Particle sorter nozzle and method of using the same

Abstract

A particle sorter nozzle is provided. The nozzle in question includes an elongated body and a gas inlet positioned radially at the proximal end of the elongated body. The gas inlet of the nozzle in question includes a radial airflow path configured to provide gas to a channel. The elongated body includes a proximal opening configured to engage in a liquid receiving relationship with a flow cell, a distal opening for discharging liquid droplets, and a channel configured to transfer liquid from the proximal end to the distal end through the elongated body. Also provided are a particle sorter having the nozzle in question, methods of using and assembling the particle sorter in question, and kits.

Description

Background Art

[0001] Flow cytometry is a technique used to characterize and often sort biological materials, such as cells in a blood sample or target particles in another type of biological or chemical sample. A flow cytometer typically includes a sample reservoir for receiving a fluid sample, such as a blood sample, and a sheath reservoir containing sheath fluid. The flow cytometer transfers particles (including cells) in the fluid sample to the flow cell as a cell stream while directing the sheath fluid to the flow cell. Light is irradiated onto the flow stream to evaluate the characteristics of the components of the flow stream.

[0002] Variations in the materials in the flow stream, such as the presence of morphological or fluorescent labels, can cause variations in the observed light, which enable characterization and separation. To evaluate the characteristics of the components in the flow stream, light must impinge on the flow stream and be collected. The light sources within a flow cytometer can vary and can include one or more broad-spectrum lamps, light-emitting diodes, and single-wavelength lasers. The light source is aligned with the flow stream, and the optical response from the irradiated particles is collected and quantified.

[0003] The isolation of biological particles is achieved by adding sorting or collection capabilities to a flow cytometer. Particles within a separated stream that are detected as having one or more desired characteristics are individually isolated from the sample stream by mechanical or electrical removal. Common flow sorting techniques utilize droplet sorting in which a fluid stream containing linearly separated particles is segmented into droplets. Droplets containing the particles of interest are electrically charged and deflected into a collection tube by passing through an electric field. Typically, linearly separated particles within the stream are characterized as they pass through an observation point located just below the nozzle tip. Once a particle is identified as meeting one or more desired criteria, the particle reaches a splitting point and the time at which the stream will split into droplets can be predicted. Ideally, just prior to the droplet containing the selected particle splitting from the stream, a small charge is applied to the fluid stream and the droplet is grounded immediately after splitting. Droplets to be sorted maintain their charge when splitting from the fluid stream while all other droplets remain uncharged.

[0004] Droplet formation at the nozzle tip has conventionally been achieved via a piezoelectric oscillator (also referred to as a piezoelectric actuator). Such a piezoelectric oscillator generates a small perturbation in the sheath flow, inducing jet flow instability and forming droplets. Figure 1 depicts a conventional cell sorter having a piezoelectric oscillator. As shown in Figure 1, a liquid sample enters at the end portion 101. The piezoelectric oscillator 102 generates a flow perturbation to form droplets, and the stream charging wire 103 applies a charge. The sample is irradiated with light in an inspection zone 104 where scattered light and fluorescent light are generated and collected for analysis. The droplets then split at the distal end of the nozzle 106, and the deflection plate 107 attracts or repels the droplets. Uncharged droplets pass into the waste 108 and charged droplets are received into the collection vessel 109. Such conventional droplet formation methods use various nozzle sizes to generate various droplet sizes. High pressure in the fluid system places stringent requirements on the construction materials and fluid seals. SUMMARY OF THE INVENTION

[0005] The inventors recognize that a conventional cell sorting system having a piezoelectric oscillator as depicted in FIG. 1 causes fluid perturbation upstream of the inspection zone, thereby degrading the quality of the data obtained therefrom. In addition, such a system requires nozzles of different diameters to generate droplets of different sizes. Thus, a single nozzle has been found to be desirable for generating droplets of different sizes that do not generate upstream fluid perturbation. Embodiments of the present invention meet this need.

[0006] Aspects of the present invention include a nozzle having an elongated body and a radially positioned gas inlet at a proximal end of the elongated body. The subject elongated body includes a proximal opening configured to engage in a liquid receiving relationship with a flow cell, a distal opening for discharging liquid droplets, and a channel configured to transfer liquid from the proximal end to the distal end through the elongated body. The subject gas inlet comprises a radial airflow path configured to provide gas to the channel. In some cases, the radial airflow path includes a height in the range of 150 μm to 250 μm (e.g., 200 μm). In certain cases, the radial airflow path includes a radius in the range of 200 μm to 300 μm (e.g., 250 μm). The gas inlet may include a plurality of radial airflow paths in some embodiments, such as where the number of radial airflow paths of the plurality of radial airflow paths ranges from 2 to 5 (e.g., 4). In some aspects, the gas inlet is configured to be operably connected to one or more gas conduits. The channel may have a diameter in the range of 150 μm to 250 μm (e.g., 200 μm) in a selected aspect, may have a constant diameter over the length of the elongated body, or may not. In certain cases, the elongated body is cylindrical and has a length in the range of 4 mm to 4.5 mm (e.g., 4.3 mm).

[0007] Aspects of the present invention additionally include a particle sorter having a nozzle of the subject matter. The particle sorter of interest includes a flow cell configured to transport particles in a flow stream, an elongate body, and a nozzle having a radially positioned gas inlet at a proximal end of the elongate body (e.g., as described above). The particle sorter of the present invention may, in embodiments, include a source of compressed gas such as a pneumatic compressor or a gas canister. In some cases, the source of compressed gas is configured to produce a gas having a pressure in the range of 2 kPa to 10 kPa. In certain aspects, the particle sorter includes one or more gas conduits configured to operably connect the source of compressed gas to the gas inlet. Embodiments of the present invention additionally include a processor operably connected to the source of compressed gas and configured to regulate the pressure of the produced gas provided to the gas inlet. In some cases, the nozzle is not operably attached to a piezoelectric actuator. The particle sorter according to some aspects also includes a light source configured to irradiate the flow stream at an inspection point, a detector configured to collect particle-modulated light from the flow cell, a plurality of receptacles configured to receive droplets emitted through an opening at a distal end of the elongate structure, and one or more of a deflection plate configured to deflect a droplet within a given receptacle of the plurality of receptacles.

[0008] Aspects of the present invention also include a method of sorting a particle sample. The method of interest includes introducing a particle sample into a cell sorter comprising a flow cell configured to transport particles in a flow stream and a nozzle of the subject matter (e.g., as described above), and sorting the particle sample by flow cytometry. In some embodiments, the method includes regulating the pressure of a gas provided to the gas inlet by a source of compressed gas. The particle sample to be sorted may, in some cases, be a biological sample such as a cell.

[0009] The elements of the present disclosure additionally include a method of assembling a particle sorter. The method in question includes operably connecting a nozzle, such as the nozzle described above, to a flow cell configured to transport particles in a flow stream. In the subject method, operably connecting the nozzle includes engaging an opening at the proximal end of the elongate body with the flow cell in a liquid receiving relationship. In some instances, the method includes gas connecting a source of compressed gas (e.g., an air compressor) to the gas inlet. In a particular instance, the method includes operably connecting one or more gas conduits to the source of compressed gas and the gas inlet. When selected, the method includes connecting a processor to the source of compressed gas, the processor being configured to regulate the pressure of the resulting gas provided to the gas inlet. The method according to a particular embodiment does not include operably attaching a piezoelectric actuator to the nozzle.

[0010] Aspects of the invention further include a kit. The kit in question includes the subject nozzle (e.g., as described above). In some cases, the kit also includes a source of compressed gas (e.g., an air compressor). The kit according to some embodiments of the invention includes one or more gas conduits configured to operably connect the source of compressed gas to the gas inlet. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] The present invention can be best understood from the following detailed description when read in conjunction with the accompanying drawings. The drawings include the following figures.

[0012]

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[0013] A particle sorter nozzle is provided. The subject nozzle includes an elongated body and a gas inlet positioned radially at the proximal end of the elongated body. The gas inlet of the subject nozzle includes a radial airflow path configured to provide gas to a channel. The elongated body includes a proximal opening configured to engage a flow cell in a liquid receiving relationship, a distal opening for discharging liquid droplets, and a channel configured to transfer liquid from the proximal end to the distal end through the elongated body. Also provided are a particle sorter having the subject nozzle, methods of using and assembling the subject particle sorter, and kits.

[0014] Before the present invention is described in more detail, it is to be understood that the present invention is not limited to the particular embodiments described, and accordingly, can of course vary. Also, since the scope of the present invention will be limited only by the appended claims, it is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0015] When a range of values is provided, unless the context clearly dictates otherwise, each intermediate value between the upper and lower limits of that range, to one tenth of the unit of the lower limit, and any other stated value or intermediate value within the stated range of this description is to be understood as being included in the present invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also included within the present invention, subject to any specific excluded limitations within the stated range. When the stated range includes one or both of the limitations, ranges excluding one or both of those included limitations are also included in the present invention.

[0016] In this specification, the term "about" is prefixed to a numerical value to present a particular range. The term "about" is used in this specification to provide literal support for the exact number that it precedes, as well as for a number that is close to or substantially the same as the number that the term precedes. In determining whether a number is close to or substantially the same as a specifically recited number, a number that is close to or substantially the same as an unrecited number may be a number that provides substantial equivalence to the specifically recited number in the context presented.

[0017] Unless defined otherwise, all technical and scientific terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Also, any methods and materials similar to or equivalent to those described herein can be used in the practice or testing of the present invention, but representative exemplary methods and materials are described below.

[0018] All publications and patents cited in this specification are hereby incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference, and incorporated by reference herein to disclose and describe the relevant methods and / or materials by which those publications are cited. Any citation of a publication is for its disclosure prior to the filing date, and the present invention should not be construed as admitting that the invention has no right to antedate such a publication by virtue of the features of the prior invention. Further, the dates of the publications provided may be different from the actual publication dates that may need to be independently confirmed.

[0019] As used in this specification and the appended claims, it should be noted that the singular forms of "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. It should further be noted that the claims may be drafted to exclude any optional element. Accordingly, this description is intended to serve as a precedent for the use of exclusive terms such as "solely", "only", etc. or the use of "negative" limitations in connection with the recitation of claim elements.

[0020] As will be apparent to those skilled in the art upon reading this disclosure, each of the separate embodiments described and illustrated herein can be readily separated from, or combined with, the features of any of the various other embodiments without departing from the scope or spirit of the present invention. Any recited method can be performed in the order of the recited events, or any other order that is logically possible.

[0021] The systems and methods are described, or are to be described, with functional descriptive flexibility, but the claims are not to be construed as necessarily limited by the construction of "means" or "step" limitations unless expressly set forth under 35 U.S.C. § 112, and are to be given the meaning ascribed to the definition provided by the claims under the principles of statutory equivalents and to the full scope of equivalents thereof, and it is expressly understood that where the claims are expressly set forth under 35 U.S.C. § 112, full statutory equivalents are to be given thereunder.

[0022] Particle sorter nozzle As described above, aspects of the present invention include a particle sorter nozzle. The subject nozzle includes an elongated body and a radially positioned gas inlet at a proximal end of the elongated body. In some cases, the nozzles described herein enable the formation of liquid droplets having any convenient diameter. In these cases, the formation of liquid droplets having different diameters can be achieved without adjusting the nozzle itself (e.g., by replacing the nozzle with another nozzle having a different diameter). For example, in some cases, the liquid droplets produced by the subject nozzle can have any convenient diameter in the range of 75 μm to 230 μm, as desired. The liquid droplets can have any convenient volume in the range of, for example, 0.25 nL to 6 nL, as desired. Additionally, the subject nozzle can reduce fluid perturbation upstream of the inspection zone by, for example, 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 100%.

[0023] In addition to the above, the nozzles of the subject matter can be used to generate liquid droplets without using a piezoelectric oscillator. The nozzles described herein can instead be used to generate droplets by using two immiscible fluids of a dispersed phase and a continuous phase. The dispersed phase is the fluid that is broken down into droplets (e.g., a flow stream containing particles). The continuous phase (e.g., air) is immiscible with the dispersed phase and is driven independently. Without being bound by theory, it has been found that when the dispersed phase and the continuous phase converge at a junction, the interaction of the two phases at the junction leads to droplet formation. Additional information regarding the physical mechanism using the continuous phase and the dispersed phase can be found in Baroud et al. Lab Chip (2010) 10, 2032-2045, the disclosure of which is incorporated herein by reference.

[0024] "Elongated body" means that the nozzle of the subject has a length greater than its width. In other words, the nozzle has a distinct proximal end and a distal end. The proximal end is the end where the nozzle receives liquid from the flow cell when the nozzle is used in a particle sorter. The distal end is the end where the liquid splits to form droplets when the nozzle is used in a particle sorter. The elongated body can have any convenient cross-sectional shape, and examples of the cross-sectional shape of interest include, but are not limited to, linear cross-sectional shapes such as square, rectangular, trapezoidal, triangular, hexagonal, etc., curved cross-sectional shapes such as circular, elliptical, and irregular shapes such as a parabolic bottom portion coupled to a planar upper portion. In an embodiment, the elongated body has a substantially circular cross-sectional shape at positions along its length. "Substantially" circular cross-section means that in an embodiment, one or more positions along the length of the nozzle can have a cross-section that deviates slightly from the circular cross-section that characterizes the rest of the structure. For example, in some aspects, the elongated structure has a cross-section of a polygon (e.g., hexagonal, pentagonal, etc.) at one or more positions along its length. In other words, in such aspects, the elongated body is not a perfect cylinder, but instead has some regions with a circular cross-sectional shape having a diameter larger than the diameter of other regions. In other cases, the elongated body is, for example, cylindrical over its entire length. The elongated body can have any convenient length such that the length is in the range of 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 a particular case, the length of the elongated body is 4.3 mm.

[0025] The nozzle of interest includes a proximal opening configured to engage in a liquid receiving relationship with a flow cell. In an embodiment, the opening is located at the geometric center of the cross-section of the nozzle at the proximal end. The "liquid receiving" relationship means that the subject nozzle is configured to contact the flow cell such that liquid being transported through the flow cell (e.g., for analysis) can be received within the nozzle at the proximal end of the elongated body. The proximal end of the elongated body can be configured to contact the flow cell in any convenient manner. For example, in certain cases, the proximal end of the elongated body contacts the flow cell in a liquid-tight and / or air-tight manner. In such cases, fluid and / or air does not enter or exit the flow cell or the nozzle at the interface between the two components. The proximal end can include a gasket or O-ring in certain cases to maintain a liquid-tight and / or air-tight seal. In other cases, the proximal end includes a groove for receiving an O-ring. The nozzle can include any convenient means for operably attaching to the flow cell, including but not limited to clamps, magnets, latches, notches, countersinks, counterbores, grooves, pins, tethers, hinges, non-permanent adhesives, or combinations thereof.

[0026] The proximal opening can have any convenient cross-sectional shape, examples of which include, but are not limited to, linear cross-sectional shapes such as square, rectangular, trapezoidal, triangular, hexagonal, etc., curved cross-sectional shapes such as circular, elliptical, and irregular shapes such as a parabolic bottom portion joined to a planar upper portion. In certain cases, the opening has a circular cross-sectional shape. In such cases, the opening can have any suitable diameter in the range of 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 a diameter of 199 μm to 201 μm. In certain embodiments, the opening has a diameter of 200 μm.

[0027] The nozzle in question also includes an opening at the distal end for discharging liquid droplets. The opening at the distal end can have any convenient cross-sectional shape, and examples of the cross-sectional shape in question include, but are not limited to, linear cross-sectional shapes such as square, rectangular, trapezoidal, triangular, hexagonal, etc., curved cross-sectional shapes such as circular, elliptical, and irregular shapes such as a parabolic bottom portion coupled to a planar upper portion. In certain cases, the opening has a circular cross-sectional shape. In such cases, the opening at the distal end can have any suitable diameter in the range of 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 a diameter of 199 μm to 201 μm. In certain embodiments, the opening has a diameter of 200 μm.

[0028] The nozzle of the subject matter also includes a channel within an elongated body extending from the proximal end to the distal end and is configured to transfer liquid therethrough. The channel can have any convenient cross-sectional shape, and examples of the cross-sectional shape in question include, but are not limited to, linear cross-sectional shapes such as square, rectangular, trapezoidal, triangular, hexagonal, etc., curved cross-sectional shapes such as circular, elliptical, and irregular shapes such as a parabolic bottom portion coupled to a planar upper portion. In certain cases, the channel has a circular cross-sectional shape. In such cases, the channel can be cylindrical. The channel can have any convenient diameter in the range of 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 a diameter of 199 μm to 201 μm. In certain embodiments, the channel has a diameter of 200 μm. The channel can have a constant or variable diameter over its length. In some cases, the channel includes a constant diameter over the entire length of the elongated body. In such cases, the opening at the proximal end, the opening at the distal end, and the entire length of the channel have the same diameter.

[0029] The nozzle in question includes a gas inlet positioned radially at the proximal end of the elongated body. "Gas inlet" means a portal that includes one or more paths through which gas from an external source can be transferred into the interior of the nozzle. The gas inlet includes a radial airflow path configured to provide gas to the channel. "Radial airflow path" means a path disposed within the gas inlet through which gas travels to the channel. The gas inlet and the radial airflow path that constitutes it extend radially with respect to an imaginary axis extending through the length of the elongated body. The gas inlet includes any suitable number of radial airflow paths. In certain embodiments, the gas inlet includes a single radial airflow path. In other embodiments, the gas inlet includes a plurality of radial airflow paths. In such embodiments, the number of radial airflow paths can range from 2 to 6, including 2 to 5. In a particular case, the gas inlet comprises four radial airflow paths. The radial airflow path can have any convenient shape. In some cases, each radial airflow path is wedge-shaped. In such cases, the opening of the radial airflow path facing the external environment is wide, and the opening of the radial airflow path facing the channel is relatively narrow. Each radial airflow path can have any convenient length. The length of each radial airflow path can be conceptualized as a radius measured from an imaginary axis extending through the length of the elongated body to the external environment (i.e., the end of the path). For example, the radial airflow path can have a radius in the range of 150 μm to 350 μm, such as 175 μm to 325 μm, such as 200 μm to 300 μm, such as 225 μm to 275 μm, such as 245 μm to 255 μm, including 249 μm to 251 μm. In a particular case, the radial airflow path has a radius of 250 μm. Each radial airflow path may have any convenient height, i.e., when measured from the upper surface of the radial airflow path to the bottom surface of the radial airflow path, the upper 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 height of the radial airflow path ranges from 100 μm to 300 μm, including 199 - 201 μm, for example from 125 μm to 275 μm, for example from 150 μm to 250 μm, for example from 175 μm to 225 μm, for example from 190 μm to 210 μm, for example from 195 μm to 205 μm. In the selected case, the radial airflow path includes a height of 200 μm.

[0030] In some embodiments, the gas inlet is configured to be operably connected to one or more gas conduits. A "gas conduit" means a channel through which gas can be provided to the gas inlet or the radial airflow path it constitutes. The radial airflow path may be configured to contact the gas conduit. For example, each radial airflow path may have a shape complementary to the shape of the gas conduit, such that the gas conduit can be releasably attached to the radial airflow path. In some embodiments, the gas conduit is gas-connected to the radial airflow path via press-fitting. In other cases, the gas conduit may be attached to the radial airflow path via a clamp, magnet, latch, notch, countersink, counterbore, groove, pin, tether, hinge, non-permanent adhesive, or a combination thereof.

[0031] The nozzle of the subject matter can be composed of any convenient material. In certain instances, the nozzle includes one or more polymeric materials. For example, in some embodiments, the nozzle includes one or more rigid plastic materials among other polymeric plastic materials such as, for example, polycarbonate, polyvinyl chloride (PVC), polyurethane, polyether, polyamide, polyimide, etc. Examples of polymeric materials include acrylonitrile butadiene styrene (ABS), polylactic acid (PLA), acrylonitrile styrene acrylonitrile (ASA), polyethylene terephthalate (PET), glycol-modified polyethylene terephthalate (PETG), polyaryletherketone (PAEK), polyetherimide (PEI), polycarbonate (PC), polypropylene (PP), aliphatic polyamide (PPA), polyoxymethylene (POM), polymethyl methacrylate (PMMA), polybutylene terephthalate (PBT), polyphenylsulfone (PPSU), polyetheretherketone (PEEK), and nylon as well as their composite materials and mixtures. In certain cases, the nozzle is composed of PEEK. In some embodiments, the nozzle includes a glass-filled polymer (i.e., having glass fibers in a matrix of a polymeric material). In such embodiments, any suitable polymer (such as those described above, for example) can be combined with glass fibers to produce a glass-filled polymer. In certain instances, the nozzle includes one or more metal components including, for example, aluminum, titanium, brass, iron, lead, nickel, steel (such as stainless steel), copper, tin, and combinations and alloys thereof.

[0032] Figure 2A depicts a nozzle according to a particular embodiment of the present invention. The nozzle 200 includes an elongated body 201 (only a partial view is shown) having an opening 202 at the proximal end. In the example of Figure 2A, a portion of the elongated body 201 is such that the channel 203 is not visible. The nozzle 200 also includes a gas inlet 204 that includes a wedge-shaped radial airflow path 205. The radial airflow path 205 is configured to receive the gas conduit 206 shown in Figure 2B in order to provide gas to the channel 203.

[0033] Particle sorter Aspects of the present invention additionally include a particle sorter. The particle sorter of interest includes an elongate body and a nozzle having a gas inlet (e.g., as described above). A "particle sorter" is any device configured to sort particles (e.g., beads, cells, etc.) by flow cytometry. The particle sorter of interest may or may not include a piezoelectric actuator. In embodiments, the nozzle is not operably attached to a piezoelectric actuator. "Not operably attached" means that the piezoelectric actuator is not present near the flow cell or nozzle such that it provides sufficient fluid perturbation to cause droplet breakup during operation. In embodiments where no piezoelectric actuator is present, droplets are formed by combining a dispersed phase that includes sheath fluid (optionally containing particles) received from a flow cell with a continuous phase that includes a gas. The continuous phase (e.g., air) is immiscible with the dispersed phase, and the interaction of the two phases at the gas inlet of the nozzle is sufficient to generate droplets at the distal end of the nozzle. Additionally, aspects of the particle sorter of interest are configured to adjust the size / volume of the generated droplets by varying the amount of pressure applied to the nozzle at the gas inlet. When selected, the size / volume of the generated droplets is adjusted without changing the nozzle or otherwise varying the diameter of the nozzle.

[0034] In addition to the nozzles described above, the particle sorter of the subject matter includes a flow cell having a flow channel for transporting particles in a flow stream therethrough from a proximal inlet to a distal outlet. As described herein, a "flow cell" is described in its conventional sense as a component that includes a flow channel having a liquid flow stream for transporting particles in a sheath fluid. In embodiments, the flow cell of the subject matter includes a cuvette. The cuvette of interest includes a container with a passage extending therein. The flow stream may include a liquid sample injected from a sample tube. The flow cell of interest includes a flow channel that is optically accessible. In some cases, the flow cell includes a transparent material (e.g., quartz) that allows light to pass through. Any convenient flow cell, such as the flow cells described herein, that propagates a fluid sample to a sample inspection region may be used. In some embodiments, the flow cell includes a cylindrical flow cell, a frustoconical flow cell, or a flow cell that includes a proximal cylindrical portion defining a longitudinal axis and a distal conical portion that terminates in a flat surface having an orifice that is transverse to the longitudinal axis.

[0035] In certain embodiments, the particle sorter includes a sample fluid source. The sample fluid source can be any suitable reservoir or container (e.g., having rigid or flexible walls) for holding the sample fluid. The sample fluid container can have a volume in the range of 1 mL to 100 mL. For example, the volume of the container can be in the range of 1 mL to 90 mL, 1 mL to 80 mL, 1 mL to 70 mL, 1 mL to 60 mL, 1 mL to 50 mL, 1 mL to 40 mL, 1 mL to 30 mL, 1 mL to 20 mL, or 1 mL to 10 mL.

[0036] In some embodiments, the particle sorter includes a sheath fluid source. The sheath fluid source can be any suitable reservoir or container (e.g., having a rigid or flexible wall) for holding the sheath fluid. In certain embodiments, the sheath fluid source is fluidly coupled to the input of the flow cell. The sheath fluid container can have a volume in the range of 1 L to 100 L. For example, the volume of the container can be in the range of 1 L to 90 L, 1 L to 80 L, 1 L to 70 L, 1 L to 60 L, 1 L to 50 L, 1 L to 40 L, 1 L to 30 L, 1 L to 20 L, or 1 L to 10 L.

[0037] In some embodiments, the flow cell includes a sample injection port configured to provide a sample from a sample fluid source to the flow cell. The sample injection port can be an orifice positioned in the wall of the internal chamber or a conduit positioned at the proximal end of the internal chamber. When the sample injection port is an orifice positioned in the wall of the internal chamber, the sample injection port orifice can be of any suitable shape, and examples of the cross-sectional shape of the object include, but are not limited to, linear cross-sectional shapes such as square, rectangular, trapezoidal, triangular, hexagonal, etc., curved cross-sectional shapes such as circular, elliptical, etc., and irregular shapes such as a parabolic bottom portion coupled to a planar upper portion. In certain embodiments, the sample injection port has a circular orifice. The size of the orifice of the sample injection port can vary depending on the shape. In certain cases, it has an opening, for example, in the range of 0.2 to 3.0 mm, such as 0.5 mm to 2.5 mm, 0.75 mm to 2.25 mm, 1 mm to 2 mm, 0.1 mm to 5.0 mm, including 1.25 mm to 1.75 mm, for example, having an opening of 1.5 mm.

[0038] In certain instances, the sample injection port is a conduit positioned at the proximal end of the flow cell internal chamber. For example, the sample injection port can be a conduit positioned such that it has an orifice of the sample injection port along the flow cell orifice. When the sample injection port is a conduit positioned along the flow cell orifice, the cross-sectional shape of the sample injection tube can be any suitable shape, and examples of the cross-sectional shape of interest include, but are not limited to, linear cross-sectional shapes such as square, rectangular, trapezoidal, triangular, hexagonal, etc., curved cross-sectional shapes such as circular, elliptical, and irregular shapes such as a parabolic bottom portion coupled to a planar upper portion. The orifice of the conduit can vary depending on the shape, and in certain instances, it has an opening in the range of 0.1 mm to 5.0 mm, such as 1.25 mm to 1.75 mm, for example, 0.2 to 3.0 mm, for example, 0.5 mm to 2.5 mm, 0.75 mm to 2.25 mm, etc., such as 1 mm to 2 mm, such as 1.5 mm. The shape of the tip of the sample injection port can be the same as or different from the cross-sectional shape of the sample injection tube. For example, the orifice of the sample injection port can include a beveled tip having a bevel angle in the range of 1 degree to 10 degrees, such as 2 degrees to 9 degrees, such as 3 degrees to 8 degrees, such as 4 degrees to 7 degrees, including a 5-degree bevel angle.

[0039] In some embodiments, the flow cell also includes a sheath fluid injection port configured to provide sheath fluid from a sheath fluid source to the flow cell. In an embodiment, the sheath fluid injection system is configured to provide a flow of sheath fluid to the flow cell internal chamber, for example, in combination with the sample, to generate a laminar flow stream of sheath fluid surrounding the sample flow stream. Depending on the desired characteristics of the flow stream, the velocity of the sheath fluid transmitted to the flow cell chamber can be in the range of 50 μL / second to 1000 μL / second, such as 75 μL / second or more to 750 μL / second, such as 25 μL / second to 2500 μL / second.

[0040] In some embodiments, the sheath fluid injection port is an orifice positioned in the wall of the internal chamber. The orifice of the sheath fluid injection port can be of any suitable shape, and the cross-sectional shape of the object is not limited to the following, for example, linear cross-sectional shapes such as square, rectangular, trapezoidal, triangular, hexagonal, etc., curved cross-sectional shapes such as circular, elliptical, and irregular shapes such as a parabolic bottom portion coupled to a planar upper portion. The size of the orifice of the sample injection port can vary depending on the shape. In certain cases, it includes 1.25 mm to 1.75 mm, for example, in the range of 0.2 to 3.0 mm, for example, 0.5 mm to 2.5 mm, 0.75 mm to 2.25 mm, etc., 1 mm to 2 mm, etc., 0.1 mm to 5.0 mm, and has an opening of, for example, 1.5 mm.

[0041] In some embodiments, the system further includes a pump in fluid communication with the flow cell to propagate a flow stream through the flow cell. Any convenient fluid pump protocol can be used to control the flow of the flow stream through the flow cell. In certain cases, the system includes a peristaltic pump, such as a peristaltic pump with a pulse damper. The pump in the subject system is configured to transfer fluid through the flow cell at a rate suitable for multi-photon counting of light from the sample in the flow stream. For example, the system can include a pump configured to flow the sample through the flow cell at a rate in the range of 1 nL / min to 250 nL / min, including 10 nL / min to 50 nL / min, for example, 1 nL / min to 100 nL / min, 2 nL / min to 90 nL / min, 3 nL / min to 80 nL / min, 4 nL / min to 70 nL / min, 5 nL / min to 60 nL / min, 1 nL / min to 500 nL / min. In certain embodiments, the flow rate of the flow stream is 5 nL / min to 6 nL / min.

[0042] The flow stream within the flow cell is configured to be irradiated with light from a light source at an inspection point. The flow stream can include a liquid sample injected from a sample tube. In certain embodiments, the flow stream can include a narrow, rapidly flowing stream of liquid arranged such that linearly separated particles transported therein are separated from each other in a single file manner. As used herein, an "inspection point" refers to, for example, a region within the flow cell where particles are irradiated by light from a light source for analysis. The size of the inspection point can vary as desired. For example, if 0 μm represents the axis of the light emitted by the light source, the inspection point can range from -15 μm to 30 μm, including -50 μm to 50 μm, such as -25 μm to 40 μm, or -100 μm to 100 μm.

[0043] The distal end of the flow cell is operably connected to the proximal end of the nozzle such that the nozzle is in a liquid receiving relationship with the flow cell. The proximal end of the elongated body can contact the flow cell in any convenient manner. For example, in certain cases, the proximal end of the elongated body contacts the flow cell in a liquid tight and / or air tight manner. In such cases, fluid and / or air does not enter or exit the flow cell or the nozzle at the interface between the two components. The proximal end can include, in certain cases, a gasket or O-ring to maintain a liquid tight and / or air tight seal. In other cases, the proximal end includes a groove for receiving an O-ring. The nozzle can include any convenient means for operably attaching to the flow cell, including, but not limited to, a clamp, magnet, latch, notch, countersink, counterbore, groove, pin, tether, hinge, non-permanent adhesive, or combinations thereof.

[0044] In certain cases, the particle sorter includes a compressed gas source. The compressed gas source may be any device that generates and / or stores gas under pressure. In some embodiments, the compressed gas source is a gas compressor. In other cases, the compressed gas source may be a gas canister in which gas is stored under pressure. Any convenient gas may be used. Examples of the target gas include, but are not limited to, O2, CO2, N2, etc., and combinations thereof. In certain cases, the gas is the atmosphere from the surrounding environment. In such cases, the gas can be purified to remove any particles that may potentially contaminate the sorted sample. For example, the compressed gas source may be gas-connected to a compressed air filter configured for particle removal. In some embodiments, the target filter may be configured to filter particles having a size in the range of 1 micron to 100 microns. In some cases where the compressed gas source is a gas canister, the gas stored in the canister can be purified. The compressed gas source may be configured to provide a gas having any convenient pressure to the nozzle. For example, in a selected embodiment, the compressed gas source is configured to generate a gas having a pressure in the range of 2 kPa to 10 kPa. The compressed gas source may be adjustable such that the pressure of the gas received at the gas inlet can vary as desired (e.g., to generate droplets having different sizes). In some cases where the compressed gas source is a gas canister, the pressure can be adjusted, for example, by a pressure regulator. When the compressed gas source is a gas compressor, the operation of the compressor can be adjusted to regulate the pressure of the gas generated therefrom.

[0045] In some embodiments, the particle sorter includes one or more gas conduits. As described above, a gas conduit is a channel through which gas can be provided to the gas inlet of the nozzle of interest or to the radial airflow path that it constitutes. In some cases, the gas conduit is configured to operably connect a compressed gas source to the gas inlet. Any convenient number of gas conduits may be used. In some cases, the particle sorter includes a single gas conduit that gas connects a compressed gas source to a single radial airflow path within the gas inlet. In other cases, the particle sorter includes a plurality of gas conduits. In some such cases, the number of gas conduits within the plurality of gas conduits ranges from 2 to 6, including 2 to 5. In a particular case, the particle sorter includes four gas conduits. The gas conduit can have any convenient cross-sectional shape, and examples of the cross-sectional shape of interest include, but are not limited to, linear cross-sectional shapes such as square, rectangular, trapezoidal, triangular, hexagonal, etc., curved cross-sectional shapes such as circular, elliptical, and irregular shapes such as a parabolic bottom portion joined to a planar upper portion. In some embodiments, the gas conduit has a rectangular cross-sectional shape.

[0046] The gas conduit can be made of any convenient material. In certain cases, the gas conduit includes one or more polymeric materials. For example, in some embodiments, the gas conduit includes one or more rigid plastic materials among other polymeric plastic materials such as, for example, polycarbonate, polyvinyl chloride (PVC), polyurethane, polyether, polyamide, polyimide, etc. Examples of polymeric materials include acrylonitrile butadiene styrene (ABS), polylactic acid (PLA), acrylonitrile styrene acrylate (ASA), polyethylene terephthalate (PET), glycol-modified polyethylene terephthalate (PETG), polyaryletherketone (PAEK), polyetherimide (PEI), polycarbonate (PC), polypropylene (PP), aliphatic polyamide (PPA), polyoxymethylene (POM), polymethyl methacrylate (PMMA), polybutylene terephthalate (PBT), polyphenylsulfone (PPSU), polyetheretherketone (PEEK), and nylon as well as their composite materials and mixtures. In certain cases, the gas conduit is composed of PEEK. In some embodiments, the gas conduit includes a glass-filled polymer (i.e., having glass fibers in a matrix of a polymeric material). In such embodiments, any suitable polymer (such as those described above, for example) can be combined with glass fibers to produce a glass-filled polymer. In certain cases, the gas conduit includes one or more metal components including, for example, aluminum, titanium, brass, iron, lead, nickel, steel (such as stainless steel), copper, tin, as well as combinations and alloys thereof.

[0047] Figure 3 depicts one view of the particle sorter 300 according to a particular embodiment of the present invention. As shown in FIG. 3, the particle sorter 300 includes a flow cell 301. Additionally, a titanium hose return portion 302 is provided through which fluid can be provided to the flow cell, for example, to generate a laminar flow. Particles traveling through the flow cell 301 are irradiated by a laser beam at an irradiation point 303. The distal end of the flow cell 301 is operably connected to the proximal end of a nozzle 304. The particle sorter 300 additionally includes a gas conduit 305 through which compressed gas is provided to the channel of the nozzle 304. Providing gas to the channel through the gas inlet at the proximal end of the nozzle is sufficient to precipitate droplet formation at the distal end of the nozzle.

[0048] In an embodiment, the particle sorter additionally includes a processor operably connected to a compressed gas source. The subject processor may be configured to adjust the pressure of the gas provided to the gas inlet. For example, the subject processor may be operated in conjunction with programmable logic implemented in hardware, software, firmware, or any combination thereof to adjust the pressure of the gas provided to the gas inlet. For example, where the programmable logic is implemented in software, the pressure adjustment may be at least partially realized by a computer-readable data storage medium containing program code including instructions configured to adjust the pressure of the gas provided to the gas inlet when executed. For example, where the compressed gas source is a gas canister, the processor may be operably connected to a regulator associated with the gas canister such that a desired level of pressure can be applied to the gas inlet of the nozzle. In an aspect where the compressed gas source is a gas compressor, the processor may be operably connected to the gas compressor such that the processor controls the activity of the compressor and a desired level of pressure is achieved. In some cases, the processor is configured to increase the pressure to decrease the droplet size and decrease the pressure to increase the droplet size.

[0049] In an embodiment, the particle sorter includes a droplet deflector configured to redirect droplets containing the analyzed cells from a stream of droplets generated from a flow stream emitted from a flow nozzle to a receiving location. Redirecting the target droplets to the receiving location can be achieved by the droplet deflector via electrostatic charging of the droplets and deflection of the charged droplets from the flow stream by application of an electrostatic field. Such an electrostatic field can be formed by deflection plates positioned adjacent to the flow stream. As used herein, the terms "deflection" or "deflected" mean that the cells can be identified and tracked in the flow stream and only those droplets of the flow stream containing those target cells are redirected and collected by the collection vessel, referring to the electrostatic deflection of the target droplets from the flow stream of the analyzed droplets. In some cases, the particle sorter includes deflection plates configured to deflect a single droplet into each collection vessel.

[0050] The deflection plates within the target sort block can be configured based on the type of cells being sorted, the speed of sorting, the voltage applied to the cells, and the number of components being sorted in the sample. In an embodiment, a suitable length of the deflection plate can be in the range of 5 mm to 100 mm, including 10 mm to 50 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. The width of the deflection plate can vary in the range of 1 mm to 25 mm, including 5 mm to 10 mm, such as 2 mm to 20 mm, such as 3 mm to 15 mm. The distance between each deflection plate can vary depending on the applied voltage and the size of the particles being sorted in the flow stream. In some embodiments, the distance between each deflection plate can be 1 mm or more, including 10 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. For example, the distance between each deflection plate can be in the range of 1 mm to 25 mm, including 5 mm to 15 mm, such as 2 mm to 22.5 mm, such as 3 mm to 20 mm, such as 4 mm to 17.5 mm. The deflection plates can also be oriented at an angle to each other in the range of 15° to 75°, including 30° to 60°, such as 20° to 70°, such as 25° to 65°.

[0051] The voltage applied to the deflection plate to deflect charged particles can be 10 mV or more, including 10000 mV or more, such as 25 mV or more, 50 mV or more, 100 mV or more, 250 mV or more, 500 mV or more, 750 mV or more, 1000 mV or more, 2500 mV or more, 5000 mV or more. In certain embodiments, the voltage applied to the deflection plate is in the range of 0.001 V to 6000 V, including 0.001 V to 5000 V, such as 0.01 V to 4000 V, 0.1 V to 3000 V, 1 V to 2000 V, 5 V to 1500 V, 10 V to 1000 V, 25 V to 750 V, including 100 V to 500 V.

[0052] The deflection plate is configured to deflect particles from the flow stream to a receiving location downstream from the deflection plate. In embodiments, the deflection plate can deflect each particle by a varying angle. In some embodiments, the deflection plate is configured to deflect each particle by an angle of 0.5 degrees or more from the longitudinal axis of the flow stream, including deflecting each particle by an angle of 10 degrees or more from the longitudinal axis of the flow stream, such as 1 degree or more, 1.5 degrees or more, 2 degrees or more, 2.5 degrees or more, 3 degrees or more, 5 degrees or more, 7.5 degrees or more. For example, each particle can be deflected by an angle of 0.1 degrees to 30 degrees from the longitudinal axis of the flow stream, including 5 degrees to 10 degrees, such as 0.5 degrees to 25 degrees, 1 degree to 20 degrees, 2 degrees to 15 degrees.

[0053] Particles within the frost stream can be deflected by any convenient deflection plate protocol, including but not limited to the cell sorting deflection plates described in U.S. Patent 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, 6,809,804, and 7,880,108, the entire disclosures of which are incorporated herein by reference. In certain embodiments, the deflection plate includes a charged plate for sorting cells in a flow stream used in flow cytometry systems such as the BD Biosciences FACSCanto™, BD Biosciences FACSVantage™, BD Biosciences FACSort™, BD Biosciences FACSAria™, BD Biosciences FACSCount™, BD Biosciences FACScan™, and BD Biosciences FACSCalibur™ systems, the BD Biosciences Influx™ cell sorter, and the like.

[0054] The particle sorter of interest may additionally include a light source configured to irradiate particles passing through the flow cell at the inspection point. Any convenient light source can be used as the light source described herein. In some embodiments, the light source is a laser. In an embodiment, the laser can be any convenient 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 cases, 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-fluoride (ArF) excimer laser, a krypton-fluoride (KrF) excimer laser, a xenon chloride (XeCl) excimer laser, or a xenon-fluoride (XeF) excimer laser, or a combination thereof. In other cases, the flow cytometer of interest includes a dye laser such as a stilbene laser, a coumarin laser, or a rhodamine laser. In yet another example, the laser of interest 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 yet another case, the flow cytometer of interest includes a solid 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 slim YAG laser, a ytterbium YAG laser, a Yb2O3 laser, or a cerium-doped laser, and combinations thereof.

[0055] A laser light source according to certain embodiments may also include one or more optical adjustment components. In certain embodiments, the optical adjustment component is located between the light source and the flow cell and can change the spatial width of the irradiation or some other characteristics of the irradiation from the light source, such as, for example, the irradiation direction, wavelength, beam width, beam intensity, and focus. The optical adjustment protocol may include any convenient device for adjusting one or more characteristics of the light source, including, but not limited to, lenses, mirrors, filters, optical fibers, wavelength separators, pinholes, slits, collimating protocols, and combinations thereof. In certain embodiments, the flow cytometer of interest includes one or more focusing lenses. The focusing lens may be, in one example, a non-magnifying lens. In still other embodiments, the flow cytometer of interest includes an optical fiber.

[0056] If the optical adjustment component is configured to move, the optical adjustment component may be configured to move continuously or at discrete intervals, including increments of 25 mm or more, such as increments of 0.05 μm or more, 0.1 μm or more, 0.5 μm or more, 1 μm or more, 10 μm or more, 100 μm or more, 500 μm or more, 1 mm or more, 5 mm or more, 10 mm or more, for example, increments of 0.01 μm or more.

[0057] Any displacement protocol can be used to move the optical adjustment component structure, such as being coupled to a movable support stage or being directly coupled to a geared translation device that uses a motorized translation stage, a lead screw translation assembly, or other types of motors, such as a stepper motor, a servo motor, a brushless electric motor, a brushed DC motor, a microstepping drive motor, a high-resolution stepper motor.

[0058] The light source and the flow cell can be positioned at any suitable distance and angle relative to the flow cell. For example, the light source and the flow cell can be separated by a distance of 0.01 mm or more, such as 0.05 mm or more, 0.1 mm or more, 0.5 mm or more, 1 mm or more, 5 mm or more, 10 mm or more, 25 mm or more, including distances of 100 mm or more. In addition, the light source can be positioned at an angle within the range of 10 degrees to 90 degrees, such as 15 degrees to 85 degrees, 20 degrees to 80 degrees, 25 degrees to 75 degrees, including angles of 30 degrees to 60 degrees, for example, at an angle of 90 degrees, relative to the flow cell at any suitable angle.

[0059] In some embodiments, the light source includes two or more lasers, such as three or more lasers, four or more lasers, five or more lasers, ten or more lasers, including fifteen or more lasers configured to provide laser light for discrete irradiation of the flow stream. Depending on the desired wavelength of the light for irradiating the flow stream, each laser can have a specific wavelength that varies from 250 nm to 1250 nm, such as 300 nm to 1000 nm, 350 nm to 900 nm, 200 nm to 1500 nm, including 400 nm to 800 nm. In certain embodiments, the lasers can include one or more of a 405 nm laser, a 488 nm laser, a 561 nm laser, and a 635 nm laser.

[0060] The particle sorter can further include one or more particle-modulated light detectors for detecting particle-modulated light intensity data. In some embodiments, the particle-modulated light detector includes one or more forward-scattered light detectors configured to detect forward-scattered light. For example, the particle sorter can include one forward-scattered light detector, or a plurality of forward-scattered light detectors, such as two or more, such as three or more, such as four or more, and five or more. In certain embodiments, the particle sorter includes one forward-scattered light detector. In other embodiments, the particle sorter includes two forward-scattered light detectors.

[0061] Any convenient detector for detecting the collected light can be used in the forward-scattered light detector described herein. Among detectors, the detector in question may include, but is not limited to, optical sensors or detectors such as active pixel sensors (APS), avalanche photodiodes, image sensors, charge-coupled devices (CCD), intensified charge-coupled devices (ICCD), light-emitting diodes, photon counters, bolometers, pyroelectric detectors, photoreistors, solar cells, photodiodes, photomultiplier tubes (PMT), phototransistors, quantum dot photoconductors, or photodiodes, as well as combinations thereof. In certain embodiments, the collected light is measured by 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. In certain embodiments, the detector is 1 cm 2 ~5 cm 2 including, 0.01 cm 2 ~10 cm 2 e.g., 0.05 cm 2 ~9 cm 2 e.g., 0.1 cm 2 ~8 cm 2 e.g., 0.5 cm 2 ~7 cm 2 a photomultiplier tube such as a photomultiplier tube having an active detection surface area in the range of each region of.

[0062] In embodiments, the forward-scattered light detector is configured to measure light continuously or at discrete intervals. In some cases, the detector in question is configured to continuously measure the collected light. In other cases, the detector in question is configured to measure light at discrete intervals, such as every 0.001 milliseconds, every 0.01 milliseconds, every 0.1 milliseconds, every 1 millisecond, every 10 milliseconds, every 100 milliseconds, or every some other interval, including every 1000 milliseconds.

[0063] In additional embodiments, one or more particle-modulated light detectors may include one or more side-scattered light detectors for detecting the side-scattered wavelengths of light (i.e., light refracted and reflected from the surface and internal structure of the particles). In some embodiments, the particle sorter includes a single side-scattered light detector. In other embodiments, the particle sorter includes a plurality of side-scattered light detectors, such as two or more, such as three or more, such as four or more, and five or more.

[0064] Any convenient detector for detecting the collected light may be used for the side-scattered light detectors described herein. Suitable detectors include, but are not limited to, optical sensors or detectors such as active pixel sensors (APS), avalanche photodiodes, image sensors, charge-coupled devices (CCD), intensified charge-coupled devices (ICCD), light-emitting diodes, photon counters, bolometers, pyroelectric detectors, photoresistors, solar cells, photodiodes, photomultiplier tubes (PMT), phototransistors, quantum dot photoconductors, or photodiodes, and combinations thereof. In certain embodiments, the collected light is measured with 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. In certain embodiments, the detector has an active detection surface area in each region in the range of 0.01 cm 2 to 5 cm 2 including, for example, 0.05 cm 2 to 10 cm 2 such as, for example, 0.05 cm 2 to 9 cm 2 such as, for example, 0.1 cm 2 to 8 cm 2 such as, for example, 0.5 cm 2 to 7 cm 2 and is a photomultiplier tube such as a photomultiplier tube having an active detection surface area in each region in the range of.

[0065] In an embodiment, the subject particle sorter also includes a fluorescence detector configured to detect one or more fluorescence wavelengths of light. In other embodiments, the particle sorter includes a plurality of fluorescence detectors, for example, two or more, for example, three or more, for example, four or more, five or more, ten or more, fifteen or more, and twenty or more.

[0066] Any convenient detector for detecting the collected light can be used in the fluorescence detectors described herein. Among the detectors, the detectors may include, but are not limited to, active pixel sensors (APS), avalanche photodiodes, image sensors, charge-coupled devices (CCD), intensified charge-coupled devices (ICCD), light-emitting diodes, photon counters, bolometers, pyroelectric detectors, photoresistors, solar cells, photodiodes, photomultiplier tubes (PMT), phototransistors, quantum dot photoconductors, or photodiodes, and combinations thereof. In certain embodiments, the collected light is measured by 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. In certain embodiments, the detector has an active detection surface area in the range of 0.01 cm 2 to 5 cm 2 including, for example, 0.05 cm 2 to 9 cm 2 such as, for example, 0.1 cm 2 to 8 cm 2 such as, for example, 0.5 cm 2 to 7 cm 2 such as, for example, 0.01 cm 2 to 10 cm 2 and is a photomultiplier tube such as a photomultiplier tube having an active detection surface area in each region of the range.

[0067] When the particle sorter of the subject includes a plurality of fluorescence detectors, each fluorescence detector may be the same, or the collection of fluorescence detectors may be a combination of different types of detectors. For example, when the particle sorter of the subject includes two fluorescence detectors, in some embodiments, the first fluorescence detector is a CCD type device, and the second fluorescence detector (or image sensor) is a CMOS type device. In other embodiments, both the first fluorescence detector and the second fluorescence detector are CCD type devices. In still other embodiments, both the first fluorescence detector and the second fluorescence detector are CMOS type devices. In still other embodiments, the first fluorescence detector is a CCD type device, and the second fluorescence detector is a photomultiplier tube (PMT). In still other embodiments, the first fluorescence detector is a CMOS type device, and the second fluorescence detector is a photomultiplier tube. In still other embodiments, both the first fluorescence detector and the second fluorescence detector are photomultiplier tubes.

[0068] In embodiments of the present disclosure, the fluorescence detector of interest is configured to measure the collected light at one or more wavelengths, including measuring the light emitted by a sample in the flow stream at two or more wavelengths, such as five or more different wavelengths, such as ten or more different wavelengths, such as twenty-five or more different wavelengths, such as fifty or more different wavelengths, such as one hundred or more different wavelengths, such as two hundred or more different wavelengths, such as three hundred or more different wavelengths, four hundred or more different wavelengths, and the like. In some embodiments, two or more detectors within the particle sorter described herein are configured to measure the same, or overlapping, wavelengths of the collected light.

[0069] In some embodiments, the fluorescence detector of interest is configured to measure light collected over a range of wavelengths (e.g., 200 nm to 1000 nm). In certain embodiments, the detector of interest is configured to collect the spectrum of light over a range of wavelengths. For example, a particle sorter can include one or more detectors configured to collect the spectrum of light over one or more of the wavelength ranges from 200 nm to 1000 nm. In still other embodiments, the detector of interest is configured to measure light emitted by a sample in a flow stream at one or more specific wavelengths. For example, a particle sorter can include one or more detectors configured to measure one or more of the light at 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 certain embodiments, the one or more detectors can be configured to pair with a specific fluorophore, such as those used with a sample in a fluorescence assay.

[0070] In some embodiments, the particle sorter includes one or more wavelength separators positioned between the flow cell and the particle modulated light detector. The term "wavelength separator" is used herein in its conventional sense to refer to an optical component configured to separate light collected from a sample into a predetermined spectral range. In some embodiments, the particle sorter includes a single wavelength separator. In other embodiments, the particle sorter includes more than 100 wavelength separators, such as, for example, two or more wavelength separators, such as, for example, three or more, such as, for example, four or more, such as, for example, five or more, such as, for example, six or more, such as, for example, seven or more, such as, for example, eight or more, such as, for example, nine or more, such as, for example, ten or more, such as, for example, fifteen or more, such as, for example, twenty-five or more, such as, for example, fifty or more, such as, for example, seventy-five or more wavelength separators. In other embodiments, the wavelength separator is configured to separate light collected from a sample into a predetermined spectral range by passing light having a predetermined spectral range and reflecting light in one or more remaining spectral ranges. In other embodiments, the wavelength separator is configured to separate light collected from a sample into a predetermined spectral range by passing light having a predetermined spectral range and absorbing light in one or more remaining spectral ranges. In still other embodiments, the wavelength separator is configured to spatially diffract light collected from a sample into a predetermined spectral range. Each wavelength separator can be any convenient light separation protocol, such as, for example, 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 certain embodiments, the wavelength separator in the subject light detection system is a dichroic mirror.

[0071] Suitable flow cytometry systems include, but are not limited to, those described in Ormerod (ed.), Flow Cytometry: A Practical Approach, Oxford Univ. Press (1997); Jaroszeski et al. (eds.), Flow Cytometry Protocols, Methods in Molecular Biology No. 91, Humana Press (1997); Practical Flow Cytometry, 3rd ed., Wiley-Liss (1995); Virgo, et al. (2012) Ann Clin Biochem. Jan; 49(pt 1):17-28; Linden, et. al., Semin Throm Hemost. 2004 Oct; 30(5):502-11, Alison, et al. J Pathol, 2010 Dec; 222(4):335-344, and Herbig, et al. (2007) Crit Rev Ther Drug Carrier Syst. 24(3):203-255, the disclosures of which are incorporated herein by reference.In certain instances, the flow cytometry system of interest includes, among others, the BD Biosciences FACSCanto™ flow cytometer, the BD Biosciences FACSCanto™ II flow cytometer, the BD Accuri™ flow cytometer, the BD Accuri™ C6 Plus flow cytometer, the BD Biosciences FACSCelesta™ flow cytometer, the BD Biosciences FACSLyric™ flow cytometer, the BD Biosciences FACSVerse™ flow cytometer, the BD Biosciences FACSymphony™ flow cytometer, the BD Biosciences LSRFortessa™ flow cytometer, the BD Biosciences LSRFortessa™ X-20 flow cytometer, the BD Biosciences FACSPresto™ flow cytometer, the BD Biosciences FACSVia™ flow cytometer, as well as the BD Biosciences FACSCalibur™ cell sorter, the BD Biosciences FACSCount™ cell sorter, the BD Biosciences FACSLyric™ cell sorter, the BD Biosciences Via™ cell sorter, the BD Biosciences Influx™ cell sorter, the BD Biosciences Jazz™ cell sorter, the BD Biosciences Aria™ cell sorter, the BD Biosciences FACSAria™ II cell sorter, the BD Biosciences FACSAria™ III cell sorter, the BD Biosciences FACSAria™ Fusion cell sorter, and the BD Biosciences FACSMelody™ cell sorter, the BD Biosciences FACSymphony™ S6 cell sorter, etc.

[0072] In some embodiments, the subject system is a flow cytometric system such as those described in U.S. Patent 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; 9,095,494; 9,092,034; 8,975,595; 8,753,573; 8,233,146; 8,140,300; 7,544,326; 7,201,875; 7,129,505; 6,821,740; 6,813,017; 6,809,804; 6,372,506; 5,700,692; 5,643,796; 5,627,040; 5,620,842; 5,602,039; 4,987,086; 4,498,766, the disclosures of which are incorporated herein by reference in their entirety.

[0073] FIG. 4 shows a system 400 for flow cytometry, according to an exemplary embodiment of the present invention. The system 400 includes a flow cytometer 410, a controller / processor 490, and a memory 495. The 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.

[0074] The excitation lasers 415a to 415c emit light in the form of laser beams. The wavelengths of the laser beams emitted from the excitation lasers 415a to 415c are 488 nm, 633 nm, and 325 nm, respectively, in the exemplary system of FIG. 4. The laser beams are first directed through one or more of the beam splitters 445a and 445b. The beam splitter 445a transmits light at 488 nm and reflects light at 633 nm. The beam splitter 445b transmits UV light (light having wavelengths in the range of 10 to 400 nm) and reflects light at 488 nm and 633 nm.

[0075] The laser beams are then directed to the focusing lens 420, which focuses the beams onto the portion of the fluid stream in the flow chamber 425 where the particles of the sample are located. The flow chamber is part of a fluidics system that directs, typically one at a time, the particles in the stream into the focused laser beam for investigation. The flow chamber can comprise a flow cell within a benchtop flow cytometer or a nozzle tip within a stream-in air cytometer.

[0076] The light from the laser beams interacts with the particles in the sample by diffraction, refraction, reflection, scattering, and absorption with re - emission at various different wavelengths depending on particle characteristics such as particle size, internal structure, and the presence of one or more fluorescent molecules attached to or naturally occurring on or in the particles. Fluorescent emission, as well as diffracted light, refracted light, reflected light, and scattered light can be routed through one or more of the beam splitters 445c to 445g, band - pass filters 450a to 450e, long - pass filters 455a to 455b, and fluorescence collection lens 440 to one or more of the forward scatter detector 430, side scatter detector 435, and one or more fluorescence detectors 460a to 460f.

[0077] The fluorescence collection lens 440 collects the light emitted from the interaction between the particle laser beams and routes the light towards one or more beam splitters and filters. Bandpass filters such as bandpass filters 450a to 450e enable a narrow wavelength range 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, the 510 / 20 filter extends 10 nm on each side of the center of the spectral band, or from 500 nm to 520 nm. Short-pass filters transmit light below a specified wavelength. Long-pass filters such as long-pass filters 455a to 455b transmit light at or above the wavelength of the specified light. For example, long-pass filter 455b, which is a 670 nm long-pass filter, transmits light with a wavelength of 670 nm or above. Filters are often selected to optimize the specificity of the detector for a particular fluorescent dye. Those filters can be configured such that the spectral band of the light transmitted to the detector is close to the emission peak of the fluorescent dye.

[0078] The forward scatter detector 430 is positioned slightly off-axis from the direct beam passing through the flow cell and is configured to detect diffracted light, excitation light that travels mostly in the forward direction through or around the particles. The intensity of the light detected by the forward scatter detector depends on the overall size of the particles. The forward scatter detector can include a photodiode. The side scatter detector 435 is configured to detect diffracted light and reflected light from the surface and internal structure of the particles, which tends to increase as the particle structure becomes more complex. Fluorescent emission from fluorescent molecules associated with the particles can be detected by one or more fluorescence detectors 460a to 460f. The side scatter detector 435 and the fluorescence detectors can include photomultiplier tubes. Signals detected by the forward scatter detector 430, the side scatter detector 435, and the fluorescence detectors can be converted by the detectors into electronic signals (voltages). This data can provide information about the sample.

[0079] One of ordinary skill in the art will recognize that a flow cytometer according to one embodiment of the present invention is not limited to the flow cytometer illustrated in FIG. 4, but 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 at various wavelengths and in various different configurations.

[0080] During operation, the operation of the cytometer is controlled by a controller / processor 490, and measurement data from the detector can 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 an output signal therefrom, and is also coupled to the electrical and electromechanical components of the flow cytometer 410 to control lasers, fluid flow parameters, etc. An input / output (I / O) function unit 497 may also be provided within the system. The memory 495, the controller / processor 490, and the I / O 497 may be provided as an integral part of the flow cytometer 410. In such an embodiment, the display may also form part of the I / O function unit 497 for presenting experimental data to the user of the cytometer 410. Alternatively, some or all of the memory 495, the controller / processor 490, and the I / O function unit may be part of one or more external devices such as a general-purpose computer. In some embodiments, some or all of the memory 495 and the controller / processor 490 can communicate wirelessly or wired with the flow cytometer 410. Together with the memory 495 and the I / O 497, the controller / processor 490 may be configured to perform various functions related to the preparation and analysis of flow cytometer experiments.

[0081] The system illustrated in FIG. 4 includes six different detectors that detect fluorescence within six different wavelength bands (which may be referred to herein as “filter windows” for a given detector), as defined by the configuration of filters and / or splitters in the beam path from the flow cell 425 to each detector. Different fluorescent molecules in the fluorescent panel used in the flow cytometry experiment emit light in their own characteristic wavelength bands. The particular fluorescent labels used in the experiment, and their associated fluorescence emission bands, can be selected to generally coincide with the filter windows of the detectors. I / O 497 can be configured to receive data related to flow cytometry experiments having a panel of fluorescent labels and multiple cell parent populations having a plurality of markers, where each cell parent population has a subset of the plurality of markers. I / O 497 can also be configured to receive biological data assigning one or more markers to one or more cell parent populations, marker concentration data, emission spectrum data, data assigning labels to one or more markers, and cytometer configuration data. Flow cytometry experiment data such as label spectral characteristics and flow cytometer configuration data can also be stored in the memory 495. The controller / processor 490 can be configured to evaluate one or more assignments of labels to markers.

[0082] In some embodiments, the subject system is a particle sorting system configured to sort particles using a sealed particle sorting module, such as that described in U.S. Patent Publication No. 2017 / 0299493, filed Mar. 28, 2017, the disclosure of which is incorporated herein by reference. In certain embodiments, sample particles (e.g., cells) are sorted using a sorting determination module having a plurality of sorting determination units, such as that described in U.S. Patent Publication No. 2020 / 0256781, filed Dec. 23, 2019, the disclosure of which is incorporated herein by reference. In some embodiments, the system for sorting components of a sample includes a particle sorting module having a deflector plate, such as that described in U.S. Patent Publication No. 2017 / 0299493, filed Mar. 28, 2017, the disclosure of which is incorporated herein by reference.

[0083] FIG. 5 shows a functional block diagram of an example of a sorting control system, such as processor 500, for analyzing and displaying biological events. Processor 500 may be configured to perform various processes for controlling a graphic display of biological events.

[0084] A flow cytometer or sorting system 502 may be configured to acquire biological event data. For example, a flow cytometer can generate flow cytometric event data (e.g., particle-modulated light data). Flow cytometer 502 may be configured to provide biological event data to processor 500. A data communication channel may be included between flow cytometer 502 and processor 500. Biological event data may be provided to processor 500 via the data communication channel.

[0085] Processor 500 may be configured to 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 be configured to provide a graphical display including a first plot of the biological event data to display device 506. Processor 500 may be further configured to render a region of interest as a gate (e.g., a first gate) around a population of biological event data shown by display device 506, for example, overlaid on the first plot. In some embodiments, the gate may be a logical combination of one or more graphical regions of interest drawn on a histogram of a single parameter or a bivariate plot. In some embodiments, the display may be used to display particle parameters or saturated detector data.

[0086] Processor 500 may be further configured to display the biological event data on display device 506 within the gate differently from other events within the biological event data outside the gate. For example, processor 500 may be configured to render the color of the biological event data included within the gate to be distinguishable from the color of the biological event data outside the gate. Display device 506 may be implemented as a monitor, a tablet computer, a smartphone, or other electronic device configured to present a graphical interface.

[0087] Processor 500 may be configured to receive a gate selection signal that identifies a gate from a first input device. For example, the first input device may be implemented as a mouse 510. This mouse 510 can initiate a gate selection signal to processor 500 that identifies a gate to be displayed or manipulated via display device 506 (e.g., by clicking on or within a desired gate when positioning a cursor there). In some implementations, the first device may be implemented as a keyboard 508, or as other means for providing an input signal to processor 500, such as a touch screen, input pen, light detector, or voice recognition system. Some input devices may include multiple input functions. In such implementations, each input function may be considered an input device. For example, as shown in FIG. 5, mouse 510 may include a right mouse button and a left mouse button, each of which may generate an activation event.

[0088] This activation event may cause processor 500 to change how data is displayed, which portions of the data are actually displayed on display device 506, and / or provide input to further processing such as selection of a population of interest for particle sorting.

[0089] In some embodiments, processor 500 may be configured to detect when a gate selection is initiated by mouse 510. Processor 500 may be further configured to automatically modify the visualization of the plot to facilitate the gate control process. This modification may be based on a particular distribution of the biological event data received by processor 500. In some embodiments, processor 500 expands the first gate such that a second gate is generated (e.g., as described above).

[0090] Processor 500 may be connected to memory device 504. This memory device 504 may be configured to receive and store biometric event data from processor 500. Memory device 504 may also be configured to receive and store flow cytometric event data from processor 500. Memory device 504 may be further configured by processor 500 to enable retrieval of biometric event data such as flow cytometric event data.

[0091] Display device 506 may be configured to receive display data from processor 500. The display data may include a plot of biometric event data and gates that outline sections of the plot. Display device 506 may be further configured to modify information presented in accordance with input received from processor 500 in conjunction with input from flow cytometer 502, memory device 504, keyboard 508, and / or mouse 510.

[0092] In some implementations, processor 500 can generate a user interface for receiving exemplary events for sorting. For example, the user interface can include a mechanism for receiving exemplary events or exemplary images. Exemplary events or images, or exemplary gates, can be provided prior to collection of event data for a sample or based on an initial set of events for a portion of the sample.

[0093] FIG. 6A is a schematic diagram of a particle sorter system 600 (e.g., flow cytometer 502) according to one embodiment presented herein. In some embodiments, the particle sorter system 600 is a cell sorter system. The nozzle 603 discharges liquid into the moving fluid column 608 at the distal end. Within the fluid conduit 601, the sheath fluid 604 hydrodynamically focuses the sample fluid 606 containing the particles 609 into the moving fluid column 608 (e.g., stream). Within the moving fluid column 608, the particles 609 (e.g., cells) are lined up in a single file and cross a monitoring area 611 (e.g., where a laser stream intersects) and are irradiated by an irradiation source 612 (e.g., laser). The nozzle 603 includes a gas inlet 646 that includes a plurality of radial air flow paths (not shown). Air from a compressed gas source 645 is provided to the gas inlet 646 via a gas conduit. Due to the interaction between the sample fluid 606 and the air, the moving fluid column 608 splits into a plurality of droplets 610, some of which contain the particles 609.

[0094] During operation, the detection station 614 (e.g., an event detector) identifies when a target particle (or a target cell) crosses the monitoring area 611. The detection station 614 supplies an input to the timing circuit 628, which then supplies an input to the flash charge circuit 630. At the droplet splitting point notified by the timed droplet delay (Δt), a flash charge can be applied to the moving fluid column 608, and thus the target droplet carries a charge. The target droplet may contain one or more particles or cells to be sorted. The charged droplet is then sorted by actuating a deflection plate (not shown) to deflect the droplet into a vessel such as a collection tube, or a multiwell or microwell sample plate where wells or microwells can be associated with specific target droplets. As shown in FIG. 6A, the droplets can be collected in the drain receiver 638. The detection system 616 (e.g., a droplet boundary detector) serves to automatically determine the phase of the droplet drive signal when a target particle passes through the monitoring area 611. An exemplary droplet boundary detector is described in U.S. Patent No. 7,679,039, which is hereby incorporated by reference in its entirety. The detection system 616 enables the device to accurately calculate the position of each detected particle in the droplet. In the example of FIG. 6A, the detection system 616 is connected to the processor 640 and provides information regarding the moving fluid column 608 thereto. The processor 640 is operably connected to the compressed gas source 645 and is configured to adjust the pressure of the gas generated by the compressed gas source 645 in response to measurements from the detection system and / or inputs from the user. In this way, the size and volume of the droplets 610 can be adjusted.

[0095] In some implementations, sorting electronics (e.g., detection system 616, detection station 614, and processor 640) can be coupled to a memory configured to store detected events and sorting decisions based thereon. Sorting decisions can be included in the event data of the particles. In some implementations, detection system 616 and detection station 614 can be implemented as a single detection unit, or can be communicatively coupled such that event measurements are collected by one of detection system 616 or detection station 614 and provided to non-collecting elements.

[0096] FIG. 6B is a schematic diagram of a particle sorter system according to one embodiment presented herein. The particle sorter system 600 shown in FIG. 6B includes deflection plates 652 and 654. Charge can be applied via a stream charging wire within the barb. This forms a stream of droplets 610 containing particles 609 for analysis. The particles can be irradiated with one or more light sources (e.g., lasers) to generate light scattering and fluorescence information. Information about the particles is analyzed, such as by sorting electronics or other detection systems (not shown in FIG. 6B). Deflection plates 652 and 654 are independently controlled to attract or repel charged droplets and direct the droplets towards a desired collection vessel (e.g., one of 672, 674, 676, or 678). As shown in FIG. 6B, deflection plates 652 and 654 are controlled to direct the particles along a first path 662 towards vessel 674 or along a second path 668 towards vessel 678. If the particle is not of interest (e.g., does not exhibit scattering or illumination information within a specified sort range), the deflection plate can allow the particle to continue to travel along flow path 664. Such uncharged droplets can be transferred into a waste container, such as via suction device 670.

[0097] Sorting electronics can be included to initiate collection of measurement values, receive fluorescence signals regarding the particles, and determine how to adjust the deflection plates to cause sorting of the particles. As an exemplary implementation of the embodiment shown in FIG. 6B, a BD FACSAria (trademark) series flow cytometer commercially available from Becton, Dickinson and Company (Franklin Lakes, NJ) can be mentioned.

[0098] Method for sorting a particle sample As discussed above, aspects of the present invention also include a method for sorting a particle sample. The method in question includes introducing a particle sample into a cell sorter comprising a flow cell configured to transport particles in a flow stream and a nozzle, and sorting the particle sample by flow cytometry. As described above, the nozzle of the subject matter includes an opening at the proximal end engaged in a liquid receiving relationship with the flow cell, an opening at the distal end for discharging liquid droplets, and a channel configured to transport liquid from the proximal end to the distal end through an elongated body, and includes the elongated body. In addition, the nozzle of the present invention includes a radially positioned gas inlet at the proximal end of the elongated body, and the gas inlet includes a radial airflow path configured to provide gas to the channel.

[0099] In some embodiments, the method includes adjusting the pressure of the gas provided to the gas inlet by a compressed gas source. In such embodiments, the user may input a desired pressure or droplet size / volume using, for example, a user interface device such as that described above with respect to FIG. 5. The input value may be received by a processor, which then adjusts the compressed gas source accordingly.

[0100] In some cases, the sample analyzed by the present method is a biological sample. The term "biological sample" is used in its conventional meaning to refer to a subset of whole organisms, plants, fungi, or, in certain cases, animal tissues, cells, or components that can be found in blood, mucus, lymph, synovial fluid, cerebrospinal fluid, saliva, bronchoalveolar lavage, amniotic fluid, amniotic cord blood, urine, vaginal fluid, and semen. Thus, a "biological sample" refers to both natural organisms or subsets of their tissues, as well as, without limitation, for example, plasma, serum, cerebrospinal fluid, lymph, skin sections, respiratory tract, gastrointestinal tract, cardiovascular, and urinary organs, tears, saliva, milk, blood cells, tumors, organs, homogenates, lysates, or extracts prepared from a subset of an organism or its tissue. A biological sample can be any type of biological tissue, including both healthy and diseased tissues (e.g., cancerous, malignant, necrotic, etc.). In certain embodiments, the biological sample is a liquid sample such as blood or a derivative thereof, e.g., plasma, tears, urine, semen, etc., and in some cases, the sample is a blood sample including whole blood, such as blood obtained by venipuncture or finger stick (the blood may or may not be combined with any reagents such as preservatives, anticoagulants, etc. prior to the assay).

[0101] In certain embodiments, the sample source is a "mammal" or "mammalian animal", and these terms are widely used to describe organisms within the class of mammals, including carnivores (e.g., dogs and cats), rodents (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 applied to samples obtained from human subjects of either sex at any stage of development (i.e., neonates, infants, juveniles, young adults, adults), and in certain embodiments, the human subject is a juvenile, young adult, or adult. It is to be understood that the present invention can be applied to samples from human subjects, but also, without limitation, to samples from other animal subjects such as birds, mice, rats, dogs, cats, livestock, and horses (i.e., "non-human subjects").

[0102] The target cells can be targeted for characterization according to various parameters, such as phenotypic characteristics identified by attaching a specific fluorescent label to the target cells. In some embodiments, the system is configured to deflect analyzed droplets determined to contain target cells. Various cells can be characterized using the methods of the subject matter. Target cells of interest include, but are not limited to, stem cells, T cells, dendritic cells, B cells, granulocytes, leukemia cells, lymphoma cells, viral cells (e.g., HIV cells), NK cells, macrophages, monocytes, fibroblasts, epithelial cells, endothelial cells, and erythrocyte cells. Target cells of interest include cells having a convenient cell surface marker or cell surface antigen that can be captured or labeled by a convenient affinity agent or complex thereof. For example, target cells can 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 alpha / beta, T cell receptor gamma / delta, CD253, CD95, CD20, CD105, CD117, CD120b, Notch4, Lgr5 (N-terminus), SSEA-3, TRA-1-60 antigen, disialoganglioside GD2, and CD71. In some embodiments, the target cells are 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 cord blood.

[0103] When practicing the methods of the subject matter, an amount of an initial fluid sample is injected into a flow cytometer. The amount of sample injected into the particle sorting module can vary in the range of 0.001 mL to 1000 mL, such as 0.005 mL to 900 mL, such as 0.01 mL to 800 mL, such as 0.05 mL to 700 mL, such as 0.1 mL to 600 mL, such as 0.5 mL to 500 mL, such as 1 mL to 400 mL, such as 2 mL to 300 mL, including samples of 5 mL to 100 mL.

[0104] The method according to embodiments of the present disclosure includes counting and sorting labeled particles (e.g., target cells) in a sample. When implementing the subject method, a fluid sample containing the particles is first introduced into the flow nozzle of the system. As the particles exit the flow nozzle, they pass substantially one by one through a sample inspection region, where each of the particles is irradiated with a light source, and measurements of light scattering parameters and, in some cases, fluorescence emission (e.g., measurements of two or more light scattering parameters and one or more fluorescence emissions) are separately recorded for each particle. Depending on the characteristics of the flow stream being inspected, 0.001 mm or more of the flow stream, such as 0.005 mm or more, 0.01 mm or more, 0.05 mm or more, 0.1 mm or more, 0.5 mm or more, can be irradiated with light, including that 1 mm or more of the flow stream can be irradiated with light. In certain embodiments, the method includes irradiating a planar cross-section of the flow stream within the sample inspection region, for example, with a laser (as described above). In other embodiments, the method includes irradiating a predetermined length of the flow stream within the sample inspection region, such as corresponding to the irradiation profile of a diffused laser beam or a lamp.

[0105] In certain embodiments, a method is provided that includes irradiating the flow stream at or near the flow cell nozzle orifice. For example, the method can include irradiating the flow stream at a position about 0.001 mm or more from the nozzle orifice, including 1 mm or more from the nozzle orifice, such as 0.005 mm or more, 0.01 mm or more, 0.05 mm or more, 0.1 mm or more, 0.5 mm or more. In certain embodiments, the method includes irradiating the flow stream directly adjacent to the flow cell nozzle orifice.

[0106] In an embodiment of the method, a detector such as a photomultiplier tube (PMT) is used to record the light passing through each particle (in certain cases, referred to as forward light scattering) when the particle passes through the sensing region and is illuminated by an energy source, the light reflected orthogonally to the direction of the flow of particles passing through the sensing region (in some cases, referred to as orthogonal or side light scattering), and the fluorescence emitted from the particle when it is labeled with a fluorescent marker. Each of forward light scattering (FSC), side light scattering (SSC), and fluorescence emission includes a separate parameter for each particle (or "event"). Thus, for example, two, three, or four parameters can be collected (and recorded) from particles labeled with two different fluorescent markers.

[0107] In some embodiments, the method includes removing an adapter from a sorting flow cytometer. In some cases, the method further includes reattaching a second adapter to the particle sorting system after a first particle sorting module has been removed. The first adapter may be cleaned and sterilized (e.g., in an autoclave) for subsequent use or may be discarded.

[0108] The data recorded for each particle is analyzed in real time if necessary or stored in a data storage and analysis means such as a computer. U.S. Patent No. 4,284,412 describes the configuration and use of a flow cytometer for an object 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.

[0109] In certain embodiments, particles are detected and uniquely identified by, optionally, exposing the particles to excitation light and measuring the fluorescence of each particle within one or more detection channels. The fluorescence emitted within the detection channels used to identify the particles and associated binding complexes may be measured following excitation with a single light source or separately following excitation with multiple light sources. Where multiple excitation light sources are used to excite the particle labels, the labels may be selected such that all labels are excitable by each of the excitation light sources used.

[0110] The method in certain embodiments also includes the acquisition, analysis, and recording of data, such as using a computer, where multiple data channels record data from each detector for light scattering and fluorescence emitted by each particle as the particles pass through the sample inspection region of the particle sorting module. In these embodiments, the analysis includes classifying and counting the particles such that each particle exists as a set of digitized parameter values. The subject system may be set to trigger on selected parameters to distinguish the particles of interest from background and noise. "Trigger" refers to a preset threshold for detecting a parameter and may be used as a means for detecting the passage of a particle through the light source. Detection of an event that exceeds the threshold of the selected parameter triggers the acquisition of the light scattering and fluorescence data of the particle. For particles or other components within the medium being analyzed that give rise to a response below the threshold, no data is acquired. The trigger parameter may be the detection of forward scattered light caused by the particle passing through the light beam. The flow cytometer then detects and collects the light scattering and fluorescence data of the particle.

[0111] Next, a specific subset of the subjects is further analyzed by "gating" based on data collected for the entire population. To select an appropriate gate, the data is plotted to obtain the best possible separation of the subset. This procedure can be carried out by plotting forward scatter (FSC) versus side (i.e., orthogonal) scatter (SSC) on a two-dimensional dot plot. Next, a subset of the particles (i.e., those cells within the gate) is selected and particles not within the gate are excluded. Optionally, the gate can be selected by using a cursor on a computer screen to draw a line around the desired subset. Then, only those particles within the gate are further analyzed, for example, by plotting other parameters of these particles such as fluorescence. Optionally, the above analysis can be configured to result in a count of the target particles in the sample.

[0112] The subject method may further include employing sorted particles in research, laboratory tests, or treatments. In some embodiments, the subject method includes obtaining individual cells prepared from a target fluid or tissue biological sample. For example, the subject method includes obtaining cells from a fluid or tissue sample used as a specimen for research or diagnosis of a disease such as cancer. Similarly, the subject method includes obtaining cells from a fluid or tissue sample used in therapy. A cell therapy protocol is, for example, a protocol that can prepare viable cell material including cells and tissues and introduce it into a subject as a therapeutic treatment. Conditions that can be treated by administration of a sample sorted by flow cytometry include, but are not limited to, blood disorders, immune system disorders, organ damage, and the like.

[0113] A typical cell therapy protocol can include the steps of sample collection, cell isolation, genetic recombination, culture, and in vitro expansion, cell harvesting, sample volume reduction and washing, biopreservation, storage, and introduction of the cells into a subject. The protocol can begin with collecting viable cells and tissues from a source tissue of the subject to generate a sample of cells and / or tissues. The sample can be collected via any suitable procedure including, for example, administering a cell mobilizing agent to the subject, collecting blood from the subject, removing bone marrow from the subject, and the like. After collecting the sample, cell enrichment can occur via several methods including, for example, centrifugation-based methods, filter-based methods, elution, magnetic separation methods, fluorescence-activated cell sorting (FACS), and the like. In some cases, the enriched cells can be genetically recombined by any convenient method, such as nuclease-mediated gene editing. The genetically recombined cells can be cultured, activated, and expanded in vitro. In some cases, the cells are retained and, for example, cryopreserved and stored for future use. For future use, the cells are thawed and then administered to a patient. For example, the cells can be injected into the patient.

[0114] Method for assembling a particle sorter As discussed above, the method of the present invention further includes a method of assembling a particle sorter. The method in question includes operably connecting a nozzle to a flow cell configured to transfer particles in a flow stream. As described above, the subject nozzle includes an elongated body having a proximal end opening engaged with the flow cell in a liquid receiving relationship, a distal end opening for discharging liquid droplets, and a channel configured to transfer liquid from the proximal end to the distal end through the elongated body. Additionally, the nozzle of the present invention includes a radially positioned gas inlet at the proximal end of the elongated body, the gas inlet having a radial gas flow path configured to provide gas to the channel. "Operably connecting" the nozzle to the flow cell means engaging the proximal end opening of the elongated body with the flow cell in a liquid receiving relationship. In certain embodiments, the method includes operably connecting the flow cell and the nozzle in a fluid tight (e.g., airtight, liquid tight) manner. In such cases, one or more gaskets and / or O-rings may be used.

[0115] In some cases, the method additionally includes gas connecting a compressed gas source to the gas inlet. "Gas connecting" the compressed gas source to the gas inlet means connecting the two elements such that gas can be exchanged between them. As discussed above, any convenient compressed gas source (e.g., a gas compressor, a gas cylinder, etc.) may be used. The method may additionally involve operably connecting (e.g., gas connecting) one or more gas conduits to the compressed gas source and the gas inlet. Each gas conduit may be in contact with the radial gas flow path within the gas inlet via any other suitable attachment mechanism including, for example, press fitting, or without limitation, clamps, magnets, latches, notches, countersinks, counterbores, grooves, pins, tethers, hinges, non-permanent adhesives, or combinations thereof. In some cases, the method includes operably connecting a plurality of gas conduits to the compressed gas source and the gas inlet.

[0116] Methods according to some embodiments also include operably connecting a processor to a compressed gas source. As discussed above, the processor of interest is configured to regulate the pressure of the resulting gas provided at the gas inlet. The processor may or may not be an existing processor within the particle sorter. If the processor already exists within the particle sorter, the processor is modified (e.g., via a plug-in) to have an additional function of regulating the compressed gas source to provide a certain amount of pressure.

[0117] Computer control system Aspects of the invention include a computer control system, the system additionally including one or more computers for full or partial automation. In some embodiments, the system includes a computer having a non-transitory computer-readable storage medium with a computer program stored thereon, the computer program including instructions that, when loaded into the computer, receive a desired droplet size and / or volume (e.g., input by a user) and actuate a change in the compressed gas source so that the desired droplet size and / or volume is achieved.

[0118] The system may include a display and an operator input device. The operator input device may be, for example, a keyboard, a mouse, etc. The processing module includes a processor that accesses a memory having instructions stored therein for performing 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, and an input / output controller, a cache memory, a data backup unit, and many other devices. The processor may be a commercially available processor or one of other processors that are available or will become available. The processor executes an operating system, and the operating system interfaces with firmware and hardware in a known manner and facilitates the processor to cooperate and execute the functions of various computer programs that may be described in various programming languages such as Java, Perl, C++, Python, other high-level or low-level languages, and combinations thereof, as is known in the art. 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 / output control, file and data management, memory management, and communication control and related services, all according to known techniques. In some embodiments, the processor includes analog electronics that provide feedback control such as, for example, negative feedback control.

[0119] The system memory can be any of various known or future memory storage devices. Examples include any commonly available random access memory (RAM), magnetic media such as resident hard disks or tapes, optical media such as read / write compact discs, flash memory devices, or other memory storage devices. The memory storage device can be any of various known or future devices, including a compact disc drive, a tape drive, or a disk drive. Memory storage devices of such types typically read from and / or write to a program storage medium (not shown), such as a compact disc. Any of these program storage media, or others currently in use or that may be developed later, can be considered a computer program product. As is understood, these program storage media typically store computer software programs and / or data. A computer software program, also referred to as computer control logic, is typically stored in the system memory and / or a program storage device used in conjunction with the memory storage device.

[0120] In some embodiments, a computer program product is described as comprising a computer-usable medium having control logic (a computer software program including program code) stored therein. The control logic, when executed by a processor, causes the computer or processor to perform the functions described herein. In other embodiments, some functions are implemented primarily in hardware, using, for example, a hardware state machine. Implementations of hardware state machines for performing the functions described herein will be apparent to those skilled in the relevant art.

[0121] The memory can be any suitable device such as a magnetic, optical, or solid-state storage device (including magnetic or optical disks, or tapes, or RAM, or any other suitable device, either fixed or portable). The processor can include a general-purpose digital microprocessor suitably programmed from a computer-readable medium carrying the necessary program code. The programming can be provided remotely to the processor via a communication channel, or can be pre-stored using any of those devices together with the memory in a computer program product such as a memory or any other portable or fixed computer-readable storage medium. For example, a magnetic or optical disk can carry the programming and can be read by a disk writer / reader. The system of the present invention also includes, for example, programming in the form of a computer program product, and algorithms for use in implementing the above methods. The programming according to the present invention can be recorded on a computer-readable medium, for example, 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 magnetic disks, hard disk storage media, and magnetic tapes, optical storage media such as CD-ROMs, electrical storage media such as RAM and ROM, portable flash drives, and hybrids of these categories such as magnetic / optical storage media.

[0122] The processor can also have access to a communication channel for communicating with a user at a remote location. A remote location means 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., a smartphone), where the user does not directly contact the system, and relays input information from an external device to an input manager.

[0123] In some embodiments, the system according to the present disclosure may be configured to 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 may be configured 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) for mobile communications).

[0124] In one embodiment, the communication interface is configured to 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 and other external devices, such as a computer terminal configured for similar complementary data communication (e.g., in a clinic or hospital environment).

[0125] In one embodiment, the communication interface is configured for infrared communication, Bluetooth® communication, or any other suitable wireless communication protocol, enabling the subject system to communicate with other devices, such as a computer terminal and / or a network, a communicable mobile phone, a personal digital assistant, or any other communication device that the user may use in combination.

[0126] In one embodiment, the communication interface is configured to provide a connection for data transfer using the Internet protocol (IP) via a cellular phone network, 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 at a Wi-Fi hotspot.

[0127] In one embodiment, the subject system is configured to wirelessly communicate with a server device via a communication interface using a common standard such as, for example, the 802.11 or Bluetooth® RF protocol, or the IrDA infrared protocol. The server device may be another portable device such as a smartphone, a personal digital assistant (PDA) or a notebook computer, or a larger device such as a desktop computer, an appliance, etc. In some embodiments, the server device has a display such as a liquid crystal display (LCD), and input devices such as buttons, a keyboard, a mouse, or a touch screen.

[0128] In some embodiments, the communication interface is configured to automatically or semi-automatically communicate data stored in a network or a server device and within the subject system, for example, within an optional data storage unit, using one or more of the communication protocols and / or mechanisms described above.

[0129] The output controller may include a controller for any of a variety of known display devices for presenting information to a user, whether human or machine, local or remote. When one of the display devices provides visual information, this information can typically be logically and / or physically organized as an array of pixels. The 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 for processing user input. The functional elements of the computer may communicate with each other via a system bus. Some of these communications may be achieved in alternative embodiments using a network or other type of remote communication. The output manager may also provide information generated by the processing module to the user at a remote location, according to known techniques, e.g., via the Internet, telephone, or satellite network. The presentation of data by the output manager can be implemented according to a variety of known techniques. As some examples, the data may include SQL, HTML, or XML documents, email or other files, or other forms of data. The data may include an Internet URL address so that the user can retrieve additional SQL, HTML, XML, or other documents or data from a remote source. One or more platforms present within the subject system may typically be of any type of known computer platform or a type to be developed in the future, but they are generally of the class of computers referred to as servers. However, they can be mainframe computers, workstations, or other computer types. They can be connected via other communication systems, including any known or future type of cable wiring or either a wired or wireless system, whether networked or not. They may be located in the same place or physically separated.Depending on the type and / or configuration of the selected computer platform, various operating systems can be used on any of the computer platforms. 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™, SGI IRIX®, Oracle Solaris®, and the like.

[0130] FIG. 7 shows a general architecture of an exemplary computing device 600 according to a particular embodiment. The general architecture of computing device 700 shown in FIG. 7 includes the arrangement of computer hardware and software components. However, not all of these generally traditional elements necessarily need to be shown in order to provide an enabling disclosure. As shown, computing device 700 includes a processing unit 710, a network interface 720, a computer-readable media drive 730, an input / output device interface 740, a display 750, and an input device 760, all of which can communicate with each other via a communication bus. Network interface 720 can provide connectivity to one or more networks or computing systems. Thus, processing unit 710 can receive information and instructions from other computing systems or services via the network. Processing unit 710 can also communicate with memory 770 and further provide output information for optional display 750 via input / output device interface 740. For example, analysis software (e.g., data analysis software or program such as FlowJo®) stored as executable instructions in the non-transitory memory of the analysis system can display flow cytometry event data to the user. Input / output device interface 740 can also receive input from optional input device 760 such as a keyboard, mouse, digital pen, microphone, touch screen, gesture recognition system, voice recognition system, game pad, accelerometer, gyroscope, or other input device.

[0131] Memory 770 may contain computer program instructions (grouped as modules or components in some embodiments) that the processing unit 710 executes in sequence to implement one or more embodiments. Memory 770 generally includes RAM, ROM, and / or other persistent, auxiliary, or non-transitory computer-readable media. Memory 770 may store an operating system 772 that provides computer program instructions for use by the processing unit 710 in the general management and operation of computing device 700. Data may be stored in data storage device 790. Memory 770 may further contain computer program instructions and other information for implementing aspects of the present disclosure.

[0132] Utility The nozzles, cell sorters, methods, and kits of the present disclosure find use, for example, when it is desirable to adjust the size of droplets within a cell sorter without exchanging the nozzles. Additionally, the present invention can be used when it is desirable to improve the data received from particle-modulated light within a cell sorter by removing a perturbation source upstream of the flow cell. Embodiments of the present invention find use in applications where cells prepared from a biological sample may be desirable for use in research, laboratory testing, or treatment. In some embodiments, the subject methods and devices may facilitate obtaining individual cells prepared from a target fluid or tissue biological sample. For example, the subject methods and systems facilitate obtaining cells from a fluid or tissue sample that is used as a specimen for research or diagnosis of diseases such as cancer. Similarly, the subject methods and systems may facilitate obtaining cells from a fluid or tissue sample used in treatment. The methods and devices of the present disclosure enable the separation and collection of cells from biological samples (e.g., organs, tissues, tissue fragments, body fluids) with improved efficiency and at lower cost compared to conventional flow cytometry systems.

[0133] Kit Aspects of the invention additionally include a kit. The kit in question includes the nozzle of the subject matter. As described above, the nozzle of the subject matter includes an elongated body having a proximal end opening engaged in a liquid receiving relationship with a flow cell, a distal end opening for discharging liquid droplets, and a channel configured to transfer liquid from the proximal end to the distal end through the elongated body. Additionally, the nozzle of the invention includes a radially positioned gas inlet at the proximal end of the elongated body, the gas inlet having a radial gas flow path configured to provide gas to the channel. The kit in question may additionally include one or more gas conduits. In certain cases, the kit includes a plurality of gas conduits. In some instances, the kit includes a compressed gas source. As discussed above, any convenient compressed gas source (e.g., a gas compressor, a gas canister, etc.) may be used.

[0134] In some aspects, the kit additionally includes a processor configured to regulate the pressure of the resulting gas provided to the gas inlet. In other cases, the kit includes instructions for modifying an existing processor on a cell sorter to regulate the pressure of the resulting gas provided to the gas inlet. The kit in some such cases includes a storage medium such as a magneto-optical disk, a CD-ROM, a CD-R, a magnetic tape, a non-volatile memory card, a ROM, a DVD-ROM, a Blu-ray disk, a solid state disk, and a network-connected storage device (NAS). Any of these program storage media, or other ones currently in use or that may be developed later, may be included in the kit of the subject matter. A plugin may be provided on the storage medium to modify the existing processor.

[0135] In addition to the above components, the subject kit may further include, for example, instructions (in some embodiments) for installing a plugin into an existing software package. These instructions can exist within the subject kit in various forms, one or more of which can be present within the kit. One form in which these instructions can exist is as printed information on a suitable medium or substrate, such as a piece of paper on which the information is printed, within the package of the kit, within an accompanying document, etc. Another form of these instructions is a computer-readable medium on which the information is recorded, such as a diskette, a compact disk (CD), a portable flash drive, etc. Yet another form of these instructions that can exist is a website address that can be used via the Internet to access the information at a remote site.

[0136] The following examples are presented by way of illustration and not by way of limitation.

[0137] Experiment Simulations were performed to demonstrate the operating principle of using a dispersed flow and a continuous flow to periodically generate droplets from a nozzle. The simulations were performed using COMSOL® Multiphysics software. The simulated nozzle was assumed to have a radial gas flow path with a diameter of 200 μm and a height of 200 μm, and an extended cylinder with a diameter of 500 μm and a length of 4.3 mm. FIG. 8 provides a depiction of the simulated nozzle. As shown in FIG. 8, the nozzle includes an elongated body 801, a proximal end opening 802 for receiving liquid from a flow cell (not shown), a channel 803, a distal end opening for discharging liquid droplets 804, and a gas inlet 805. The volumetric flow rate through the simulated nozzle was set to 6.3 ml / min (105 μl / sec).

[0138] Three different simulations were performed using the nozzle described above. In each simulation, one of three air pressures, namely 4 kPa (0.58 psi), 5 kPa (0.73 psi), and 6 kPa (0.87 psi), was applied to the gas inlet 805. Figure 9 depicts the results of these simulations. As shown in Figure 9, the simulation performed at 4 kPa resulted in droplets having a diameter of approximately 130 μm, the simulation performed at 5 kPa resulted in droplets having a diameter of approximately 100 μm, and the simulation performed at 6 kPa resulted in droplets having a diameter of approximately 80 μm. The results indicate that the droplet size can be adjusted by the inlet air pressure of the air stream.

[0139] Notwithstanding the appended claims, the present disclosure is also defined by the following appendices. 1. A particle sorter comprising: a flow cell configured to transfer particles in a flow stream; a nozzle wherein the nozzle has an elongated body having an opening at a proximal end engaged in a liquid receiving relationship with the flow cell; an opening at a distal end for discharging liquid droplets; and a channel configured to transfer liquid from the proximal end to the distal end through the elongated body; and a gas inlet positioned radially at the proximal end of the elongated body; wherein the gas inlet comprises a radial air flow path configured to provide gas to the channel. A particle sorter. 2. The particle sorter according to appendix 1, wherein the radial air flow path includes a height in the range of 150 μm to 250 μm. 3. The particle sorter according to appendix 1 or 2, wherein the radial air flow path includes a radius in the range of 200 μm to 300 μm. 4. The particle sorter according to any one of appendices 1 to 3, wherein the gas inlet includes a plurality of radial air flow paths. 5. The number of radial airflow paths in the plurality of radial airflow paths is in the range of 2 to 5, the particle sorter according to appendix 4.

[0140] 6. The particle sorter according to any one of appendices 1 to 5, further comprising a compressed gas source. 7. The compressed gas source is an air compressor, the particle sorter according to appendix 6. 8. The compressed gas source is configured to generate a gas having a pressure in the range of 2 kPa to 10 kPa, the particle sorter according to appendix 6 or 7. 9. The particle sorter according to any one of appendices 6 to 8, further comprising a gas conduit configured to operably connect the compressed gas source to the gas inlet. 10. The particle sorter according to appendix 9, wherein the particle sorter comprises a plurality of gas conduits.

[0141] 11. The particle sorter according to any one of appendices 6 to 10, further comprising a processor operably connected to the compressed gas source and configured to adjust the pressure of the generated gas provided to the gas inlet. 12. The channel has a diameter in the range of 150 μm to 250 μm, the particle sorter according to any one of appendices 1 to 11. 13. The channel has a constant diameter throughout the length of the elongated body, the particle sorter according to appendix 12. 14. The elongated body is cylindrical, the particle sorter according to any one of appendices 1 to 13. 15. The length of the elongated body is in the range of 4 mm to 4.5 mm, the particle sorter according to any one of appendices 1 to 14.

[0142] 16. The nozzle is not operably attached to a piezoelectric actuator, the particle sorter according to any one of appendices 1 to 15. 17. The particle sorter according to any one of appendices 1 to 16, further comprising a light source configured to irradiate the flow stream at the inspection point. 18. The particle sorter according to appendix 17, further comprising a detector configured to collect particle-modulated light from the flow cell. The particle sorter according to any one of appendices 1 to 18, further comprising a plurality of receptacles configured to receive droplets discharged through an opening at the distal end of the elongated structure. The particle sorter according to appendix 19, further comprising a deflector configured to deflect droplets into a given receptacle of the plurality of receptacles.

[0143] 21. A method of sorting a particle sample, comprising: (a) a cell sorter comprising: a flow cell configured to transfer particles in a flow stream, and a nozzle, the nozzle comprising: an elongated body having: an opening at the proximal end engaged in a liquid receiving relationship with the flow cell, an opening at the distal end for discharging liquid droplets, and a channel configured to transfer liquid from the proximal end to the distal end through the elongated body; a gas inlet positioned radially at the proximal end of the elongated body; and introducing a particle sample into the cell sorter, wherein the gas inlet comprises a radial airflow path configured to provide gas to the channel; (b) sorting the particle sample by flow cytometry. A method as described above. 22. The method according to appendix 21, wherein the radial airflow path includes a height in the range of 150 μm to 250 μm. 23. The method according to appendix 21 or 22, wherein the radial airflow path includes a radius in the range of 200 μm to 300 μm. 24. The method according to any one of appendices 21 to 23, wherein the gas inlet comprises a plurality of radial airflow paths. 25. The method according to appendix 24, wherein the number of radial airflow paths in the plurality of radial airflow paths is in the range of 2 to 5.

[0144] 26. The method according to any one of appendices 21 to 25, wherein the cell sorter further comprises a compressed gas source. 27. The method according to appended claim 26, wherein the compressed gas source is an air compressor. 28. The method according to appended claim 26 or 27, wherein the compressed gas source is configured to generate a gas having a pressure in the range of 2 kPa to 10 kPa. 29. The method according to any one of appended claims 26 to 28, wherein the particle sorter further comprises a gas conduit configured to operably connect the compressed gas source to the gas inlet. 30. The method according to appended claim 29, wherein the particle sorter comprises a plurality of gas conduits.

[0145] 31. The method according to any one of appended claims 26 to 30, further comprising adjusting the pressure of the gas provided to the gas inlet by the compressed gas source. 32. The method according to any one of appended claims 21 to 31, wherein the channel has a diameter in the range of 150 μm to 250 μm. 33. The method according to appended claim 32, wherein the channel has a constant diameter throughout the length of the elongated body. 34. The method according to any one of appended claims 21 to 33, wherein the elongated body has a cylindrical shape. 35. The method according to any one of appended claims 21 to 34, wherein the length of the elongated body is in the range of 4 mm to 4.5 mm.

[0146] 36. The method according to any one of appended claims 21 to 35, wherein the nozzle is not operably attached to a piezoelectric actuator. 37. The method according to any one of appended claims 21 to 36, wherein the particle sorter further comprises a light source configured to irradiate the flow stream at an inspection point. 38. The method according to appended claim 37, wherein the particle sorter further comprises a detector configured to collect particle-modulated light from the flow cell. 39. The method according to any one of appended claims 21 to 38, wherein the particle sorter further comprises a plurality of receptacles configured to receive droplets discharged through an opening at the distal end of the elongated structure. 40. The method according to appended claim 39, wherein the particle sorter further comprises a deflector plate configured to deflect the droplets into a given receptacle of the plurality of receptacles.

[0147] 41. The sample is a biological sample, and is prepared by the method described in any one of Supplementary Notes 21 to 40. 42. The sample contains cells, and is prepared by the method described in Supplementary Note 41.

[0148] 43. A nozzle, having an elongated body, a proximal opening configured to engage with a flow cell in a liquid receiving relationship, a distal opening for discharging liquid droplets, and an elongated body having a channel configured to transfer liquid from the proximal end to the distal end through the elongated body, at the proximal end of the elongated body, a gas inlet positioned radially, and comprising, the gas inlet comprising a radial airflow path configured to provide gas to the channel. 44. The nozzle according to Supplementary Note 43, wherein the radial airflow path includes a height in the range of 150 μm to 250 μm. 45. The nozzle according to Supplementary Note 44, wherein the radial airflow path includes a height of 200 μm. 46. The nozzle according to any one of Supplementary Notes 43 to 45, wherein the radial airflow path includes a radius in the range of 200 μm to 300 μm. 47. The nozzle according to Supplementary Note 46, wherein the radial airflow path includes a radius of 250 μm.

[0149] 48. The nozzle according to any one of Supplementary Notes 43 to 47, wherein the gas inlet comprises a plurality of radial airflow paths. 49. The nozzle according to Supplementary Note 48, wherein the number of radial airflow paths in the plurality of radial airflow paths is in the range of 2 to 5. 50. The nozzle according to Supplementary Note 49, wherein the gas inlet comprises four radial airflow paths. 51. The nozzle according to any one of Supplementary Notes 43 to 50, wherein the gas inlet is configured to be operably connected to a gas conduit. 52. The gas inlet is the nozzle according to appended claim 51, configured to be operably connected to a plurality of gas conduits.

[0150] 53. The channel is the nozzle according to any one of appended claims 43 to 52, including a diameter in the range of 150 μm to 250 μm. 54. The channel is the nozzle according to appended claim 53, including a diameter of 200 μm. 55. The channel is the nozzle according to appended claim 53 or 54, including a constant diameter throughout the length of the elongated body. 56. The elongated body is the nozzle according to any one of appended claims 43 to 55, having a cylindrical shape. 57. The length of the elongated body is the nozzle according to any one of appended claims 43 to 56, in the range of 4 mm to 4.5 mm. 58. The length of the elongated body is the nozzle according to appended claim 57, being 4.3 mm.

[0151] 59. A method of assembling a particle sorter, including operably connecting a nozzle to a flow cell configured to transfer particles in a flow stream, the nozzle having an elongated body having a proximal opening, a distal opening for discharging liquid droplets, and a channel configured to transfer liquid from the proximal end to the distal end through the elongated body, and a gas inlet positioned radially at the proximal end of the elongated body and comprising, the gas inlet comprising a radial gas flow path configured to provide gas to the channel, wherein operably connecting the nozzle includes engaging the opening at the proximal end of the elongated body with the flow cell in a liquid receiving relationship. 60. The radial gas flow path is the method according to appended claim 59, including a height in the range of 150 μm to 250 μm. 61. The radial gas flow path is the method according to appended claim 59 or 60, including a radius in the range of 200 μm to 300 μm. 62. The gas inlet is the method according to any one of Appendices 59 to 61, comprising a plurality of radial gas flow paths. 63. The method according to Appendix 62, wherein the number of the radial gas flow paths in the plurality of radial gas flow paths is in the range of 2 to 5.

[0152] 64. The method according to any one of Appendices 59 to 63, further comprising gas-connecting a compressed gas source to the gas inlet. 65. The method according to Appendix 64, wherein the compressed gas source is an air compressor. 66. The method according to Appendix 64 or 65, wherein the compressed gas source is configured to generate a gas having a pressure in the range of 2 kPa to 10 kPa. 67. The method according to any one of Appendices 64 to 66, further comprising operably connecting a gas conduit to the compressed gas source and the gas inlet. 68. The method according to Appendix 67, comprising operably connecting a plurality of gas conduits to the compressed gas source and the gas inlet.

[0153] 69. The method according to any one of Appendices 64 to 68, further comprising operably connecting a processor to the compressed gas source, wherein the processor is configured to adjust the pressure of the generated gas provided to the gas inlet. 70. The method according to any one of Appendices 59 to 69, wherein the channel includes a diameter in the range of 150 μm to 250 μm. 71. The method according to Appendix 70, wherein the channel includes a constant diameter throughout the length of the elongated body. 72. The method according to any one of Appendices 59 to 71, wherein the elongated body is cylindrical. 73. The method according to any one of Appendices 59 to 72, wherein the length of the elongated body is in the range of 4 mm to 4.5 mm. 74. The method according to any one of Appendices 59 to 73, not including operably attaching a piezoelectric actuator to the nozzle.

[0154] 75. A kit, comprising a nozzle, the nozzle being an elongated body, a proximal opening configured to engage in a flow cell and liquid receiving relationship, a distal opening for discharging liquid droplets, and an elongate body having a channel configured to transfer liquid from the proximal end to the distal end through the elongate body, a gas inlet positioned radially at the proximal end of the elongate body and comprising, a kit, wherein the gas inlet comprises a radial airflow path configured to provide gas to the channel. 76. The kit according to appendix 75, wherein the radial airflow path includes a height in the range of 150 μm to 250 μm. 77. The kit according to appendix 76, wherein the radial airflow path includes a height of 200 μm. 78. The kit according to any one of appendices 75 to 77, wherein the radial airflow path includes a radius in the range of 200 μm to 300 μm. 79. The kit according to appendix 78, wherein the radial airflow path includes a radius of 250 μm.

[0155] 80. The kit according to any one of appendices 75 to 79, wherein the gas inlet comprises a plurality of radial airflow paths. 81. The kit according to appendix 80, wherein the number of radial airflow paths in the plurality of radial airflow paths is in the range of 2 to 5. 82. The kit according to appendix 81, wherein the gas inlet comprises four radial airflow paths. 83. The kit according to any one of appendices 75 to 82, further comprising a compressed gas source. 84. The kit according to appendix 83, wherein the compressed gas source is an air compressor.

[0156] 85. The kit according to appendix 83 or 84, wherein the compressed gas source is configured to generate a gas having a pressure in the range of 2 kPa to 10 kPa. 86. The kit according to any one of appendices 75 to 85, further comprising a gas conduit configured to operably connect the compressed gas source to the gas inlet. 87. The kit according to appended claim 86, comprising a plurality of gas conduits. 88. The kit according to any one of appended claims 75 to 87, wherein the channel has a diameter in the range of 150 μm to 250 μm. 89. The kit according to claim 88, wherein the channel has a diameter of 200 μm.

[0157] 90. The kit according to claim 88 or 89, wherein the channel has a constant diameter throughout the length of the elongated body. 91. The kit according to any one of appended claims 75 to 90, wherein the elongated body is cylindrical in shape. 92. The kit according to any one of appended claims 75 to 91, wherein the length of the elongated body is in the range of 4 mm to 4.5 mm. 93. The kit according to claim 92, wherein the length of the elongated body is 4.3 mm.

[0158] Although the above invention has been described in some detail by way of illustration and example for the purpose of clear understanding, it will be readily apparent to those skilled in the art that, in light of the teachings of the present invention, certain changes and modifications can be made to those inventions without departing from the spirit or scope of the appended claims.

[0159] Accordingly, the foregoing merely illustrates the principles of the present invention. It will be appreciated by those skilled in the art that, although not explicitly described or illustrated herein, various arrangements embodying the principles of the present invention and falling within its spirit and scope can be devised. Further, all examples and conditional language recited herein are principally intended to aid the reader in understanding the principles of the present invention and the concepts contributed by the inventors to further the art, and are to be construed as not being limited to such specifically recited examples and conditions. Moreover, all descriptions in this specification of the principles, aspects, and embodiments of the present invention, as well as specific examples thereof, are intended to encompass both structural and functional equivalents thereof. Additionally, such equivalents are intended to include both currently known equivalents and equivalents developed in the future, i.e., any elements developed to perform the same function regardless of structure. Further, nothing disclosed herein is intended to be dedicated to the public whether or not such disclosure is explicitly recited in the claims.

[0160] Accordingly, it is not intended that the scope of the present invention be limited to the exemplary embodiments shown and described herein. Rather, the scope and spirit of the present invention are embodied by the appended claims. In the claims, 35 U.S.C. § 112(f) or 35 U.S.C. § 112, ¶ 6 is explicitly defined as being invoked for a claim limitation only when the exact phrase "means for" or the exact phrase "step for" is recited at the beginning of such a limitation in the claim, and 35 U.S.C. § 112(f) or 35 U.S.C. § 112, ¶ 6 is not operative when such exact phrase is not used in the claim limitation.

[0161] Cross - Reference to Related Applications This application claims priority under 35 U.S.C. § 119(e) to the filing date of U.S. Provisional Patent Application No. 63 / 342,724, filed May 17, 2022, the entire disclosure of which is incorporated herein by reference.

Claims

1. A particle sorter, comprising: a flow cell configured to transport particles in a flow stream; a nozzle; The nozzle is an elongated body having: an opening at a proximal end engaged in a liquid receiving relationship with the flow cell; an opening at a distal end for discharging liquid droplets; and a channel configured to transport liquid from the proximal end to the distal end through the elongated body; a gas inlet positioned radially at the proximal end of the elongated body; The particle sorter, wherein the gas inlet comprises a radial airflow path configured to provide gas to the channel.

2. The particle sorter according to claim 1, wherein the gas inlet comprises a plurality of radial airflow paths.

3. The particle sorter according to claim 1 or 2, further comprising a compressed gas source.

4. The particle sorter according to claim 3, further comprising a gas conduit configured to operably connect the compressed gas source to the gas inlet.

5. The particle sorter according to claim 4, wherein the particle sorter comprises 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 the compressed gas source and configured to adjust the pressure of the generated gas provided to the gas inlet.

7. The particle sorter according to any one of the preceding claims, wherein the channel has a diameter in the range of 150 μm to 250 μm.

8. The particle sorter according to claim 7, wherein the channel has a constant diameter throughout the length of the elongated body.

9. The particle sorter according to any one of the preceding claims, wherein the elongated body has a cylindrical shape.

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

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

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

13. The particle sorter according to any one of the preceding claims, further comprising a plurality of receptacles configured to receive the liquid droplets discharged by the opening at the distal end of the elongated body.

14.

15. ​ ​ The particle sorter according to claim 13, further comprising a deflector configured to deflect the liquid droplets into a given receptacle of the plurality of receptacles.

15. A method for sorting a particle sample, comprising: (a) a cell sorter, comprising: a flow cell configured to transfer particles in a flow stream, and a nozzle, wherein the nozzle comprises: an elongated body having: an opening at a proximal end engaged with the flow cell in a liquid receiving relationship, an opening at a distal end for discharging liquid droplets, and a channel configured to transfer liquid from the proximal end to the distal end through the elongated body; a gas inlet positioned radially at the proximal end of the elongated body; and introducing the particle sample into the cell sorter, wherein the gas inlet comprises a radial air flow path configured to provide gas to the channel; and (b) sorting the particle sample by flow cytometry. A method.

16. A nozzle, comprising: an elongated body having: an opening at a proximal end configured to engage with a flow cell in a liquid receiving relationship, an opening at a distal end for discharging liquid droplets, and a channel configured to transfer liquid from the proximal end to the distal end through the elongated body; a gas inlet positioned radially at the proximal end of the elongated body; and wherein the gas inlet comprises a radial air flow path configured to provide gas to the channel.

17. A method for assembling a particle sorter, comprising: operatively connecting a nozzle to a flow cell configured to transfer particles in a flow stream, wherein the nozzle comprises: an elongated body having: an opening at a proximal end, an opening at a distal end for discharging liquid droplets, and a channel configured to transfer liquid from the proximal end to the distal end through the elongated body; a gas inlet positioned radially at the proximal end of the elongated body; and wherein the gas inlet comprises a radial air flow path configured to provide gas to the channel, and operatively connecting the nozzle comprises engaging the opening at the proximal end of the elongated body with the flow cell in a liquid receiving relationship. A method.

18. A kit, comprising: a nozzle, wherein the nozzle comprises: an elongated body having: an elongated body... A proximal opening configured to engage in a flow cell and liquid receiving relationship, A distal opening for discharging liquid droplets, and An elongate body having a channel configured to transfer liquid from the proximal end to the distal end through the elongate body, A gas inlet positioned radially at the proximal end of the elongate body, and Comprising, A kit, wherein the gas inlet comprises a radial airflow path configured to provide gas to the channel.

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