle sorter includes multiple side scattered light detectors, such as 2 or more, such as 3 or more, such as 4 or more, including 5 or more.

[0075] The side scattered light detector described in the present invention may use any suitable detector for detecting the collected light. The target detector may include, but is not limited to, an optical sensor or detector, such as an active pixel sensor (APS), an avalanche photodiode, an image sensor, a charge coupled device (CCD), an enhanced charge coupled device (ICCD), a light emitting diode, a photon counter, a bolometer, a pyroelectric detector, a photoresistor, a photovoltaic cell, a photodiode, a photomultiplier tube (PMT), a phototransistor, a quantum dot photoconductor or a photodiode and a combination thereof, and other detectors. In some embodiments, a charge coupled device (CCD), a semiconductor charge coupled device (CCD), an active pixel sensor (APS), a complementary metal oxide semiconductor (CMOS) image sensor, or an N-type metal oxide semiconductor (NMOS) image sensor is used to measure the collected light. In some embodiments, the detector is a photomultiplier tube, for example, the active detection surface area of ​​each region ranges from 0.01 cm 2 -10 cm 2 (e.g. 0.05 cm 2 -9 cm 2 , for example 0.1 cm 2 -8 cm 2 , for example 0.5 cm 2 -7 cm 2 , including 1cm 2 -5 cm 2 ) of the photomultiplier tube.

[0076] In an embodiment, the subject particle sorter further comprises a fluorescence detector that can detect one or more fluorescence wavelengths. In other embodiments, the particle sorter comprises a plurality of fluorescence detectors, such as 2 or more, such as 3 or more, such as 4 or more, 5 or more, 10 or more, 15 or more, including 20 or more.

[0077] The fluorescence detector described in the present invention may use any suitable detector for detecting the collected light. The target detector may include, but is not limited to, an optical sensor or detector, such as an active pixel sensor (APS), an avalanche photodiode, an image sensor, a charge coupled device (CCD), an enhanced charge coupled device (ICCD), a light emitting diode, a photon counter, a bolometer, a pyroelectric detector, a photoresistor, a photovoltaic cell, a photodiode, a photomultiplier tube (PMT), a phototransistor, a quantum dot photoconductor or a photodiode and a combination thereof, and other detectors. In some embodiments, a charge coupled device (CCD), a semiconductor charge coupled device (CCD), an active pixel sensor (APS), a complementary metal oxide semiconductor (CMOS) image sensor, or an N-type metal oxide semiconductor (NMOS) image sensor is used to measure the collected light. In some embodiments, the detector is a photomultiplier tube, for example, the active detection surface area of ​​each region ranges from 0.01 cm 2 -10 cm 2 (e.g. 0.05 cm 2 -9 cm 2 , for example 0.1 cm 2 -8 cm 2 , for example 0.5 cm 2 -7 cm 2 , including 1 cm 2 -5cm 2 ) of the photomultiplier tube.

[0078] When the subject particle sorter includes multiple fluorescence detectors, each fluorescence detector can be identical, or the collection of fluorescence detectors can be a combination of different types of detectors. For example, when the subject particle sorter includes two fluorescence detectors, in some embodiments, the first fluorescence detector is a CCD type device and the second fluorescence detector (or imaging sensor) is a CMOS type device. In other embodiments, the first and second fluorescence detectors are both CCD type devices. And in other embodiments, the first and second fluorescence detectors are both CMOS type devices. In other embodiments, the first fluorescence detector is a CCD type device and the second fluorescence detector is a photomultiplier tube (PMT). In other embodiments, the first fluorescence detector is a CMOS type device and the second fluorescence detector is a photomultiplier tube. And in other embodiments, the first and second fluorescence detectors are both photomultiplier tubes.

[0079] In embodiments of the present disclosure, the fluorescence detector may measure the collected light at one or more wavelengths (e.g., 2 or more wavelengths, 5 or more different wavelengths, 10 or more different wavelengths, 25 or more different wavelengths, 50 or more different wavelengths, 100 or more different wavelengths, 200 or more different wavelengths, 300 or more different wavelengths), including measuring the light emitted by the sample in the flow stream at 400 or more different wavelengths. In certain embodiments, two or more detectors in the particle sorter described herein may measure the same or overlapping wavelengths of the collected light.

[0080] In some embodiments, the target fluorescence detector can measure light collected within a certain wavelength range (e.g., 200 nm-1000 nm). In some embodiments, the target detector can collect a spectrum of light within a certain wavelength range. For example, a particle sorter may include one or more detectors that can collect one or more spectra of light within a wavelength range of 200 nm-1000 nm. In other embodiments, the target detector can measure light emitted by a sample in a flow stream at one or more specific wavelengths. For example, a particle sorter may include one or more detectors that can measure light at wavelengths of 450 nm, 518 nm, 519 nm, 561 nm, 578 nm, 605 nm, 607 nm, 625 nm, 650 nm, 660 nm, 667 nm, 670 nm, 668 nm, 695 nm, 710 nm, 723 nm, 780 nm, 785 nm, 647 nm, 617 nm, and any combination thereof. In some embodiments, one or more detectors can be paired with a specific fluorophore, such as a fluorophore used with a sample in a fluorescence analysis method.

[0081] In certain embodiments, the particle sorter includes one or more wavelength separators, which are located between the flow chamber and the particle modulated light detector. The term "wavelength separator" used in the present invention refers to an optical element in its conventional sense that can separate the light collected from the sample into a predetermined spectral range. In certain embodiments, the particle sorter includes a single wavelength separator. In other embodiments, the particle sorter includes a plurality of wavelength separators, such as 2 or more wavelength separators, such as 3 or more, such as 4 or more, such as 5 or more, such as 6 or more, such as 7 or more, such as 8 or more, such as 9 or more, such as 10 or more, such as 15 or more, such as 25 or more, such as 50 or more, such as 75 or more, including 100 or more wavelength separators. In certain embodiments, the wavelength separator can separate the light collected from the sample into a predetermined spectral range by allowing light with a predetermined spectral range to pass and reflecting light of one or more remaining spectral ranges. In other embodiments, the wavelength separator can separate the light collected from the sample into a predetermined spectral range by allowing light with a predetermined spectral range to pass and absorbing light of one or more remaining spectral ranges. In other embodiments, the wavelength separator can diffract the light collected from the sample into a predetermined spectral range in space. Each wavelength separator can be any suitable light separation scheme, such as one or more dichroic mirrors, bandpass filters, diffraction gratings, beam splitters or prisms. In some embodiments, the wavelength separator is a prism. In other embodiments, the wavelength separator is a diffraction grating. In some embodiments, the wavelength separator in the subject light detection system is a dichroic mirror.

[0082] Suitable flow cytometer systems may include, but are not limited to, those described in Ormerod (ed.), Flow Cytometry: A Practical Approach, Oxford University Press (1997); Jaroszeski et al. (eds.), Flow Cytometry Protocols, Methods in Molecular Biology, Vol. 91, Humana Press (1997); Practical Flow Cytometry, 3rd ed., Wiley-Liss (1995); Virgo et al. (2012), Annals of Clinical Biochemistry, January; 49 (pt. 1):17-28; Linden et al., Seminars in Thrombosis and Hemostasis, October 2004; 30(5):502-11; Alison et al., Journal of Pathology, December 2010; 222(4):335-344; and Herbig et al. (2007), Critical Reviews of Therapeutic Drug Delivery Systems, 24(3):203-255; the disclosures of the above references are incorporated herein by reference. In some cases, the flow cytometry system of interest comprises a BD Biosciences FACSCantoTM Flow cytometer, BD Biosciences FACSCanto TM II flow cytometer, BD Accuri TM Flow cytometer, BD Accuri TM C6 Plus flow cytometer, BD Biosciences FACSCelesta TM Flow cytometer, BD Biosciences FACSLyric TM Flow cytometer, BD Biosciences FACSVerse TM Flow cytometer, BD Biosciences FACSymphony TM Flow cytometer, BD Biosciences LSRFortessa TM Flow cytometer, BD Biosciences LSRFortessa TM X-20 flow cytometer, BD Biosciences FACSPresto TM Flow cytometer, BD Biosciences FACSVia TM Flow cytometer and BD Biosciences FACSCalibur TM Cell sorter, BD Biosciences FACSCount TM Cell sorter, BD Biosciences FACSLyric TM Cell sorter, BD Biosciences Via TM Cell Sorters, BD Biosciences Influx™ Cell Sorter, BD Biosciences Jazz™ Cell Sorter, BD Biosciences Aria™ Cell Sorter, BD Biosciences FACSAria™ II Cell Sorter, BD Biosciences FACSAria™ III Cell Sorter, BD Biosciences FACSAria™ Fusion Cell Sorter, and BD Biosciences FACSMelody™ Cell Sorter, BD Biosciences FACSymphony TM S6 cell sorter, etc.

[0083] In some embodiments, the subject system is a flow cytometric system, such as those described in U.S. Pat. Nos. 10,663,476; 10,620,111; 10,613,017; 10,605,713; 10,585,031; 10,578,542; 10,578,469; 10,481, 074; 10,302,545; 10,145,793; 10,113,967; 10,006,852; 9,952,076; 9,933,341; 9,726,527; 9,453,789; 9,200,334; 9,097,640; 9,09 No. 5,494; No. 9,092,034; No. 8,975,595; No. 8,753,573; No. 8,233,146; No. 8,140,300; No. 7,544,326; No. 7,201,875; No. 7,129,505; No. 6,821,740; No. 6,813,017; No. 6,809 ,804; No. 6,372,506; No. 5,700,692; No. 5,643,796; No. 5,627,040; No. 5,620,842; No. 5,602,039; No. 4,987,086; No. 4,498,766; the disclosures of the aforementioned U.S. patents are incorporated herein by reference in their entirety and become a part of the present invention.

[0084] Figure 4 A flow cytometry system 400 is shown in accordance with an illustrative embodiment of the invention. System 400 includes a flow cytometer 410, a controller / processor 490, and a memory 495. Flow cytometer 410 includes one or more excitation lasers 415a-415c, a focusing lens 420, a flow chamber 425, a forward scatter detector 430, a side scatter detector 435, a fluorescence collection lens 440, one or more beam splitters 445a-445g, one or more bandpass filters 450a-450e, one or more long pass ("LP") filters 455a-455b, and one or more fluorescence detectors 460a-460f.

[0085] The excitation lasers 415a-c emit light in the form of laser beams. Figure 4In the example system of FIG. 4 , the wavelengths of the laser beams emitted from the excitation lasers 415a-415c are 488 nm, 633 nm, and 325 nm, respectively. The laser beams are first directed through one or more beam splitters 445a and 445b. Beam splitter 445a transmits 488 nm light and reflects 633 nm light. Beam splitter 445b transmits ultraviolet light (light with a wavelength in the range of 10-400 nm) and reflects 488 nm and 633 nm light.

[0086] The laser beam is then directed to a focusing lens 420 which focuses the beam onto the portion of the fluid stream where the sample particles are located in a flow chamber 425. The flow chamber is the portion of the fluid system that directs particles in the stream (usually one at a time) to the focused laser beam for interrogation. The flow chamber may include a flow chamber in a benchtop cytometer or a nozzle tip in an air flow cytometer.

[0087] Light from the laser beam interacts with particles in the sample at various wavelengths by diffraction, refraction, reflection, scattering, and absorption and re-emission, depending on the characteristics of the particles, such as size, internal structure, and the presence or absence of one or more fluorescent molecules attached to or naturally present on or in the particles. Fluorescent emission, as well as diffracted, refracted, reflected, and scattered light, can be directed to one or more of a forward scatter detector 430 and a side scatter detector 435, and one or more fluorescence detectors 460a-460f, via one or more of beam splitters 445c-445g, bandpass filters 450a-450e, longpass filters 455a-455b, and fluorescence collection lens 440.

[0088] Fluorescence collection lens 440 collects light emitted by the interaction of the particles with the laser beam and directs it to one or more beam splitters and filters. Bandpass filters, such as bandpass filters 450a-450e, allow a narrow range of wavelengths to pass through the filter. For example, bandpass filter 450a is a 510 / 20 filter. The first number represents the center of the spectral band. The second number provides the range of the spectral band. Thus, a 510 / 20 filter extends 10 nm on each side of the center of the spectral band, or extends from 500 nm to 520 nm. Shortpass filters transmit light less than or equal to a specified wavelength. Longpass filters (such as longpass filters 455a-455b) transmit light greater than or equal to a specified wavelength. For example, longpass filter 455b, i.e., a 670 nm longpass filter, transmits light greater than or equal to 670 nm. Filters are typically selected to optimize the specificity of a detector for a particular fluorescent dye. The filter can cause the spectral band of light transmitted to the detector to be close to the emission peak of the fluorescent dye.

[0089] The forward scatter detector 430 is slightly offset from the axis of the direct beam through the flow chamber, detecting diffracted light and excitation light that mainly propagates through or around the particles in the forward direction. The intensity of the light detected by the forward scatter detector depends on the overall size of the particle. The forward scatter detector may include a photodiode. The side scatter detector 435 detects refracted light and reflected light from the surface and internal structure of the particle, and the refracted light and reflected light tend to increase with the increase of the complexity of the particle structure. One or more fluorescence detectors 460a-460f can be used to detect the fluorescence emission from the fluorescent molecules associated with the particles. The side scatter detector 435 and the fluorescence detector may include a photomultiplier tube. The signals detected at the forward scatter detector 430, the side scatter detector 435 and the fluorescence detector can be converted into electronic signals (voltage) by the detector. This data can provide information about the sample.

[0090] Those skilled in the art will appreciate that the flow cytometer according to the embodiments of the present invention is not limited to Figure 4 The flow cytometer shown, instead, may include any flow cytometer known in the art. For example, the flow cytometer may have any number of lasers, beam splitters, filters, and detectors of various wavelengths and various different configurations.

[0091] During operation, the flow cytometer is controlled by a controller / processor 490, and measurement data from the detector may be stored in a memory 495 and processed by the controller / processor 490. Although not explicitly shown, the controller / processor 490 is coupled to the detector to receive output signals therefrom, and may also be coupled to the electrical and electromechanical components of the flow cytometer 410 to control lasers, fluid flow parameters, etc. The system may also have input / output (I / O) capabilities 497. The memory 495, the controller / processor 490, and the I / O 497 may all be integral to the flow cytometer 410. In such embodiments, the display may also constitute part of the I / O capabilities 497 for presenting experimental data to a user of the cytometer 410. Alternatively, some or all of the memory 495 and the controller / processor 490 and the I / O capabilities may be part of one or more external devices (e.g., a general purpose computer). In some embodiments, some or all of the memory 495 and the controller / processor 490 may communicate wirelessly or wired with the cytometer 410. Controller / processor 490, together with memory 495 and I / O 497, performs various functions related to the preparation and analysis of flow cytometric experiments.

[0092] Figure 4The system shown includes six different detectors that detect fluorescence in six different bands (which may be referred to in the present invention as "filter windows" for a given detector), as defined by the configuration of filters and / or beam splitters in the beam path from the flow chamber 425 to each detector. Different fluorescent molecules in the set of fluorochromes used in a flow cytometer experiment will emit light in their own unique wavelength bands. In order to be generally consistent with the filter windows of the detectors, the specific fluorescent labels and their associated fluorescence emission bands used in the experiment can be selected. I / O 497 can receive data about a flow cytometer experiment, the experiment comprising a set of fluorescent labels and a plurality of cell populations with a plurality of labels, each cell population having a subset of the plurality of labels. I / O 497 can also receive biological data assigning one or more labels to one or more cell populations, label density data, emission spectral data, data assigning labels to one or more labels, and cytometer configuration data. Flow cytometer experiment data, such as label spectral characteristics and flow cytometer configuration data, can also be stored in memory 495. Controller / processor 490 can evaluate one or more assignment results of labels in the labels.

[0093] In some embodiments, the subject system is a particle sorting system, which can sort particles through a closed particle sorting module, such as the particle sorting module described in U.S. Patent Publication No. 2017 / 0299493 filed on March 28, 2017, the disclosure of which is incorporated by reference in this invention. In some embodiments, a sorting decision module having multiple sorting decision units is used to sort particles (e.g., cells) of a sample, such as the sorting decision module described in U.S. Patent Publication No. 2020 / 0256781 filed on December 23, 2019, the disclosure of which is incorporated by reference in this invention. In some embodiments, the sample component sorting system includes a particle sorting module having a deflection plate, such as described in U.S. Patent Publication No. 2017 / 0299493 filed on March 28, 2017, the disclosure of which is incorporated by reference in this invention.

[0094] Figure 5 5 is a functional block diagram of one example of a sorting control system, such as processor 500, for analyzing and displaying biological events. Processor 500 may implement various processes for controlling the graphical display of biological events.

[0095] The flow cytometer or sorting system 502 can collect biological event data. For example, the flow cytometer can generate flow cytometric event data (e.g., particle modulated light data). The flow cytometer 502 can provide the biological event data to the processor 500. A data communication channel can be provided between the flow cytometer 502 and the processor 500. The biological event data can be provided to the processor 500 through the data communication channel.

[0096] Processor 500 may receive biological event data from flow cytometer 502. The biological event data received from flow cytometer 502 may include flow cytometric event data. Processor 500 may provide a graphical display to display device 506, including a first graph of the biological event data. Processor 500 may further present a target region as a gate (e.g., a first gate) around a set of biological event data overlaid on the first graph as shown by display device 506. In some embodiments, the gate may be a logical combination of one or more target graphical regions plotted on a single parameter histogram or a bivariate graph. In some embodiments, the display may be used to display particle parameters or saturated detector data.

[0097] Processor 500 may further display the biological event data inside the gate on display device 506, which is different from other events in the biological event data outside the gate. For example, processor 500 may present the biological event data contained inside the gate in a color different from the color of the biological event data outside the gate. Display device 506 may be implemented as a monitor, a tablet computer, a smart phone, or other electronic device that presents a graphical interface.

[0098] The processor 500 may receive a door selection signal from a first input device identifying a door. For example, the first input device may be implemented as a mouse 510. The mouse 510 may send a door selection signal to the processor 500 to identify a door to be displayed on the display device 506 or manipulated through the display device (e.g., when the cursor is on or in the desired door, just click here). In some embodiments, the first device may be implemented as a keyboard 508 or other device for providing input signals to the processor 500, such as a touch screen, a stylus, an optical detector, or a voice recognition system. Some input devices may include multiple input functions. In such embodiments, each input function may be considered an input device. For example, if Figure 5 As shown, the mouse 510 may include a right mouse button and a left mouse button, each of which may generate a trigger event.

[0099] The triggering event may cause the processor 500 to change the manner in which the data is displayed and the portion of the data actually displayed on the display device 506, and / or provide input information for further processing, such as selecting a target population for particle sorting.

[0100] In some embodiments, processor 500 can detect when a gate selection is initiated by mouse 510. Processor 500 can further automatically modify the chart visualization to facilitate the gating process. The modification can be based on a particular distribution of bio-event data received by processor 500. In some embodiments, processor 500 expands the first gate to generate a second gate (e.g., as described above).

[0101] Processor 500 may be coupled to storage device 504. Storage device 504 may receive and store bio-event data from processor 500. Storage device 504 may also receive and store flow cytometric event data from processor 500. Storage device 504 may further allow retrieval of bio-event data, such as flow cytometric event data, by processor 500.

[0102] The display device 506 can receive display data from the processor 500. The display data can include a graph of the biological event data and gates outlining portions of the graph. The display device 506 can further change the information presented based on input information received from the processor 500, in combination with input information from the flow cytometer 502, the storage device 504, the keyboard 508, and / or the mouse 510.

[0103] In some embodiments, the processor 500 may generate a user interface to receive sample events for sorting. For example, the user interface may include a mechanism for receiving sample events or sample images. Sample events or images or sample gates may be provided before collecting event data for a sample, or based on an initial set of events for a portion of the sample.

[0104] Fig. 6A 600 (e.g., flow cytometer 502) according to one embodiment of the present invention. In some embodiments, particle sorter system 600 is a cell sorter system. Nozzle 603 emits liquid in a moving liquid column 608 at a distal end. In fluid conduit 601, sheath fluid 604 fluidically focuses sample fluid 606 containing particles 609 into the moving liquid column 608 (e.g., liquid stream). In the moving liquid column 608, particles 609 (e.g., cells) are arranged in a single file, traverse a monitoring area 611 (e.g., where a laser and liquid stream meet), and are irradiated by an irradiation source 612 (e.g., a laser). Nozzle 603 includes an air inlet 646, which includes a plurality of radial air flow paths (not shown). Air from a compressed gas source 645 is provided to the air inlet 646 through a gas conduit. The interaction between the sample fluid 606 and the air causes the moving liquid column 608 to break into a plurality of droplets 610 , some of which contain particles 609 .

[0105] During operation, the time at which a target particle (or target cell) traverses the monitoring zone 611 is determined by a detection station 614 (e.g., an event detector). The detection station 614 feeds into a timing circuit 628, which in turn feeds into a flash charging circuit 630. At the droplet breakoff point, the moving liquid column 608 can be flash charged, prompted by a timed droplet delay (Δt), to charge the target droplet. The target droplet may include one or more particles or cells to be sorted. The charged droplets may then be sorted by activating a deflection plate (not shown) to deflect the charged droplets into a container such as a collection tube or a multi-porous or micro-well sample plate, in which a well or micro-well may be associated with a particular target droplet. As Fig. 6A As shown, the droplets can be collected in a discharge container 638. When the target particle passes through the monitoring area 611, the detection system 616 (e.g., a droplet boundary detector) functions to automatically determine the phase of the droplet drive signal. An exemplary droplet boundary detector is described in U.S. Patent No. 7,679,039, which is incorporated herein by reference in its entirety. The detection system 616 allows the instrument to accurately calculate the position of each detected particle in the droplet. Fig. 6A In the example of FIG. 6 , the detection system 616 is connected to the processor 640 and provides information about the moving liquid column 608 to the processor 640. The processor 640 is operably connected to the compressed gas source 645 and can adjust the gas pressure generated by the compressed gas source 645 in response to the measurement results of the detection system and / or the input from the user. The size and volume of the droplet 610 can be adjusted in this way.

[0106] In some embodiments, the sorting electronics (e.g., detection system 616, detection station 614, and processor 640) may be coupled to a memory that can store detected events and sorting decisions based on the events. The sorting decision may be included in the event data of the particle. In some embodiments, the detection system 616 and the detection station 614 may be implemented as a single detection unit or may be coupled in a communication manner so that event measurements can be collected by the detection system 616 or the detection station 614 and provided to a non-collecting component.

[0107] Figure 6B is a schematic diagram of a particle sorting machine system according to an embodiment of the present invention. Figure 6B The particle sorter system 600 shown includes deflection plates 652 and 654. Charge can be applied through the liquid flow charging wires in the barbs. This forms a droplet stream 610 containing the particles to be analyzed 609. The particles can be irradiated with one or more light sources (such as lasers) to generate light scattering and fluorescence information. The particles can be detected by sorting electronics or other detection systems ( Figure 6BThe deflector plates 652 and 654 can be independently controlled to attract or repel charged droplets and direct the droplets to a destination collection container (e.g., one of 672, 674, 676, or 678). Figure 6B As shown, the deflection plates 652 and 654 can be controlled to direct the particles to flow along the first path 662 to the container 674 or along the second path 668 to the container 678. If the particle is not a target particle (e.g., does not show scattering or illumination information within a specified sorting range), the deflection plates can allow the particle to continue to flow along the flow path 664. Such uncharged droplets can enter a waste container via an aspirator 670 or the like.

[0108] Sorting electronics may be included to initiate measurement collection, receive the fluorescence signal from the particles, and determine how to adjust the deflection plates to achieve particle sorting. Figure 6B Example implementations of the illustrated embodiments include BD FACSAria™ series flow cytometers available from BD Biosciences (Franklin Lakes, NJ).

[0109] Particle sample sorting method

[0110] As described above, various aspects of the present invention also include a particle sample sorting method. The target method includes introducing a particle sample into a cell sorter (the cell sorter includes a flow chamber that can transport particles in a flow stream and a nozzle), and performing flow cytometry sorting on the particle sample. As described above, the subject nozzle includes an elongated body, the elongated body includes a proximal opening that engages with the flow chamber to establish a liquid receiving relationship, a distal opening for emitting droplets, and a channel that can transport liquid from the proximal end to the distal end through the elongated body. In addition, the nozzle of the present invention includes an air inlet radially arranged at the proximal end of the elongated body, wherein the air inlet includes a radial air flow path that can supply air to the channel.

[0111] In some embodiments, the method includes adjusting the pressure of the gas provided to the gas inlet using a compressed gas source. In such embodiments, a user may input a desired pressure or droplet size / volume, for example using a user interface device, such as described above with respect to Figure 5 The input value may be received by a processor, which may then adjust the compressed gas source accordingly.

[0112] In some cases, the sample analyzed in the present method is a biological sample. The term "biological sample" in its conventional sense refers to a subset of a whole organism, plant, fungus or animal tissue, cell or component, which in some cases may be present in blood, mucus, lymph, synovial fluid, cerebrospinal fluid, saliva, bronchoalveolar lavage fluid, amniotic fluid, amniotic umbilical blood, urine, vaginal fluid and semen. Therefore, a "biological sample" refers to a subset of a protozoan or its tissue, as well as a homogenate, lysate or extract prepared from an organism or a subset of its tissue, including but not limited to plasma, serum, cerebrospinal fluid, lymph, skin slices, respiratory tract, gastrointestinal tract, cardiovascular, urogenital tract, tears, saliva, milk, blood cells, tumors, organs, etc. A biological sample can be any type of organism tissue, including healthy tissue and diseased tissue (e.g., cancer tissue, malignant tissue, necrotic tissue, etc.). In certain embodiments, the biological sample is a liquid sample, such as blood or its derivatives, such as plasma, tears, urine, semen, etc., wherein in some cases, the sample is a blood sample, including whole blood, such as blood obtained by venipuncture or finger stick (wherein, the blood may or may not be mixed with any reagents such as preservatives and anticoagulants before analysis).

[0113] In certain embodiments, the source of the sample is a "mammal" or "mammalian", wherein these terms are broadly used to describe organisms within the class Mammalia, including the orders Carnivora (e.g., dogs and cats), Rodentia (e.g., mice, guinea pigs, and rats), and Primates (e.g., humans, chimpanzees, and monkeys). In some cases, the subject is a human. The method can be used for samples from human subjects of both sexes and at any stage of development (i.e., newborns, infants, juveniles, adolescents, adults), wherein, in certain embodiments, the human subject is a juvenile, adolescent, or adult. Although the present invention can be used for samples from human subjects, it should be understood that the method can also be used for samples from other animal subjects (i.e., "non-human subjects", such as, but not limited to, birds, mice, rats, dogs, cats, livestock, and horses).

[0114] Target cells can be targeted for characterization based on various parameters, such as phenotypic characteristics determined by attaching specific fluorescent tags to the target cells. In certain embodiments, the system can deflect an analysis droplet determined to include a target cell. A variety of cells can be characterized using the subject method. Target cells include, but are not limited to, stem cells, T cells, dendritic cells, B cells, granulocytes, leukemia cells, lymphoma cells, viral cells (such as HIV cells), NK cells, macrophages, monocytes, fibroblasts, epithelial cells, endothelial cells, and erythroid cells. Target cells include cells with appropriate cell surface markers or antigens, which can be captured or labeled using appropriate affinity agents or conjugates thereof. For example, the target cell may include cell surface antigens such as CD11b, CD123, CD14, CD15, CD16, CD19, CD193, CD2, CD25, CD27, CD3, CD335, CD36, CD4, CD43, CD45RO, CD56, CD61, CD7, CD8, CD34, CD1c, CD23, CD304, CD235a, T cell receptor α / β, T cell receptor γ / δ, CD253, CD95, CD20, CD105, CD117, CD120b, Notch4, Lgr5 (N-terminal), SSEA-3, TRA-1-60 antigen, disialylganglioside GD2, and CD71. In certain embodiments, the target cell is selected from HIV-containing cells, Treg cells, antigen-specific T cell populations, tumor cells, or hematopoietic progenitor cells (CD34+) from whole blood, bone marrow, or umbilical cord blood.

[0115] When implementing the subject method, a certain amount of initial fluid sample is injected into the flow cytometer. For example, the amount of sample injected into the particle sorting module may vary, ranging from 0.001 mL-1000 mL, such as 0.005 mL-900 mL, such as 0.01 mL-800 mL, such as 0.05 mL-700 mL, such as 0.1 mL-600 mL, such as 0.5 mL-500 mL, such as 1 mL-400 mL, such as 2 mL-300 mL, including 5 mL-100 mL of sample.

[0116] The method described in accordance with an embodiment of the present disclosure includes counting and sorting labeled particles (such as target cells) in a sample. When implementing the subject method, a fluid sample including particles is first introduced into the flow nozzle of the system. After flowing out of the flow nozzle, the particles pass through the sample interrogation zone substantially one by one, in which each particle is irradiated by a light source, and the light scattering parameter measurement value of each particle is recorded separately, and in some instances, the required fluorescence emission measurement value (for example, two or more light scattering parameters and one or more fluorescence emission measurement values) is recorded separately. Depending on the characteristics of the flow stream being interrogated, the flow stream of 0.001 mm or more, such as 0.005 mm or more, such as 0.01 mm or more, such as 0.05 mm or more, such as 0.1 mm or more, such as 0.5 mm or more, including 1 mm or more of the flow stream can be irradiated with light. In some embodiments, the method includes irradiating a planar cross section of the flow stream in the sample interrogation zone, for example, irradiating with a laser (as described above). In other embodiments, methods include irradiating a predetermined length of the flow stream within the sample interrogation zone, for example corresponding to an irradiation profile of a diffuse laser beam or laser lamp.

[0117] In certain embodiments, the method includes irradiating the flow stream at or near a flow chamber nozzle orifice. For example, the method may include irradiating the flow stream at a distance of about 0.001 mm or more (such as 0.005 mm or more, such as 0.01 mm or more, such as 0.05 mm or more, such as 0.1 mm or more, such as 0.5 mm or more, including 1 mm or more from the nozzle orifice). In certain embodiments, the method includes irradiating the flow stream proximate to the flow chamber nozzle orifice.

[0118] In embodiments of the methods, a detector (e.g., a photomultiplier tube (PMT)) is used to record light passing through each particle (in some cases referred to as forward light scatter), light reflected orthogonal to the direction of particle flow through the sensing region (in some cases referred to as orthogonal or side light scatter), and fluorescence emitted by the particle as it passes through the sensing region and is illuminated by an energy source (if the particle is labeled with a fluorescent marker). Each of forward light scatter (FSC), side scatter (SSC), and fluorescence emission comprises a separate parameter for each particle (or each "event"). Thus, two, three, or four parameters may be collected (and recorded) from particles labeled with two different fluorescent markers.

[0119] In some embodiments, the method includes removing the adapter from the sorting flow cytometer. In some cases, the method further includes reattaching the second adapter to the particle sorting system after the first particle sorting module has been removed. The first adapter can be cleaned and sterilized using an autoclave for subsequent use, or can be discarded.

[0120] The recorded data of each particle is analyzed in real time or stored in a data storage and analysis device (such as a computer) as needed. U.S. Patent No. 4,284,412 describes the configuration and use of a target flow cytometer equipped with a single light source, while U.S. Patent No. 4,727,020 describes the configuration and use of a flow cytometer equipped with two light sources.

[0121] In certain embodiments, the particles are exposed to excitation light and the amount of fluorescence from each particle is measured in one or more detection channels, as desired, to detect and uniquely identify the particles. Fluorescence emitted in the detection channels used to identify the particles and their associated binding complexes can be measured after excitation by a single light source or can be measured separately after excitation by different light sources. If separate excitation light sources are used to excite the particle tags, the tags can be selected so that all tags are excited by each excitation light source used.

[0122] The method in certain embodiments also includes using a computer to perform data acquisition, analysis and recording, wherein multiple data channels can record data from each detector for light scatter and fluorescence emitted by each particle when passing through the sample interrogation zone of the particle sorting module. In these embodiments, the analysis includes classifying and counting the particles so that each particle exists as a set of digitized parameter values. The subject system can be set to trigger on a selected parameter to distinguish target particles from background and noise. "Trigger" refers to a preset threshold for parameter detection, which can be used as a means to detect the passage of particles through a light source. When an event exceeding a selected parameter threshold is detected, the collection of particle light scatter and fluorescence data is triggered. For particles or other components in the measured medium that cause a response below the threshold, no data is collected. The trigger parameter can be the detection of forward scattered light caused by the passage of a particle through a light beam. The flow cytometer then detects and collects the light scatter and fluorescence data of the particles.

[0123] Based on the data collected for the entire population of particles, specific subpopulations of interest are then further analyzed by "gating." In order to select the appropriate gate, the data needs to be plotted to achieve the best possible separation of subpopulations. This is accomplished by plotting forward light scatter (FSC) versus side (i.e., orthogonal) light scatter (SSC) on a 2D dot plot. Subpopulations of particles (i.e., cells within the gate) are then selected and particles that are not within the gate are excluded. If desired, a gate can be selected by drawing a line around the desired subpopulation with the cursor on the computer screen. Only the particles within the gate can then be further analyzed by plotting other parameters of these particles, such as fluorescence. If desired, the above analysis can produce a count of the target particles in the sample.

[0124] The subject method may further include using the sorted particles in research, laboratory experiments or treatment. In certain embodiments, the subject method includes obtaining single cells made from a target fluid or tissue biological sample. For example, the subject method includes obtaining cells from a fluid or tissue sample for use as a research or diagnostic specimen for a disease such as cancer. Similarly, the subject method includes obtaining cells from a fluid or tissue sample for treatment. Cell therapy protocols include: viable cell materials, including cells and tissues, can be prepared, and then the cell materials are introduced into the subject as a therapeutic treatment. Conditions that can be treated by administering flow cytometry sorted samples include, but are not limited to, blood diseases, immune system diseases, organ damage, etc.

[0125] A typical cell therapy protocol may include the following steps: sampling, cell separation, genetic modification, culture, in vitro expansion, cell harvesting, sample volume reduction and washing, biological preservation, storage, and introduction of cells into a subject. The protocol may first collect viable cells and tissues from a source tissue of a subject to produce a cell and / or tissue sample. The sample may be collected by any suitable procedure, such as administering a cell mobilization agent to a subject, drawing blood from a subject, extracting bone marrow from a subject, and the like. After collecting the sample, cells may be enriched by a variety of methods, including centrifugation, filtration, elutriation, magnetic separation, fluorescence activated cell sorting (FACS), and the like. In some cases, the enriched cells may be genetically modified by any convenient method (such as nuclease-mediated gene editing). Genetically modified cells may be cultured, activated, and expanded in vitro. In some cases, cells are preserved, such as cryopreserved, and stored for future use, i.e., the cells are thawed and then administered to the patient, such as by injecting the cells into the patient.

[0126] Particle sorter assembly method

[0127] As described above, the method of the present invention further includes a particle sorter assembly method. The target method includes operably connecting a nozzle to a flow chamber that can transport particles in a flow stream. As described above, the subject nozzle includes an elongated body, the elongated body including a proximal opening that is engaged with the flow chamber to establish a liquid receiving relationship, a distal opening for emitting droplets, and a channel through which the liquid can be transported from the proximal end to the distal end through the elongated body. In addition, the nozzle of the present invention includes an air inlet radially arranged at the proximal end of the elongated body, wherein the air inlet includes a radial air flow path that can supply air to the channel. The so-called "operably connecting" the nozzle to the flow chamber refers to engaging the proximal opening of the elongated body with the flow chamber to establish a liquid receiving relationship. In some versions, the method includes operably connecting the flow chamber and the nozzle in a fluid-tight manner (such as airtight, liquid-tight). In this case, one or more gaskets and / or O-rings can be used.

[0128] In some cases, the method further includes connecting a compressed gas source to the air inlet in a gaseous manner. The so-called "gaseous connection" of the compressed gas source to the air inlet refers to connecting the two parts so that gas can be exchanged between the two parts. As described above, any suitable compressed gas source (such as a gas compressor, a gas storage tank, etc.) can be used. The method may further include operably connecting (e.g., gaseously connecting) one or more gas conduits to the compressed gas source and the air inlet. Each gas conduit can be connected to the radial air flow path in the air inlet, for example, by a press fit or any other suitable fixing mechanism, including but not limited to a clamp, a magnet, a latch, a notch, a countersunk hole, a countersunk hole, a groove, a pin, a tether, a hinge, a non-permanent adhesive, or a combination thereof. In some cases, the method includes operably connecting multiple gas conduits to the compressed gas source and the air inlet.

[0129] The method according to some embodiments further comprises operably connecting a processor to a source of compressed gas. As described above, the target processor can adjust the pressure of the generated gas provided to the gas inlet. The processor can be a processor already in the particle sorter, or it can be a processor not already in the particle sorter. In the case where the processor is already present in the particle sorter, the processor is modified (e.g., by a plug-in) to have the additional function of adjusting the source of compressed gas to provide a certain amount of pressure.

[0130] Computer control system

[0131] Various aspects of the present invention further include a computer control system, wherein the system includes one or more computers that implement full or partial automation. In some embodiments, the system includes a computer having a non-transitory computer readable storage medium having a computer program stored thereon, and when loaded on the computer, the computer program includes instructions for receiving a desired droplet size and / or volume (e.g., input by a user), and instructions for driving a compressed gas source to change to achieve the desired droplet size and / or volume.

[0132] The system may include a display and an operator input device. For example, the operator input device may be a keyboard, a mouse, etc. The processing module includes a processor that can access a memory containing instructions for executing the steps of the subject method. The processing module may include an operating system, a graphical user interface (GUI) controller, a system memory, a memory storage device, an input and output controller, a cache memory, a data backup unit, and many other devices. The processor may be a commercially available processor or may be another processor that is available now or in the future. The processor executes an operating system that interfaces with firmware and hardware in a known manner to help the processor coordinate and execute the functions of various computer programs, which may be in various programming languages, such as Java, Perl, C++, Python, other high-level or low-level languages ​​known in the art, and combinations thereof. The operating system typically cooperates with the processor to coordinate and execute the functions of other components of the computer. The operating system also provides scheduling, input and output control, file and data management, memory management, and communication control and related services, all of which are based on known technologies. In some embodiments, the processor includes analog electronics that provide feedback control (e.g., negative feedback control).

[0133] The system memory may be any of a variety of known or future memory storage devices. Examples include any commonly used random access memory (RAM), magnetic media (such as a resident hard disk or tape), optical media (such as a read-write optical disk), flash memory devices, or other memory storage devices. The memory storage device may be any of a variety of known or future devices, including an optical drive, a tape drive, or a floppy disk drive. Such memory storage devices typically read and / or write to program storage media such as optical disks (not shown). Any of these program storage media, or other program storage media currently in use or that may be developed in the future, may be considered a computer program product. It should be understood that these program storage media typically store computer software programs and / or data. Computer software programs, also known as computer control logic, are typically stored in system memory and / or program storage devices used in conjunction with memory storage devices.

[0134] In some embodiments, the present invention describes a computer program product that includes a computer usable medium containing control logic (computer software program, including program code). When executed by a processor and a computer, the control logic causes the processor to perform the functions described in the present invention. In other embodiments, some functions are implemented in hardware, primarily (for example) through a hardware state machine. It will be apparent to a person skilled in the relevant art that implementing a hardware state machine can perform the functions described in the present invention.

[0135] The memory may be any suitable device from which the processor can store and retrieve data, such as a magnetic, optical or solid-state storage device (including a disk, optical disk, tape or RAM, or any other suitable fixed or portable device). The processor may include a general-purpose digital microprocessor, which is appropriately programmed by a computer-readable medium carrying the necessary program code. The programming may be provided to the processor remotely via a communication channel, or previously stored in a computer program product (such as a memory or other portable or fixed computer-readable storage medium using any memory-related device). For example, a disk or optical disk may carry the programming and may be read by a disk writer / reader. The system of the present invention also includes, for example, programming of the algorithm for implementing the above-mentioned method in the form of a computer program product. The programming according to the present invention may be recorded on a computer-readable medium (such as any medium that can be directly read and accessed by a computer). Such media include, but are not limited to: magnetic storage media, such as floppy disks, hard disk storage media and tapes; optical storage media, such as CD-ROMs; electrical storage media, such as RAM and ROM; portable flash drives; and hybrid media of these categories, such as magnetic / optical storage media.

[0136] The processor may also access a communication channel to communicate with a user at a remote location. A remote location is one where the user is not directly interacting with the system and relays input information to the input manager from an external device such as a computer connected to a wide area network ("WAN"), telephone network, satellite network, or any other suitable communication channel, including a mobile phone (i.e., smart phone).

[0137] In some embodiments, the system according to the present disclosure may include a communication interface. In some embodiments, the communication interface includes a receiver and / or a transmitter for communicating with a network and / or another device. The communication interface can be used for wired or wireless communication, including but not limited to radio frequency (RF) communication (e.g., radio frequency identification (RFID)), Zigbee communication protocol, Wi-Fi, infrared, wireless universal serial bus (USB), ultra-wideband (UWB), Bluetooth® communication protocol, and cellular communication, such as code division multiple access (CDMA) or global system for mobile communications (GSM).

[0138] In one embodiment, the communication interface may include one or more communication ports, such as physical ports or interfaces, such as a USB port, a USB-C port, an RS-232 port, or any other suitable electrical connection port to enable data communication between the subject system (e.g., in a doctor's office or hospital environment) and other external devices such as a computer terminal for similar supplemental data communications.

[0139] In one embodiment, the communication interface is used for infrared communication, Bluetooth® communication, or any other suitable wireless communication protocol, so that the subject system can communicate with a computer terminal and / or network, a communication-enabled mobile phone, a personal digital assistant, or other devices, or any other communication device available for use by the user.

[0140] In one embodiment, the communication interface provides a connection for data transfer using the Internet Protocol (IP) through a cellular network, a short message service (SMS), a wireless connection to a personal computer (PC) on a local area network (LAN) connected to the Internet, or a Wi-Fi connection to the Internet on a Wi-Fi hotspot.

[0141] In one embodiment, the subject system communicates via a communication interface (e.g., using 802.11 or Bluetooth ® The server device may be another portable device, such as a smart phone, a personal digital assistant (PDA), or a laptop computer; or a larger device, such as a desktop computer, a home appliance, etc. In some embodiments, the server device has a display, such as a liquid crystal display (LCD), and an input device, such as a button, a keyboard, a mouse, or a touch screen.

[0142] In some embodiments, the communication interface automatically or semi-automatically transfers data stored in the subject system (eg, optional data storage unit) via a network or server using one or more of the communication protocols and / or mechanisms described above.

[0143] The output controller may include a controller for any of a variety of known display devices, which are used to present information to a user (whether a person or a machine, whether local or remote). If one of the display devices provides visual information, the information can usually be logically and / or physically organized into a series of pixels. A graphical user interface (GUI) controller may include any of a variety of known or future software programs for providing a graphical input and output interface between the system and the user and processing user input information. The functional components of the computer can communicate with each other via a system bus, and in alternative embodiments, some of the communications can be completed using a network or other type of remote communication. The output manager can also provide information generated by the processing module to a user at a remote location based on known techniques, such as through the Internet, telephone or satellite network. The presentation of data by the output manager can be based on various known techniques. For example, the data may include SQL, HTML or XML documents, emails or other files or other forms of data. The data may include an Internet URL address so that the user can retrieve more SQL, HTML, XML or other documents or data from a remote source. Although one or more platforms present in the subject system generally belong to the general category of computers commonly known as servers, they can also be any type of known computer platform or a type developed in the future. However, they may also be rack computers, workstations, or other computer types. These platforms may be networked or otherwise connected by any known or future type of wiring or other communication system, including wireless systems. They may be co-located or physically separate. A variety of operating systems may be applied to any computer platform, which may depend on the type and / or model of computer platform selected. Suitable operating systems include Windows ® NT ® , Windows ® XP, Windows ® 7. Windows ® 8. Windows ® 10. iOS ® , macOS ® , Linux ® , Ubuntu ® , Fedora ® 、OS / 400 ® 、i5 / OS ® 、IBM i ® 、Android TM 、SGI IRIX ® , Oracle Solaris ® wait.

[0144] Figure 7The general architecture of an example computing device 600 is shown in accordance with some embodiments. Figure 7 The general architecture of the computing device 700 shown includes the arrangement of computer hardware and software components. However, it is not necessary to show all conventional components for public purposes. As shown, the computing device 700 includes a processing unit 710, a network interface 720, a computer-readable medium drive 730, an input / output device interface 740, a display 750 and an input device 760, all of which can communicate with each other through a communication bus. The network interface 720 can provide connectivity with one or more networks or computing systems. Therefore, the processing unit 710 can receive information and instructions from other computing systems or services through the network. The processing unit 710 can also communicate with the memory 770 and further provide output information to the optional display 750 through the input / output device interface 740. For example, analysis software (such as FlowJo) stored as executable instructions in the non-transient memory of the analysis system ® The input / output device interface 740 can also receive input information from an optional input device 760 (e.g., a keyboard, mouse, digital pen, microphone, touch screen, gesture recognition system, voice recognition system, game controller, accelerometer, gyroscope or other input device).

[0145] Memory 770 may contain computer program instructions (grouped in some embodiments in modules or components) executed by processing unit 710 to implement one or more embodiments. Memory 770 generally includes RAM, ROM, and / or other permanent, auxiliary, or non-transitory computer-readable media. Memory 770 may store an operating system 772 that provides computer program instructions for use by processing unit 710 in general management and operation of computing device 700. Data may be stored in data storage device 790. Memory 770 may further include computer program instructions and other information for implementing aspects of the present disclosure.

[0146] Useful functions

[0147] The nozzles, cell sorters, methods and kits disclosed herein are suitable for adjusting the size of droplets in a cell sorter without having to replace the nozzle. In addition, the present invention can also be used in cases where it is desired to improve the data received from the particle modulated light in the cell sorter by eliminating the source of disturbance upstream of the flow chamber. Embodiments of the present invention can be used in applications where it may be necessary to use cells made from biological samples for research, laboratory experiments or treatment. In some embodiments, the subject methods and devices can help obtain single cells made from target fluid or tissue biological samples. For example, the subject methods and systems help obtain cells from fluid or tissue samples for use as research or diagnostic samples for diseases such as cancer. Similarly, the subject methods and systems can help obtain cells from fluid or tissue samples for treatment. Compared to traditional flow cytometer systems, the methods and devices described in the present disclosure allow cells to be separated and collected from biological samples (such as organs, tissues, tissue fragments, fluids) with high efficiency and low cost.

[0148] Kits

[0149] Various aspects of the present invention further include a kit. The target kit includes a subject nozzle. As described above, the subject nozzle includes an elongated body, the elongated body including a proximal opening that engages with the flow chamber to establish a liquid receiving relationship, a distal opening for emitting droplets, and a channel through which the liquid can be transported from the proximal end to the distal end through the elongated body. In addition, the nozzle of the present invention includes an air inlet radially arranged at the proximal end of the elongated body, wherein the air inlet includes a radial air flow path that can supply air to the channel. The target kit may further include one or more gas conduits. In some cases, the kit includes multiple gas conduits. In some instances, the kit includes a compressed gas source. As described above, any suitable compressed gas source (such as a gas compressor, a gas storage tank, etc.) can be used.

[0150] In some versions, the kits additionally include a processor that can adjust the pressure of the generated gas provided to the gas inlet. In other cases, the kits include instructions for modifying an existing processor on a cell sorter to adjust the pressure of the generated gas provided to the gas inlet. In some such cases, the kits include storage media such as magneto-optical disks, CD-ROMs, CD-Rs, magnetic tapes, non-volatile memory cards, ROMs, DVD-ROMs, Blu-ray disks, solid-state drives, and network attached storage (NAS). Any of these program storage media, or other program storage media currently in use or that may be developed in the future, may be included in the subject kits. Plug-ins may be provided on the storage media for modifying existing processors.

[0151] In addition to the above components, the theme suite may further include (in some embodiments) instructions for installing the plug-in into an existing software package. These instructions may be present in the theme suite in various forms, wherein one or more forms may be present in the suite. One form of these instructions is information printed in a suitable medium or substrate (e.g., one or more sheets of paper with information printed thereon), in the suite packaging, in a package instruction sheet, etc. Another form of these instructions is a computer-readable medium containing information, such as a floppy disk, a compact disc (CD), a portable flash drive, etc. Another form of these instructions is a website address that can be used to access information on the removed site via the Internet.

[0152] The following examples are illustrative rather than limiting.

[0153] Experimental Examples

[0154] To demonstrate the working principle of periodic droplet generation from a nozzle using dispersed flow and continuous flow, a simulation was performed. ® The simulation was performed using Multiphysics software. The diameter of the simulated nozzle was 200 µm, the height of the radial air flow path was 200 µm, and the diameter of the extended cylinder was 500 µm and the length was 4.3 mm. Figure 8 The simulated nozzle is described. Figure 8 As shown, the nozzle includes an elongated body 801, a proximal opening 802 for receiving liquid from a flow chamber (not shown), a channel 803, a distal opening 804 for emitting droplets, and an air inlet 805. The volume flow rate through the simulated nozzle was set to 6.3 ml / min (105 µl / s).

[0155] Three different simulations were performed using the above nozzle. In each simulation, a gas pressure of 4 kPa (0.58 psi), 5 kPa (0.73 psi), and 6 kPa (0.87 psi) was applied to the gas inlet 805. Fig. 9 The results of these simulations are shown in Fig. 9 As shown in the figure, the simulation conducted at 4 kPa pressure produced droplets with a diameter of about 130 µm, the simulation conducted at 5 kPa pressure produced droplets with a diameter of about 100 µm, and the simulation conducted at 6 kPa pressure produced droplets with a diameter of about 80 µm. The results show that the size of the droplets can be adjusted by the inlet pressure of the air flow.

[0156] Notwithstanding the appended claims, the present disclosure is defined by the following terms:

[0157] 1. A particle sorter, comprising:

[0158] a flow chamber that transports particles in a flow stream;

[0159] Nozzle, including:

[0160] The long strip body comprises:

[0161] a proximal opening engageable with the flow chamber to establish a liquid receiving relationship;

[0162] a distal opening for emitting droplets;

[0163] a channel, which can transport liquid from the proximal end to the distal end through the elongated body;

[0164] An air inlet is radially disposed at the proximal end of the elongated body, wherein the air inlet includes a radial air flow path that can supply air to the channel.

[0165] 2. A particle sorter according to claim 1, wherein the height of the radial air flow path ranges from 150 µm to 250 µm.

[0166] 3. A particle sorter according to clause 1 or 2, wherein the radius of the radial air flow path ranges from 200 µm to 300 µm.

[0167] 4. A particle sorter according to any of the preceding clauses, wherein the air inlet comprises a plurality of radial air flow paths.

[0168] 5. A particle sorter according to clause 4, wherein the number of radial air flow paths in the plurality of radial air flow paths ranges from 2 to 5.

[0169] 6. A particle sorter according to any one of clauses 1 or 5, further comprising a compressed gas source.

[0170] 7. A particle sorter according to clause 6, wherein the compressed gas source is an air compressor.

[0171] 8. A particle sorter according to clause 6 or 7, wherein the compressed gas source can generate gas with a pressure range of 2 kPa-10 kPa.

[0172] 9. A particle sorter according to any one of clauses 6 to 8, further comprising a gas conduit which can operably connect a compressed gas source to the gas inlet.

[0173] 10. A particle sorter according to clause 9, wherein the particle sorter comprises a plurality of gas conduits.

[0174] 11. A particle sorter according to any one of clauses 6 to 10, further comprising a processor operably connected to a source of compressed gas and capable of regulating the pressure of the generated gas provided to the gas inlet.

[0175] 12. A particle sorter according to any of the preceding clauses, wherein the diameter of the channel is in the range of 150 µm-250 µm.

[0176] 13. A particle sorter according to clause 12, wherein the diameter of the channel is constant throughout the entire length of the elongated body.

[0177] 14. A particle sorter according to any of the preceding clauses, wherein the elongated body is cylindrical.

[0178] 15. A particle sorter according to any one of the preceding clauses, wherein the length of the elongated body is in the range of 4 mm to 4.5 mm.

[0179] 16. A particle sorter according to any of the preceding clauses, wherein the nozzle is not operably secured to a piezoelectric actuator.

[0180] 17. A particle sorter according to any of the preceding clauses, further comprising a light source operable to irradiate the flow stream at the interrogation point.

[0181] 18. The particle sorter according to clause 17, further comprising a detector capable of collecting the particle modulated light from the flow chamber.

[0182] 19. The particle sorter according to any of the preceding clauses, further comprising a plurality of containers capable of receiving droplets emitted from the distal end opening of the elongated structure.

[0183] 20. The particle sorter of clause 19, further comprising a deflection plate that can deflect droplets into a given container of the plurality of containers.

[0184] 21. A particle sample sorting method, the method comprising:

[0185] (a) introducing a particle sample into a cell sorter, the cell sorter comprising:

[0186] a flow chamber that transports particles in a flow stream;

[0187] Nozzle, including:

[0188] The long strip body comprises:

[0189] a proximal opening engageable with the flow chamber to establish a liquid receiving relationship;

[0190] a distal opening for emitting droplets;

[0191] a channel, which can transport liquid from the proximal end to the distal end through the elongated body;

[0192] an air inlet radially disposed at a proximal end of the elongated body, wherein the air inlet includes a radial air flow path capable of supplying air to the passage;

[0193] (b) Particle samples were sorted by flow cytometry.

[0194] 22. The method of clause 21, wherein the radial air flow path has a height in the range of 150 µm - 250 µm.

[0195] 23. A method according to clause 21 or 22, wherein the radial air flow path has a radius in the range of 200 µm - 300 µm.

[0196] 24. The method of any of clauses 21-23, wherein the air inlet comprises a plurality of radial air flow paths.

[0197] 25. The method of clause 24, wherein the number of radial air flow paths in the plurality of radial air flow paths ranges from 2 to 5.

[0198] 26. A method according to any one of clauses 21-25, wherein the cell sorter further comprises a source of compressed gas.

[0199] 27. The method of clause 26, wherein the source of compressed gas is an air compressor.

[0200] 28. A method according to clause 26 or 27, wherein the source of compressed gas can produce gas at a pressure in the range of 2 kPa-10 kPa.

[0201] 29. A method according to any one of clauses 26 to 28, wherein the particle sorter further comprises a gas conduit which operably connects a source of compressed gas to the gas inlet.

[0202] 30. The method of clause 29, wherein the particle sorter comprises a plurality of gas conduits.

[0203] 31. The method of any of clauses 26-30, further comprising regulating the pressure of the gas provided to the gas inlet using a compressed gas source.

[0204] 32. A method according to any of clauses 21-31, wherein the channel has a diameter in the range of 150 µm-250 µm.

[0205] 33. A method according to clause 32, wherein the diameter of the channel is constant throughout the length of the elongated body.

[0206] 34. A method according to any one of clauses 21-33, wherein the elongated body is cylindrical.

[0207] 35. A method according to any one of clauses 21-34, wherein the length of the elongated body is in the range of 4 mm to 4.5 mm.

[0208] 36. A method according to any of clauses 21-35, wherein the nozzle is not operably secured to a piezoelectric actuator.

[0209] 37. A method according to any of clauses 21 to 36, wherein the particle sorter further comprises a light source operable to irradiate the flow stream at an interrogation point.

[0210] 38. The method of clause 37, wherein the particle sorter further comprises a detector that collects particle modulated light from the flow chamber.

[0211] 39. The method according to any one of clauses 21-38, wherein the particle sorter further comprises a plurality of containers capable of receiving droplets emitted from the distal end opening of the elongated structure.

[0212] 40. The method of clause 39, wherein the particle sorter further comprises a deflection plate that can deflect droplets into a given container of the plurality of containers.

[0213] 41. A method according to any of clauses 21-40, wherein the sample is a biological sample.

[0214] 42. The method of clause 41, wherein the sample comprises cells.

[0215] 43. A nozzle comprising:

[0216] The long strip body comprises:

[0217] a proximal opening engageable with the flow chamber to establish a liquid receiving relationship;

[0218] a distal opening for emitting droplets;

[0219] a channel, which can transport liquid from the proximal end to the distal end through the elongated body;

[0220] An air inlet is radially disposed at the proximal end of the elongated body, wherein the air inlet includes a radial air flow path that can supply air to the channel.

[0221] 44. A nozzle according to clause 43, wherein the height of the radial air flow path ranges from 150 µm to 250 µm.

[0222] 45. A nozzle according to clause 44, wherein the height range of the radial air flow path is 200 μm.

[0223] 46. ​​A nozzle according to any of clauses 43-45, wherein the radius of the radial air flow path is in the range of 200 µm-300 µm.

[0224] 47. A nozzle according to clause 46, wherein the radius of the radial air flow path is 250 μm.

[0225] 48. The nozzle of any of clauses 43-47, wherein the air inlet comprises a plurality of radial air flow paths.

[0226] 49. The nozzle of clause 48, wherein the number of radial air flow paths in the plurality of radial air flow paths ranges from 2 to 5.

[0227] 50. The nozzle of clause 49, wherein the air inlet comprises four radial air flow paths.

[0228] 51. A nozzle according to any of clauses 43-50, wherein the gas inlet is operably connected to a gas conduit.

[0229] 52. A nozzle according to clause 51, wherein the gas inlet is operably connected to a plurality of gas conduits.

[0230] 53. A nozzle according to any of clauses 43-52, wherein the diameter of the channel is in the range of 150 µm-250 µm.

[0231] 54. A nozzle according to clause 53, wherein the diameter of the channel is 200 µm.

[0232] 55. A nozzle according to clause 53 or 54, wherein the diameter of the channel is constant throughout the length of the elongated body.

[0233] 56. The nozzle of any one of clauses 43-55, wherein the elongated body is cylindrical.

[0234] 57. The nozzle according to any one of clauses 43-56, wherein the length of the elongated body is in the range of 4 mm to 4.5 mm.

[0235] 58. The nozzle of clause 57, wherein the length of the elongated body is 4.3 mm.

[0236] 59. A particle sorting apparatus assembly method, the method comprising:

[0237] A nozzle is operably connected to a flow chamber, the flow chamber being capable of delivering particles in a flow stream, wherein the nozzle comprises:

[0238] The long strip body comprises:

[0239] proximal opening;

[0240] a distal opening for emitting droplets;

[0241] a channel, which can transport liquid from the proximal end to the distal end through the elongated body;

[0242] an air inlet radially disposed at a proximal end of the elongated body, wherein the air inlet includes a radial air flow path capable of supplying air to the passage;

[0243] The operably connecting the nozzle includes engaging the proximal opening of the elongated body with the flow chamber to establish a liquid receiving relationship.

[0244] 60. The method of clause 59, wherein the radial air flow path has a height in the range of 150 µm - 250 µm.

[0245] 61. A method according to clause 59 or 60, wherein the radial air flow path has a radius in the range of 200 µm - 300 µm.

[0246] 62. The method of any of clauses 59-61, wherein the air inlet comprises a plurality of radial air flow paths.

[0247] 63. The method of clause 62, wherein the number of radial air flow paths in the plurality of radial air flow paths ranges from 2 to 5.

[0248] 64. The method of any of clauses 59-63, further comprising gaseously connecting a source of compressed gas to the gas inlet.

[0249] 65. A method according to clause 64, wherein the compressed gas source is an air compressor.

[0250] 66. A method according to clause 6 or 65, wherein the compressed gas source can produce gas at a pressure range of 2 kPa-10 kPa.

[0251] 67. The method of any one of clauses 64-66, further comprising operably connecting a gas conduit to a compressed gas source and a gas inlet.

[0252] 68. A method according to clause 67, wherein the method includes operably connecting a plurality of gas conduits to a compressed gas source and a gas inlet.

[0253] 69. The method of any of clauses 64-68, further comprising operably connecting a processor to a source of compressed gas, wherein the processor can regulate the pressure of the generated gas provided to the gas inlet.

[0254] 70. A method according to any of clauses 59-69, wherein the channel has a diameter in the range of 150 µm-250 µm.

[0255] 71. A method according to clause 70, wherein the diameter of the channel is constant throughout the length of the elongated body.

[0256] 72. A method according to any one of clauses 59-71, wherein the elongated body is cylindrical.

[0257] 73. A method according to any one of clauses 59-72, wherein the length of the elongated body ranges from 4 mm to 4.5 mm.

[0258] 74. A method according to any of clauses 59-73, wherein the method does not include operably connecting a piezoelectric actuator to the nozzle.

[0259] 75. A kit comprising:

[0260] Nozzle, including:

[0261] The long strip body comprises:

[0262] a proximal opening engageable with the flow chamber to establish a liquid receiving relationship;

[0263] a distal opening for emitting droplets;

[0264] a channel, which can transport liquid from the proximal end to the distal end through the elongated body;

[0265] An air inlet is radially disposed at the proximal end of the elongated body, wherein the air inlet includes a radial air flow path that can supply air to the channel.

[0266] 76. A kit according to clause 75, wherein the height of the radial air flow path ranges from 150 µm to 250 µm.

[0267] 77. A kit according to clause 76, wherein the height range of the radial air flow path is 200 μm.

[0268] 78. A kit according to any of clauses 75-77, wherein the radial air flow path has a radius in the range of 200 µm-300 µm.

[0269] 79. A kit according to clause 78, wherein the radius of the radial air flow path is 250 μm.

[0270] 80. A kit according to any of clauses 75-79, wherein the air inlet comprises a plurality of radial air flow paths.

[0271] 81. The kit of clause 80, wherein the number of radial air flow paths in the plurality of radial air flow paths ranges from 2 to 5.

[0272] 82. The kit of clause 81 , wherein the air inlet comprises four radial air flow paths.

[0273] 83. A kit according to any of clauses 75 or 82, further comprising a source of compressed gas.

[0274] 84. A kit according to clause 83, wherein the compressed gas source is an air compressor.

[0275] 85. A kit according to clause 83 or 84, wherein the compressed gas source can produce gas at a pressure range of 2 kPa-10 kPa.

[0276] 86. A kit according to any of clauses 75-85, further comprising a gas conduit which can operably connect a compressed gas source to the gas inlet.

[0277] 87. A kit according to clause 86, wherein the kit includes a plurality of gas conduits.

[0278] 88. A kit according to any of clauses 75-87, wherein the diameter of the channel is in the range of 150 µm-250 µm.

[0279] 89. A kit according to clause 88, wherein the diameter of the channel is 200 µm.

[0280] 90. A kit according to clause 88 or 89, wherein the diameter of the channel is constant throughout the length of the elongated body.

[0281] 91. A kit according to any one of clauses 75-90, wherein the elongated body is cylindrical.

[0282] 92. A kit according to any one of clauses 75-91, wherein the length of the elongated body ranges from 4 mm to 4.5 mm.

[0283] 93. A kit according to claim 92, wherein the length of the elongated body is 4.3 mm.

[0284] Although the above invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be apparent to one of ordinary skill in the art, based on the teachings of the present invention, that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

[0285] Therefore, the above content only illustrates the principle of the present invention. It should be understood that those skilled in the art will be able to design various arrangements, which, although not explicitly described or shown in the present invention, embody the principles of the present invention and are included in its spirit and scope. In addition, all examples and conditional language listed in the present invention are mainly intended to help readers understand the principles of the present invention and the concepts provided by the inventors to deepen the field, and should be interpreted as not being limited to these specifically listed examples and conditions. In addition, all statements recording the principles, aspects and embodiments of the present invention and their specific examples in the present invention are intended to cover their structural and functional equivalents. In addition, it is intended that the equivalents include currently known equivalents and equivalents developed in the future, i.e., any part (regardless of its structure) that performs the same function developed. In addition, any content disclosed in the present invention is not intended to be open to the public specifically, regardless of whether such disclosure is explicitly listed in the claims.

[0286] Therefore, the scope of the present invention is not intended to be limited to the exemplary embodiments shown and described herein. Instead, the appended claims embody the scope and spirit of the present invention. In the claims, 35 U.S. Patent Act Section 112(f) or 35 U.S. Patent Act Section 112(6) is expressly defined as a limitation in a claim only if such limitation begins with the exact phrase "means" or the exact phrase "step" in the claim; if such exact phrase is not used in the claim, 35 U.S. Patent Act Section 112(f) or 35 U.S. Patent Act Section 112(6) is not cited.

Claims

1. A particle sorter, comprising: a flow chamber configured to transport particles in a flow stream; and Nozzle, including: The long strip body comprises: A proximal opening engaged with the flow chamber to establish a liquid receiving relationship; a distal opening for emitting droplets; and a channel configured to transport a liquid from a proximal end to a distal end through the elongated body; and An air inlet is radially disposed at a proximal end of the elongated body, wherein the air inlet includes a radial air flow path configured to supply air to the channel.

2. The particle sorter according to claim 1, wherein: The air intake includes a plurality of radial air flow paths.

3. The particle sorter according to any one of claims 1 or 2, further comprising a compressed gas source. 4 . The particle sorter of claim 3 , further comprising a gas conduit configured to operably connect a compressed gas source to the gas inlet.

5. The particle sorting apparatus according to claim 4, wherein: The particle sorter includes a plurality of gas conduits.

6. The particle sorter according to any one of claims 3 to 5, further comprising a processor operably connected to a compressed gas source and configured to adjust the pressure of the generated gas provided to the gas inlet.

7. A particle sorter according to any one of the preceding claims, wherein: The diameter of the channels ranges from 150 µm to 250 µm.

8. The particle sorting apparatus according to claim 7, wherein: The diameter of the channel is constant throughout the entire length of the elongated body.

9. A particle sorter according to any one of the preceding claims, wherein: The long strip body is cylindrical.

10. A particle sorter according to any one of the preceding claims, wherein: The nozzle is not operably secured to the piezoelectric actuator.

11. A particle sorter according to any one of the preceding claims, further comprising a light source configured to irradiate the flow stream at an interrogation point.

12. The particle sorter of claim 11, further comprising a detector configured to collect the particle modulated light from the flow chamber.

13. The particle sorting apparatus according to any one of the preceding claims, further comprising a plurality of containers configured to receive droplets emitted from the distal end opening of the elongated structure.

14. The particle sorter of claim 13, further comprising a deflection plate configured to deflect a droplet into a given container of the plurality of containers.

15. A particle sample sorting method, the method comprising: (a) introducing a particle sample into a cell sorter, the cell sorter comprising: a flow chamber configured to transport particles in a flow stream; and Nozzle, including: The long strip body comprises: A proximal opening engaged with the flow chamber to establish a liquid receiving relationship; a distal opening for emitting droplets; and a channel configured to transport a liquid from a proximal end to a distal end through the elongated body; and an air inlet radially disposed at a proximal end of the elongated body, wherein the air inlet includes a radial air flow path configured to supply air to the passage; (b) Particle samples were sorted by flow cytometry.

16. A nozzle, comprising: The long strip body comprises: a proximal opening configured to engage the flow chamber in a liquid receiving relationship; a distal opening for emitting droplets; and a channel configured to transport a liquid from a proximal end to a distal end through the elongated body; and An air inlet is radially disposed at a proximal end of the elongated body, wherein the air inlet includes a radial air flow path configured to supply air to the channel.

17. A particle sorting apparatus assembly method, the method comprising: A nozzle is operably connected to a flow chamber, the flow chamber being configured to deliver particles in a flow stream, wherein the nozzle comprises: The long strip body comprises: proximal opening; a distal opening for emitting droplets; and a channel configured to transport a liquid from a proximal end to a distal end through the elongated body; and an air inlet radially disposed at a proximal end of the elongated body, wherein the air inlet includes a radial air flow path configured to supply air to the passage; The operably connecting the nozzle includes engaging the proximal opening of the elongated body with the flow chamber to establish a liquid receiving relationship.

18. A kit comprising: Nozzle, including: The long strip body comprises: a proximal opening configured to engage the flow chamber in a liquid receiving relationship; a distal opening for emitting droplets; and a channel configured to transport a liquid from a proximal end to a distal end through the elongated body; and An air inlet is radially disposed at a proximal end of the elongated body, wherein the air inlet includes a radial air flow path configured to supply air to the channel.

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