Flow cell with optimized high voltage electrodes, flow cytometer with flow cell, and methods of use thereof

Abstract

To provide a droplet deflector having an optimized deflection plate.SOLUTION: Droplet deflectors of interest include droplet deflectors comprising deflection plates configured for high angle deflection of a droplet flow stream. Droplet deflectors of interest further include those comprising deflection plates having a shape corresponding to the path of the deflected droplet flow stream, or deflection plates configured to apply a deflection force to the flow stream at a plurality of different angles. Droplet deflectors of interest further include droplet deflectors that are configured to apply a constant deflection force to the deflected droplet flow stream from a plurality of different lateral positions, e.g., wherein the deflector plate is a segmented deflector plate. Further provided are flow cytometers comprising the subject droplet deflectors, as well as methods of using and assembling or configuring the same.SELECTED DRAWING: None

Description

[Background technology]

[0001] Characterization of analytes in biological fluids has become an important part of biological research, medical diagnostics, and the assessment of a patient's overall health and well-being. Detecting analytes in biological fluids, such as human blood or blood-derived products, can yield results that may be relevant to determining treatment protocols for patients with various medical conditions.

[0002] Flow cytometry is a technique used to characterize and often sort biological materials, such as cells in a blood sample or particles of interest 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 a sheath fluid. The flow cytometer carries particles (including cells) in the fluid sample as a cell stream to a flow cell, while the sheath fluid is directed toward the flow cell. To characterize components in the flow stream, light is irradiated onto the flow stream. Changes in the biological materials in the flow stream, such as morphology or the presence of fluorescent labels, can alter the observed light, enabling characterization and separation. To characterize components in a flow stream, light must be directed onto the flow stream and collected. The light source for a flow cytometer can be a variety of light sources, including one or more broad-spectrum lamps, light-emitting diodes, and single-wavelength lasers. The light source is aligned with the flow stream, and optical responses from the illuminated particles are collected and quantified.

[0003] Separation of biological particles has been achieved by adding a sorting or collection function to a flow cytometer. Particles present in the separated stream and detected as having one or more desired properties are individually separated from the sample stream by mechanical or electrical removal. A common flow sorting technique utilizes droplet sorting, in which a fluid stream containing linearly separated particles is split into droplets. Droplets containing particles of interest are electrically charged and deflected into a collection tube by passing through an electric field. Typically, linearly separated particles in a stream are characterized as they pass an observation point directly below the nozzle tip. Once a particle is identified as meeting one or more desired criteria, it is possible to predict the time at which the particle will reach the droplet breakoff point and separate from the stream into droplets. Ideally, the fluid stream is briefly charged just before droplets containing selected particles separate from the fluid stream, and then grounded immediately after the droplets separate. The droplets to be sorted maintain their charge as they separate from the fluid stream, while all other droplets remain uncharged. Summary of the Invention [Problem to be solved by the invention]

[0004] Current high-speed cell sorters can measure multiple parameters per cell, e.g., 100 parameters per cell. Such high-speed cell sorters can measure parameters at high speeds, e.g., 10,000–25,000 cells / second. Due to the panel complexity associated with current high-speed cell sorters, multiple color panels, e.g., 50 color panels, may be utilized for characterization of particles such as cells. Related to this aspect of current cell sorters, there is a need for the ability to sort many different particles, e.g., cells, during sorting, e.g., during operation of the high-speed cell sorter in connection with experimental data collection. The sorting direction of existing high-speed cell sorters is limited by the distance a particle, e.g., a cell or droplet, can deviate from the center of the droplet flow stream, i.e., the longitudinal axis of the droplet flow stream. That is, droplet deflection needs to be optimized or improved, and in some cases this means extending the droplet deflector to deflect droplets at higher angles relative to the longitudinal axis of the droplet flow stream, or increasing the deposition distance from the longitudinal axis of the droplet flow stream, while keeping the droplet deflector length (i.e., the length of the deflection plate along the longitudinal axis of the droplet flow stream) constant or substantially constant, for example for compatibility with existing cell sorters.

[0005] The inventors have recognized a need for optimizing the deflection of droplets in a flow stream. In particular, there is a need for a droplet deflector capable of high-angle deflection of a droplet flow stream. Embodiments of the present invention fulfill this need. In particular, embodiments of the present invention provide high-angle deflection of droplets in a flow stream. Such enhancements expand the range of possible sorting categories of sample particles, e.g., cells, by providing the potential for a wider variety of sorting categories, e.g., configured to sort into more tubes or wells of a multi-well plate. High-speed cell sorters equipped with droplet deflectors of the present invention configured for high-angle deflection may provide the additional advantage of requiring less charge to be applied to sorted droplets in a flow stream. Such an advantage may increase sorting purity and yield. [Means for solving the problem]

[0006] Aspects of the present disclosure include droplet deflectors including deflection plates configured for high-angle deflection of droplet flow streams. Deflection plates of interest have a shape that corresponds to the path of the deflected droplet flow stream. In other cases, the deflection plates have a shape that minimizes the distance between the deflected droplet flow stream and the deflection plates of the droplet deflector over the length of the deflection plates. In still other cases, the deflection plates have a shape that maintains a constant buffer region between the deflected droplet flow stream and the deflection plates of the droplet deflector over the length of the deflection plates. In embodiments, the deflection plates are configured to maximize the deflection force applied to the droplet flow stream at multiple different downstream locations. In some embodiments, the deflection plates have a shape that is configured to prevent the deflected droplet stream from colliding with the deflection plates. In other embodiments, the droplet deflector is configured such that the distance between the deflection plates increases minimally at each of multiple downstream locations.

[0007] Deflector plates of interest have nonlinear surfaces or splines. In some cases, the deflector plates are configured to apply deflection forces to the flow stream at a number of different angles. In such embodiments, twisted deflector plates may be provided. In such cases, the twist angle may be 5 degrees or more, or 30 degrees or more, or 60 degrees or more, e.g., 90 degrees.

[0008] In embodiments, the droplet deflector is configured to apply a constant deflection force to the deflected droplet flow stream from a plurality of different lateral locations. In some cases, the different lateral locations are different distances from the centerline of the deflection plates. In other cases, the distance between the deflection plates increases at different downstream locations. In still other cases, the droplet deflector is configured so that different voltage potentials are applied to the deflection plates at different downstream locations. In embodiments, each deflection plate has two or more segments. In some cases, the split deflector plates have corresponding aligned segments. Each segment may be electrically insulated from the other segments. In still other cases, the droplet deflector is configured so that each segment of the split deflector plate receives a different electrical potential. In further cases, each segment is separated from the other segments by an electrical insulator. In some embodiments, the droplet deflector further includes a plurality of resistors connected in series. In some cases, each resistor of the plurality of resistors is electrically connected to a segment of the split deflector plate. In other cases, each segment is separated from the other segments by a resistive dielectric. In still other cases, each segment of the split deflector plate is configured to receive a different magnitude of electrical potential based on the distance between corresponding segments of the electrode. In some embodiments, a voltage source operably connected to the deflector plate is further provided. In other embodiments, a voltage divider having a plurality of resistors connected in series is further provided. In some cases, each resistor of the plurality of resistors is electrically connected to a segment of the split deflector plate.

[0009] In embodiments of a droplet deflector having deflection plates configured for high-angle deflection of a droplet flow stream, the droplet deflector is configured to apply a deflection force sufficient to deflect particles by 5 mm or more, or 15 mm or more, or 30 mm or more, or within a range of 5 mm to 100 mm. In some cases, the droplet deflector is configured to deposit droplets of the deflected droplet flow stream into each well in a column of a 96-well plate. Embodiments include deflector plates comprising metal plates. In some cases, the deflector plates have a width in a range of 0.5 mm to 10 mm, or a length in a range of 1 mm to 25 mm, or are spaced apart by 1 mm or more, or 3 mm or more, or within a range of 1 mm to 10 mm. In some embodiments, the deflector plates are rectangular. Also provided are particle sorting modules including droplet deflectors according to embodiments, and systems including droplet deflectors according to embodiments. Also provided are methods, including methods for sorting particles using droplet deflectors according to embodiments. [Brief explanation of the drawings]

[0010] The present disclosure can be best understood from the following detailed description when read in conjunction with the accompanying drawing figures, which include:

[0011] [Figure 1A] FIG. 1 illustrates a prior art droplet deflector. [Figure 1B] FIG. 2 shows deflection plates of another prior art droplet deflector. [Figure 1C] 1A-1C illustrate exemplary shapes of deflection plates of a droplet deflector. [Figure 1D] FIG. 1 illustrates a droplet deflector including deflection plates configured for high-angle deflection of a droplet flow stream according to one embodiment. [Figure 1E] FIG. 1 illustrates a droplet deflector with twisted deflection plates configured for high-angle deflection of a droplet flow stream according to one embodiment. [Figure 1F] FIG. 1 illustrates a droplet deflector with segmented deflection plates configured for high-angle deflection of a droplet flow stream according to one embodiment. [Figure 1G] FIG. 10 illustrates another droplet deflector with segmented deflection plates configured for high-angle deflection of a droplet flow stream in accordance with an embodiment. [Figure 2] FIG. 1 illustrates a flow cytometry system according to an embodiment. [Figure 3-1] FIG. 1 illustrates an image-enabled particle sorter according to an embodiment. [Figure 3-2] FIG. 1 illustrates an image-enabled particle sorter according to an embodiment. [Figure 4] FIG. 1 is a functional block diagram illustrating a particle analysis system according to an embodiment. [Figure 5] FIG. 1 is a functional block diagram illustrating an example of a control system according to an embodiment. [Figure 6A] FIG. 1 is a schematic diagram illustrating a particle sorting system according to an embodiment. [Figure 6B] FIG. 1 is a schematic diagram illustrating a particle sorting system according to an embodiment. [Figure 7] FIG. 1 illustrates a computer control system according to an embodiment. [Figure 8A] 1A-1C illustrate an exemplary technique for optimizing the shape of deflection plates of a droplet deflector according to an embodiment. [Figure 8B] 1A-1C illustrate an exemplary technique for optimizing the shape of deflection plates of a droplet deflector according to an embodiment. [Figure 9] 1 is an exemplary flowchart for optimizing the shape of deflection plates of a droplet deflector according to an embodiment. [Figure 10A] 10 is a diagram showing the results of numerical calculations of deflection plate geometries and associated droplet deflection channels. [Figure 10B] 10 is a diagram showing the results of numerical calculations of deflection plate geometries and associated droplet deflection channels. [Figure 10C] 10 is a diagram showing the results of numerical calculations of deflection plate geometries and associated droplet deflection channels. [Figure 11] FIG. 1 illustrates a torsional deflection plate configured for high angle deflection of a droplet flow stream according to one embodiment. [Figure 12A] FIG. 1 illustrates an experimental droplet deflector setup with deflection plates configured for high-angle deflection of a droplet flow stream according to one embodiment. [Figure 12B] FIG. 1 illustrates an experimental droplet deflector setup with deflection plates configured for high-angle deflection of a droplet flow stream according to one embodiment. [Figure 12C] 1 is a chart illustrating performance results of a droplet deflector including deflection plates configured for high-angle deflection of a droplet flow stream according to an embodiment. [Figure 12D] FIG. 1 illustrates an experimental droplet deflector setup with deflection plates configured for high-angle deflection of a droplet flow stream according to one embodiment. [Figure 12E] FIG. 1 illustrates an experimental droplet deflector setup with deflection plates configured for high-angle deflection of a droplet flow stream according to one embodiment. [Figure 12F] FIG. 1 illustrates an experimental droplet deflector setup with deflection plates configured for high-angle deflection of a droplet flow stream according to one embodiment. [Figure 12G] FIG. 1 illustrates an experimental droplet deflector setup with deflection plates configured for high-angle deflection of a droplet flow stream according to one embodiment. [Figure 12H] 1 is a chart illustrating performance results of a droplet deflector including deflection plates configured for high-angle deflection of a droplet flow stream according to an embodiment.

[0012] In the drawings, elements with the same or similar reference numbers have the same or similar features unless otherwise stated. DETAILED DESCRIPTION OF THE INVENTION

[0013] Droplet deflectors with optimized deflection plates are provided. Droplet deflectors of interest include droplet deflectors with deflection plates configured for high-angle deflection of a droplet flow stream. Droplet deflectors of interest also include droplet deflectors with deflection plates having a shape corresponding to the path of the deflected droplet flow stream, or deflection plates configured to apply a deflection force to the flow stream at a plurality of different angles. Droplet deflectors of interest also include droplet deflectors configured to apply a constant deflection force to the deflected droplet flow stream from a plurality of different lateral positions, e.g., the deflection plates are segmented deflector plates. Flow cytometers including the subject droplet deflectors, as well as methods of use and assembly or configuration thereof, are also provided.

[0014] Before the present disclosure is described in more detail, it is to be understood that this disclosure is not limited to the particular embodiments described, as such may, of course, vary. The scope of the present disclosure will be limited only by the appended claims, and it is to be further 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 given, it is understood that each intervening value between the upper and lower limits of that range, to the tenth of the unit of the lower limit unless the context clearly indicates otherwise, and any other stated or intervening value in that stated range, is encompassed within the disclosure. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also encompassed within the disclosure.

[0016] In this specification, a range is presented with the term "about" before the numerical values. The term "about" is used herein to literally support the exact number that it precedes, as well as a number that is close to or approximately the number that it precedes. When determining whether a number is close to or approximately a specifically stated number, the unstated number that is close or approximately the number may be a number that, in the context in which the specifically stated number is presented, provides a substantial equivalent to the specifically stated number.

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

[0018] All publications and patents cited herein are incorporated by reference to the same extent as if each individual publication or patent was specifically and individually indicated to be incorporated by reference, and are incorporated by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present disclosure is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates, which may need to be independently confirmed.

[0019] It should be noted that, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. It should be further noted that the claims may be drafted to exclude any optional element. Accordingly, this statement is intended to serve as a predicate for use of exclusive terminology such as "solely," "only," and the like, or for use of a "negative" limitation 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 individual embodiments described and illustrated herein comprises separate components and features which may be readily separated from or combined with any of the features of the other multiple embodiments without departing from the scope or spirit of the disclosure. Any recited method may be carried out in the order of events recited or in any other order which is logically possible.

[0021] Although the systems and methods have been or will be described for grammatical fluidity with functional descriptions, it should be clearly understood that the claims, unless expressly recited under 35 U.S.C. 112, should not be construed as necessarily limited in any way by limitations of "means" or "step" construction, but should be accorded the full scope of the meaning and equivalents of the definition given by the claims under the judicial theory of equivalents, and that if a claim is expressly recited under 35 U.S.C. 112, it should be accorded the full legal equivalents under 35 U.S.C. 112.

[0022] As summarized above, the present disclosure provides a droplet deflector including deflection plates configured for high-angle deflection of a droplet flow stream. In further describing embodiments of the present disclosure, the droplet deflector configured for high-angle deflection of a droplet flow stream will first be described in more detail. Next, a particle sorting module and system for separating particles in a sample will be described. A method for sorting droplets in a flow stream is also provided.

[0023] Droplet deflector for high-angle deflection of droplet flow streams As summarized herein, aspects of the present disclosure include a droplet deflector comprising a deflection plate configured for high-angle deflection of a droplet flow stream. The deflection plate may further be referred to as a deflector plate, or in some cases as a metal plate, or in some cases as an electrode. The term "deflection" is used herein in its conventional sense to refer to applying a force that deflects droplets in a flow stream from flowing along their normal trajectory (i.e., the trajectory in the absence of a deflection force) to a different trajectory along the longitudinal axis of the flow stream. Furthermore, the term "high-angle deflection" is used herein to refer to applying a force that deflects droplets in a flow stream at a higher or larger angle relative to the path of the droplet's normal trajectory than is available using existing technology, or applying a deflection force to a stream of droplets using a deflection plate having a shape optimized for deflection in accordance with the technology described herein. In some cases, droplet deflectors of the present invention can be used to deflect droplets in a flow stream at a substantially higher or larger angle relative to the path of the droplet's normal trajectory than is obtainable using existing technology. In this manner, the droplet deflector of the present invention can be used to deflect droplets farther from their normal trajectory along the longitudinal axis of the flow stream compared to existing techniques, which provide advantages such as allowing the separation of a sample into more compartments, e.g., tubes or wells, or reducing the charge applied to droplets in connection with sorting particles of the sample.

[0024] Droplets in a flow stream may be deflected from their normal trajectory along the longitudinal axis of the flow stream using a drop deflector according to embodiments of the present disclosure by a distance of at least 0.001 mm, such as at least 0.005 mm, for example at least 0.01 mm, such as at least 0.05 mm, for example at least 0.1 mm, such as at least 0.5 mm, for example at least 1 mm, such as at least 2 mm, for example at least 5 mm, such as at least 10 mm, for example at least 15 mm, such as at least 20 mm, for example at least 25 mm, for example at least 30 mm, for example at least 35 mm, for example at least 50 mm, measured radially in a plane perpendicular to the longitudinal axis of the flow stream. For example, droplets in the flow stream may be deflected a distance in the range of 0.001 mm to 100 mm, such as 0.005 mm to 95 mm, for example 0.001 mm to 90 mm, for example 0.05 mm to 85 mm, for example 0.01 mm to 80 mm, for example 0.05 mm to 75 mm, for example 0.1 mm to 70 mm, for example 0.5 mm to 65 mm, for example 1 mm to 60 mm, for example 5 mm to 55 mm, for example 10 mm to 50 mm. Thus, droplets in the flow stream may be deflected by the deflection force at an angle in the range of 0.01° to 90°, for example 0.05° to 85°, for example 0.1° to 80°, for example 0.5° to 75°, for example 10° to 70°, for example 15° to 65°, for example 20° to 60°, for example 25° to 55°, for example 30° to 50° from the longitudinal axis of the flow stream.

[0025] As described in more detail herein, the subject droplet deflectors may be configured to sort particles in a sample, such as cells in a biological sample. In these embodiments, the droplet deflectors are configured to apply a deflection force sufficient to deflect particles flowing through the flow stream into one or more sample collection vessels. In embodiments, as will be appreciated by those skilled in the art, the droplet deflectors are configured for high-angle deflection of the droplet flow stream, such that particles in the sample are deflected into sample collection vessels that are spaced relatively farther apart compared to those used in existing technology, and so that more sample collection vessels can be used to sort particles of the sample into more compartments. Thus, the droplet deflector may be configured to apply a deflection force to deflect particles in the flow stream into a sample collection vessel that is 0.001 mm or more, such as 0.005 mm or more, for example 0.01 mm or more, such as 0.05 mm or more, for example 0.1 mm or more, such as 0.5 mm or more, for example 1 mm or more, such as 2 mm or more, for example 5 mm or more, such as 10 mm or more, for example 15 mm or more, such as 20 mm or more, for example 25 mm or more, such as 30 mm or more, for example 35 mm or more, such as 50 mm or more away from the longitudinal axis of the flow stream. For example, the droplet deflector may be configured to deflect particles in the flow stream to a sample collection vessel a distance away from the longitudinal axis of the flow stream in a range of 0.001 mm to 100 mm, such as 0.005 mm to 95 mm, for example 0.001 mm to 90 mm, such as 0.05 mm to 85 mm, for example 0.01 mm to 80 mm, such as 0.05 mm to 75 mm, for example 0.1 mm to 70 mm, such as 0.5 mm to 65 mm, for example 1 mm to 60 mm, such as 5 mm to 55 mm, for example 10 mm to 50 mm.

[0026] In an embodiment of the present disclosure, the droplet deflector comprises two or more metal plates, such as two or more opposing, opposed or parallel metal plates configured to generate an electric field therebetween. The voltage applied to the deflector plates to deflect the charged particles may be 10 mV or more, such as 25 mV or more, for example 50 mV or more, such as 100 mV or more, for example 250 mV or more, such as 500 mV or more, for example 750 mV or more, such as 1000 mV or more, for example 2500 mV or more, for example 5000 mV or more, such as 10000 V or more, for example 15000 V or more, for example 25000 V or more, such as 50000 V or more, for example 100000 V or more. In one embodiment, the voltage applied to each pair of metal plates is in the range of 0.5 kV to 15 kV, for example, 1 kV to 15 kV, for example, 1.5 kV to 12.5 kV, for example, 2 kV to 10 kV. In one embodiment, the voltage applied to each pair of metal plates is in the range of 0.5 kV to 15 kV, for example, 1 kV to 15 kV, for example, 1.5 kV to 12.5 kV, for example, 2 kV to 10 kV. Depending on the voltage applied to the metal plates, the electric field strength between the metal plates is in the range of 0.001 V / m to 1×10 7 V / m, e.g., 0.01 V / m ~5×10 6 V / m, e.g., 0.1V / m~1×10 6 V / m, e.g., 0.5 V / m ~5×10 5 V / m, e.g., 1V / m ~1×10 5 V / m, e.g., 5V / m ~5×10 4 V / m, e.g., 10 V / m~1×10 4 V / m, e.g., 50 V / m ~5×10 3 V / m, e.g., 1×10 5 V / m ~2×10 6 It may vary within the range of V / m.

[0027] In embodiments, the droplet deflector comprises two metal plates spaced apart by a distance sufficient to generate an electric field therebetween. For example, the metal plates may be spaced apart by 0.01 mm or more, such as 0.05 mm or more, for example 0.1 mm or more, for example 0.5 mm or more, for example 1 mm or more, for example 1.5 mm or more, for example 2 mm or more, for example 2.5 mm or more, for example 3 mm or more, for example 3.5 mm or more, for example 4 mm or more, for example 4.5 mm or more, for example 5 mm or more, for example 10 mm or more, for example 15 mm or more, for example 20 mm or more, for example 25 mm or more. In some cases, the metal plates are spaced apart by a distance in the range of 0.01 mm to 50 mm, for example 0.05 mm to 45 mm, for example 0.1 mm to 40 mm, for example 0.5 mm to 35 mm, for example 1 mm to 30 mm, for example 1.5 mm to 25 mm, for example 2 mm to 20 mm, for example 3 mm to 15 mm.

[0028] As described in more detail herein, the subject droplet deflectors may comprise a metal plate having two or more segments, e.g., three or more, e.g., four or more, e.g., five or more, e.g., six or more, e.g., seven or more, e.g., eight or more, e.g., nine or more, e.g., ten or more, each segment corresponding to an area on each deflector plate, the segments on the deflector plates being positioned directly opposite one another. In some embodiments, applying voltages to the segments of the metal plate exerts a deflecting force on droplets in the flow stream, resulting in the creation of an electric field that accelerates and deflects the trajectory of targeted droplets away from the longitudinal axis of the flow stream into one or more sample collection vessels. The voltages applied to each segment of the metal plate may be the same or different. In some embodiments, the voltage applied to each segment of the metal plate may increase as the distance between the metal plates increases, i.e., as the lateral distance between the metal plates (i.e., deflector plate segments) increases. When different voltages are applied to each segment of the metal plate, the difference between the applied voltages may be 0.01 mV or more, such as 0.05 mV or more, for example 0.1 mV or more, for example 0.5 mV or more, for example 1 mV or more, for example 5 mV or more, such as 10 mV or more, for example 25 mV or more, for example 50 mV or more, for example 75 mV or more, for example 100 mV or more, for example 250 mV or more, for example 500 mV or more, for example 750 mV or more, for example 1 V or more, for example 2.5 V or more, for example 5 V or more, for example 10 V or more, for example 25 V or more, for example 50 V or more, for example 100 V or more, for example 500 V or more, for example 1000 V or more. In an embodiment, the difference between the applied voltages may be in the range of 0.5 kV to 15 kV, for example 1 kV to 15 kV, for example 1.5 kV to 12.5 kV, for example 2 kV to 10 kV.

[0029] Depending on the applied voltage, the electric field strength between each segment of the metal plate may be the same or different. In some embodiments, the electric field strength between each segment of the metal plate is the same or substantially the same, and droplets flowing in the flow stream are subjected to a constant electric field through the droplet deflector (even though the deflection plate segments may be separated from one another by a number of different distances). In other embodiments, the electric field strength between each segment of the metal plate is different, and the electric field strength is 0.001 V / m or more, such as 0.01 V / m or more, for example 0.1 V / m or more, for example 0.5 V / m or more, for example 1 V / m or more, for example 2 V / m or more, for example 5 V / m or more, for example 10 V / m or more, for example 25 V / m or more, for example 50 V / m or more, for example 100 V / m or more, for example 500 V / m or more, for example 1×10 3 V / m or more difference.

[0030] The metal plates of the subject droplet deflectors may be formed from any suitable metal capable of generating an electric field, including aluminum, brass, chromium, cobalt, copper, gold, indium, iron, lead, nickel, platinum, palladium, tin, steel (e.g., stainless steel), silver, zinc, and combinations and alloys thereof, such as aluminum alloys, aluminum-lithium alloys, aluminum-nickel-copper alloys, aluminum-copper alloys, aluminum-magnesium alloys, aluminum-magnesium oxide alloys, aluminum-silicon alloys, aluminum-magnesium-chromium alloys, and aluminum-lithium alloys. copper-platinum alloy, cobalt alloy, cobalt-chromium alloy, cobalt-tungsten alloy, cobalt-molybdenum-carbon alloy, cobalt-chromium-nickel-molybdenum-iron-tungsten alloy, copper alloy, copper-arsenic alloy, copper-beryllium alloy, copper-silver alloy, copper-zinc alloy (e.g. brass), copper-tin alloy (e.g. bronze), copper-nickel alloy, copper-tungsten alloy, copper-gold-silver alloy, copper-nickel-iron alloy, copper-manganese-tin alloy, copper-aluminum-zinc-tin alloy, copper-gold alloy, gold alloy, gold-silver alloy, indium alloy, indium-tin alloy, Indium-tin oxide alloys, iron alloys, iron-chromium alloys (e.g., steel), iron-chromium-nickel alloys (e.g., stainless steel), iron-silicon alloys, iron-chromium-molybdenum alloys, iron-carbon alloys, iron-boron alloys, iron-magnesium alloys, iron-manganese alloys, iron-molybdenum alloys, iron-nickel alloys, iron-phosphorus alloys, iron-titanium alloys, iron-vanadium alloys, lead alloys, lead-antimony alloys, lead-copper alloys, lead-tin alloys, lead-tin-antimony alloys, nickel alloys, nickel-manganese-aluminum-silicon alloys, nickel-chromium alloys, nickel-copper alloys, nickel, molybdenum-chromium-tungsten alloys, nickel-copper-iron-manganese alloys, nickel-carbon alloys, nickel-chromium-iron alloys, nickel-silicon alloys, nickel-titanium alloys, silver alloys, silver-copper alloys (e.g., sterling silver), silver-copper-germanium alloys (e.g., argentium sterling silver), silver-gold alloys, silver-copper-gold alloys, silver-platinum alloys, tin alloys, tin-copper-antimony alloys, tin-lead-copper alloys, tin-lead-antimony alloys, titanium alloys, titanium-vanadium-chromium alloys, titanium-aluminum alloys,The alloys may include, but are not limited to, titanium-aluminum-vanadium alloys, zinc alloys, zinc-copper alloys, zinc-aluminum-magnesium-copper alloys, zirconium alloys, zirconium-tin alloys, or combinations thereof.

[0031] The metal plates of the subject droplet deflectors may have surfaces of any suitable shape (e.g., the surface of the metal plate having the largest surface area, i.e., the surface that may be referred to as the "front surface" of the metal plate, faces toward the droplet flow stream, as opposed to the shape of the metal plate along the longitudinal axis of the flow stream, e.g., a cross-section along such axis, as described herein), such as a circle, oval, semicircle, crescent, star, square, triangle, diamond, pentagon, hexagon, heptagon, octagon, rectangle, or other suitable polygon with respect to such surface of the metal plate. In some embodiments, such surface of the metal plate is rectangular or rounded rectangular. As described in more detail herein, in some cases the metal sheet is twisted, for example the twisted rectangle has a twist angle of 5° or more, such as 10° or more, for example 15° or more, such as 20° or more, for example 25° or more, such as 30° or more, for example 35° or more, such as 40° or more, for example 45° or more, such as 50° or more, for example 55° or more, such as 60° or more, for example 90° or more.

[0032] The dimensions may vary depending on the shape of the "front" of the metal plate, i.e., the surface of the metal plate having the largest surface area. In some embodiments, each metal plate has a width in the range of 0.5 mm to 10 mm, e.g., 1 mm to 9.5 mm, e.g., 1.5 mm to 9 mm, e.g., 2 mm to 8.5 mm, e.g., 2.5 mm to 8 mm, e.g., 3 mm to 7.5 mm, e.g., 3.5 mm to 7 mm, e.g., 4 mm to 6.5 mm, e.g., 4.5 mm to 6 mm. The length also varies in the range of 10 mm to 500 mm, e.g., 15 mm to 450 mm, e.g., 20 mm to 400 mm, e.g., 25 mm to 350 mm, e.g., 30 mm to 300 mm, e.g., 35 mm to 250 mm, e.g., 40 mm to 200 mm, e.g., 45 mm to 150 mm, e.g., 50 mm to 100 mm. In some embodiments, the metal plate is an asymmetric polygon, with a first end having a smaller width than a second end. The width at each end may be in the range of 0.01 mm to 10 mm, such as 0.05 mm to 9.5 mm, for example 0.1 mm to 9 mm, for example 0.5 mm to 8.5 mm, for example 1 mm to 8 mm, for example 2 mm to 8 mm, for example 2.5 mm to 7.5 mm, for example 3 mm to 6 mm. In embodiments, the surface area of ​​the "front face" of each metal plate may vary as desired, and may be in the range of 0.25 to 15 cm. 2 , e.g. 0.5 ~14cm 2 , e.g. 0.75~13cm 2 , for example, 1 to 12 cm 2 , e.g. 1.5 ~ 11cm 2 , for example, 2 to 10 cm 2 may be in the range of

[0033] As described herein, in embodiments of the droplet deflector of the present invention, the droplet deflector includes a deflection plate configured for high-angle deflection of a droplet flow stream. In some cases, the deflection plate has a shape corresponding to the path of the deflected droplet flow stream. "Shape" refers to the shape of the deflection plate along the longitudinal axis of the droplet flow stream; in some cases, "shape" refers to the shape of the deflection plate along the longitudinal axis of the droplet flow stream or the shape of the deflection plate relative to the path of the deflected droplet flow stream. In some cases, the shape of the deflection plate refers to the distance between the droplets of the droplet flow stream and the deflection plate (e.g., for a deflection plate configuration that includes the distance from the droplet flow stream in one axis (e.g., the x-axis) perpendicular to the longitudinal axis of the flow stream, or in two axes (e.g., the x-axis and the y-axis) both perpendicular to the longitudinal axis of the flow stream, e.g., a spiral configuration), in some cases. In other words, unless the context indicates otherwise, references to the "shape" of a deflection plate typically refer to the cross-sectional shape of the deflection plate along the path of the deflected droplet flow stream, i.e., along the longitudinal axis of the droplet flow stream, and in some cases, this may refer to the cross-sectional shape along the length of the deflection plate (as opposed to the shape of the "front" of the deflection plate, i.e., the surface of the deflection plate with the greatest surface area). For example, in some cases, the deflection plate has a shape that minimizes the distance between the deflected droplet flow stream and the deflection plate of the droplet deflector along the length of the deflection plate. In an embodiment, the deflection plates have a shape that minimizes the distance between the deflected droplet flow stream and the deflection plates of the droplet deflector by at least 1%, for example at least 10%, for example at least 20%, for example at least 30%, for example at least 40%, for example at least 50%, for example at least 60%, for example at least 70%, for example at least 80%, for example at least 90% of the length of the droplet deflector along the longitudinal axis of the droplet flow stream.In embodiments, any convenient distance may be applied as the minimum distance between the deflection plate and the droplet flow stream, such as 0.01 mm or less, 0.1 mm or less, 0.2 mm or less, 0.3 mm or less, 0.4 mm or less, 0.5 mm or less, 0.6 mm or less, 0.7 mm or less, 0.8 mm or less, 0.9 mm or less, 1.0 mm or less, 2.0 mm or less, 3.0 mm or less, 4.0 mm or less, 5.0 mm or less, or 10 mm or less.

[0034] Similarly, in some cases, the deflection plates have a shape that maintains a constant buffer region (i.e., a constant minimum distance) between the deflected droplet flow stream and the droplet deflector's deflection plates along the length of the deflection plates. That is, the deflection plates may have a shape such that the deflected droplet flow stream is at a constant minimum distance from the droplet deflector's deflection plates along the droplet flow stream's longitudinal axis along the length of the deflection plates. In embodiments, the deflection plates may have a shape such that the deflected droplet flow stream is at a constant minimum distance from the droplet deflector's deflection plates along the droplet flow stream's longitudinal axis for 1% or more, e.g., 10% or more, e.g., 20% or more, e.g., 30% or more, e.g., 40% or more, e.g., 50% or more, e.g., 60% or more, e.g., 70% or more, e.g., 80% or more, e.g., 90% or more of the droplet deflector's length. In embodiments, any convenient distance may be applied as a fixed minimum distance between the deflection plate and the droplet flow stream, such as 0.01 mm or less, 0.1 mm or less, 0.2 mm or less, 0.3 mm or less, 0.4 mm or less, 0.5 mm or less, 0.6 mm or less, 0.7 mm or less, 0.8 mm or less, 0.9 mm or less, 1.0 mm or less, 2.0 mm or less, 3.0 mm or less, 4.0 mm or less, 5.0 mm or less, or 10 mm or less.

[0035] As described herein, a droplet deflector is configured to deflect droplets of a droplet flow stream. The droplet deflector deflects droplets of the droplet flow stream by applying a deflection force to the droplets of the droplet flow stream. In embodiments of the invention, the droplet deflector includes deflection plates configured to maximize the deflection force applied to the droplet flow stream at multiple different downstream locations. That is, the deflection plates are shaped to maximize the deflection force applied to the droplet flow stream by the deflection plates at multiple locations along the longitudinal axis of the droplet flow stream or multiple locations along the longitudinal axis or length of the deflection plates. In embodiments, such force is maximized by maximizing the electric field between the deflector plates at multiple locations along the longitudinal axis of the droplet flow stream or multiple locations along the longitudinal axis or length of the deflection plates. In embodiments, the electric field may be maximized by decreasing the distance between the deflector plates and / or increasing the voltage applied to the deflector plates (i.e., increasing the voltage applied to different segments of the split deflector plates).

[0036] In embodiments, the deflection plates have a shape configured to prevent the deflected droplet stream from colliding with the deflection plates. That is, given the charged droplets in the droplet flow stream and the voltage applied to the deflection plates of the droplet deflector, the deflection plates have a shape that allows the charged droplets to be maximally deflected without colliding with the deflection plates, e.g., at the maximum possible angle or the maximum possible deflection distance. In some cases, the droplet deflector is configured such that the distance between the deflection plates is minimally increased at each of a plurality of downstream positions. Minimally increasing the distance between the deflection plates in some cases means that the distance between the deflection plates is increased by the minimum amount necessary to prevent the charged droplets from colliding with the deflection plates. In some embodiments, the deflection plates have nonlinear surfaces. For example, in some cases, the deflection plates have a spline shape.

[0037] As described herein, in some cases, the deflector plates are configured to apply a deflection force to the flow stream at a plurality of different angles. In certain embodiments, the metal plates are twisted. In these embodiments, the metal plates have the same twist angle and remain spaced apart from one another, sometimes to varying degrees, along the length of the metal plate. In some embodiments, the applied deflection force remains constant along the length of the twisted metal plate (e.g., in a segmented twisted deflector plate). In other embodiments, the applied deflection force varies along the length of the twisted metal plate. Depending on the desired deflection angle, the twisted metal plate may have a variety of twist angles, such as 5° or more, e.g., 10° or more, e.g., 15° or more, e.g., 20° or more, e.g., 25° or more, e.g., 30° or more, e.g., 35° or more, e.g., 40° or more, e.g., 45° or more, e.g., 50° or more, e.g., 55° or more, e.g., 60° or more. For example, the twist angle may be within the range of 1° to 90°, for example, 2° to 85°, for example, 3° to 80°, for example, 4° to 75°, for example, 5° to 70°, for example, 10° to 60°, for example, 15° to 45°, for example, 20° to 40°.

[0038] In some embodiments, the twisted metal plate is twisted by 1 helical twist or less, such as 0.9 helical twist or less, for example 0.8 helical twist or less, for example 0.7 helical twist or less, for example 0.6 helical twist or less, for example 0.5 helical twist or less. In certain embodiments, the twisted metal plate has a twist configuration such that the proximal end of the twisted metal plate is oriented at an angle in the range of 1° to 90°, for example 2° to 85°, for example 3° to 80°, for example 4° to 75°, for example 5° to 70°, for example 10° to 60°, for example 15° to 45°, for example 20° to 40°, relative to the distal end of the twisted metal plate. In certain embodiments, the proximal end of the twisted metal plate is oriented perpendicular to the distal end.

[0039] As described, in some embodiments, droplet deflectors of interest include a divided metal plate having a first metal plate segment and a second metal plate segment disposed downstream (along the flow path of the flow stream) from the first metal plate segment. In these embodiments, the shape and size of the first metal plate segment may be the same as or different from the second metal plate segment. In some embodiments, the shape of the first metal plate segment is the same as the shape of the second metal plate segment (e.g., both are rectangular). In other embodiments, the shape of the first parallel metal plate segment is different from the shape of the second parallel metal plate segment (e.g., the first metal plate segment is square and the second parallel metal plate segment is rectangular). In some cases, the dimensions of the first metal plate segment are the same as the second metal plate segment. In one example, the width of the first metal plate segment is the same as the second metal plate segment. In other cases, the length of the first metal plate segment is the same as the second metal plate segment. In yet other cases, the width and length of the first segment of the metal plate are the same as the second segment of the metal plate. In some examples, the dimensions of the first segment of the metal plate are different from the second segment of the metal plate. In one example, the width of the first segment of the metal plate is different from the second segment of the metal plate. In another example, the length of the first segment of the metal plate is different from the second segment of the metal plate. In yet another example, both the width and length of the first segment of the metal plate are different from the second segment of the metal plate. In some cases, the deflection plates have angled deflection plates, and the first segment has an upper or proximal region with parallel plates and the second segment has a lower or distal region with angled plates.

[0040] When the droplet deflector comprises two or more segments of a metal plate, each segment of the metal plate is configured to deflect the trajectory of the targeted droplets by a predetermined distance from the longitudinal axis of the flow stream. Each segment of the segmented deflector plate may be configured to deflect the trajectory of the targeted droplets by the same angle. In an embodiment, the droplet deflector comprises two segments of parallel metal plates, a first segment of the parallel metal plate configured to deflect targeted droplets in the flow stream by a distance in the range of 0.001 mm to 100 mm, such as 0.005 mm to 95 mm, for example 0.001 mm to 90 mm, for example 0.05 mm to 85 mm, such as 0.01 mm to 80 mm, for example 0.05 mm to 75 mm, for example 0.1 mm to 70 mm, such as 0.5 mm to 65 mm, for example 1 mm to 60 mm, for example 5 mm to 55 mm, for example 10 mm to 50 mm, and a second segment of the metal plate configured to deflect targeted droplets in the flow stream by a distance in the range of 0.001 mm to 100 mm, for example 0.005 mm to 95 mm, for example 0.001 mm to 90 mm, for example 0.05 mm to 85 mm, for example 0.01 mm to 80 mm, for example 0.05 mm to 75 mm, for example 0.1 mm to 70 mm, for example 0.5 mm to 65 mm, for example 1 mm to 60 mm, for example 5 mm to 55 mm, for example 10 mm to 50 mm. The metal plate may be configured to deflect the beam by a distance in the range of 0.001 V / m or more, e.g., 0.01 V / m or more, e.g., 0.05 V / m or more, e.g., 0.01 V / m or more, e.g., 0.05 V / m or more, e.g., 0.05 V / m or more, e.g., 0.1 V / m or more, e.g., 0.5 V / m or more, e.g., 10 V / m or more, e.g., 25 V / m or more. In some cases, the electric field between the first segments of the metal plates is greater than the electric field between the second segments of the metal plates, and in other cases, the electric field between the first segments of the metal plates is less than the electric field between the second segments of the metal plates.

[0041] In droplet deflector embodiments of the present invention, the droplet deflector is configured to apply a constant deflection force to the deflected droplet flow stream from a plurality of different lateral positions. In some such embodiments, the different lateral positions are at different distances from the centerline of the deflector plates. That is, the different lateral positions are at different distances from the longitudinal axis of the droplet flow stream. In some such embodiments, the distance between the deflector plates increases at different downstream positions. In some such embodiments, the droplet deflector is configured such that different voltage potentials are applied to the deflector plates at the different downstream positions.

[0042] For example, in embodiments, each deflection plate of a droplet deflector has two or more segments. In some cases, the segmented deflector plates have corresponding aligned segments. In embodiments, each segment is electrically isolated from the other segments. In other embodiments, the droplet deflector is configured so that each segment of the segmented deflector plate receives a different electric potential. For example, each segment may be configured to receive an electric potential selected such that the electric field (and thus the deflection force) between different segments remains substantially constant despite differences in distance between the different segments. In some embodiments, each segment is separated from the other segments by an electrical insulator, such as a resistive dielectric.

[0043] Embodiments of the present invention include droplet deflectors comprising a plurality of resistors connected in series. In such embodiments, each resistor of the plurality of resistors may be electrically connected to a segment of a split deflector plate. In some cases, each segment is separated from the other segments by a resistive dielectric. Any convenient resistive dielectric may be applied to separate electrode segments, i.e., segments, of the deflector plate. In embodiments, the electrode segments typically face each other directly across the longitudinal axis of the droplet flow stream. In such embodiments, the resistive dielectric typically has spaces configured to evenly space or evenly separate the deflector plate segments. In embodiments comprising split deflector plates, each segment of the split deflector plate is configured to receive a potential with a different magnitude based on the distance between corresponding electrode segments, i.e., different electric field strengths are applied at different lateral positions. In some embodiments, a voltage source operably connected to the deflector plate is further provided. In some cases, embodiments further include a voltage divider having a plurality of resistors connected in series. In such cases, each resistor of the plurality of resistors is electrically connected to a segment of the split deflector plate.

[0044] In existing technology, droplet deflectors include parallel deflection plates. FIG. 1A shows an exemplary droplet deflector. Droplet deflector 100 includes parallel deflection plates 101a and 101b spaced a distance d apart. Deflector plate 101a has proximal ends 101a-p and distal ends 101a-d located downstream from proximal ends 101a-p along a longitudinal axis 198 of the flow stream. Deflector plate 101b has proximal ends 101b-p and distal ends 101b-d located downstream from proximal ends 101b-p along a longitudinal axis 198 of the flow stream. Flow stream 102 exits flow nozzle 199 at nozzle orifice 199a. The droplet deflector 100 can deflect the droplet flow stream according to a trajectory 102 that corresponds to a distance Δ from the longitudinal axis of the droplet flow stream (i.e., the trajectory of the droplet flow stream in the absence of a deflection force). The deflected droplets are collected in a sample collection reservoir (not shown) downstream of the deflection plates 101 a, 101 b.

[0045] For a droplet deflector 100 with parallel deflection plates 101 a, 101 b, the electric field between the deflection plates 101 a, 101 b may be approximated as E=V / d, where V is the plate voltage and d is the separation distance between the deflection plates 101 a, 101 b. Based on the characteristics of this electric field, the droplet mass M, and the droplet charge q, the deflection force can be calculated, and then the acceleration, velocity, and position of the deflected droplet in the flow stream as a function of time can be calculated as follows: First, the electric field and deflection force can be characterized as follows: E=V / d and F=qE=qV / d Therefore, the acceleration of the deflected droplets in the flow stream can be characterized as follows: a=F / M=qV / Md Therefore, the horizontal displacement (ie, the displacement perpendicular to the longitudinal axis of the droplet flow stream) can be characterized as follows:

[0046]

number

[0047] As shown by the above characteristics of the flow path of the droplet deflector 100 with parallel deflection plates 101a, 101b, the trajectory of the deflected droplet flow stream is parabolic.

[0048] In addition to the parallel deflection plates 101a, 101b of the droplet deflector 100, other shapes of deflection plates of the droplet deflector may be applied. FIG. 1B shows a shape of deflection plate 105 based on existing technology, which has a parallel portion and an angled portion following the parallel portion. The shape of deflection plate 105 has a short straight portion followed by an angled portion (i.e., a portion angled away from the longitudinal axis of the droplet flow stream to avoid deflected droplets, i.e., a portion angled to prevent deflected droplets from colliding with deflection plate 105). Other possible shapes of deflection plates are shown in FIG. 1C. Deflection plates 110a, 110b have a slanted shape. Deflection plates 111a, 111b have an angled shape. Deflection plates 112a, 112b have a parabolic shape. Deflection plates 113a, 113b have a hyperbolic cosine shape. Each of the shapes of deflector plates 110a, 110b, 111a, 111b, 112a, 112b, 113a, and 113b is not optimized for droplet deflection because each of these exemplary shapes may "understeer" droplets (i.e., position the deflected droplets "too far" from the deflector plate) at some times (i.e., at some locations along the longitudinal axis of the droplet flow stream) and "oversteer" droplets (i.e., cause the droplets to intersect or collide with the deflector plate) at other times (i.e., at other locations along the longitudinal axis of the droplet flow stream). "Understeer" droplets means that the deflector plate is shaped such that droplets are further away from the deflector plate than if the deflector plate had a different shape, which reflects a non-optimized design. Such a scenario reflects a sub-optimal design, as a closer distance between the deflector plates would result in a greater deflection force being exerted on the charged droplets in the flow stream.

[0049] In an embodiment of the present invention, deflection plates are provided with a shape that maximizes the electric field by optimizing the distance between the deflection plates by bringing the plates close together but not so close that charged droplets cross or collide with the deflection plates. In an embodiment of the present invention, deflection plates are provided with a shape that closely mimics the path of deflected droplets. FIG. 1D illustrates a droplet deflector configured for high-angle deflection of a droplet flow stream according to an embodiment of the present invention. Droplet deflector 115 includes deflection plates 116a and 116b configured for high-angle deflection of droplet flow stream 117. Deflection plate 116a has proximal ends 116a-p and distal ends 116a-d located downstream from proximal end 116a-p along a longitudinal axis 198 of the flow stream. Deflector plate 116b has a proximal end 116b-p and a distal end 116b-d disposed downstream from proximal end 116b-p along flow stream longitudinal axis 198. Deflector plates 116a, 116b are shaped with inner surfaces 119a, 119b configured to cause a high angle deflection of droplet flow stream 117 relative to droplet flow stream longitudinal axis 198 (i.e., the path of the droplet flow stream in the absence of any deflecting forces). (An example of how the term "shape" is typically used herein is the shape of the inner surfaces 119a, 119b of the deflector plates 116a, 116b.) The deflector plates 116a, 116b have a shape on their inner surfaces 119a, 119b that generates an electric field that deflects the flow stream 117 along a path that is substantially parallel to or follows the deflector plates 116a, 116b, particularly a path that is parallel to the inner surfaces 119a, 119b. That is, the inner surfaces 119a, 119b have a shape such that the deflected droplet flow stream 117 is at a constant distance from the deflector plates 116a as it deflects along the length of the deflector plates 116a, 116b. Such a shape is configured to position the deflector plates 116a, 116b as close together as possible across the longitudinal axis 198 (thereby maximizing the deflection force applied to the deflected droplet flow stream 117) while simultaneously maintaining a distance that ensures that the deflected droplet flow stream 117 does not collide with the deflector plates 116a, 116b.

[0050] In droplet deflector 115, deflector plates 116a, 116b remain substantially opposed to one another. That is, the shape of deflector plates 116a, 116b is optimized with respect to one dimension (the distance between the flow stream and the deflector plates in one plane) or with respect to plates that do not twist about their longitudinal axis 198. FIG. 1E illustrates another embodiment of a droplet deflector 120 configured for high-angle deflection of droplet flow streams in accordance with the present invention. Droplet deflector 120 includes deflector plates 121a, 121b that twist about their longitudinal axis 198. Deflector plate 121a has proximal ends 121a-p and distal ends 121a-d disposed downstream along the longitudinal axis 198 of the flow stream from proximal end 121a-p. Deflector plate 121b has proximal end 121b-p and distal end 121b-d disposed downstream from proximal end 121b-p along flow stream longitudinal axis 198. As deflector plates 121a, 121b extend along longitudinal axis 198 (i.e., move relatively distally along longitudinal axis 198), the distance between deflector plates 121a, 121b increases in two dimensions so that deflector plates 121a, 121b remain as close as possible in two dimensions, similar to droplet deflector 115, yet are still far enough apart so that deflected droplets of the deflected droplet flow stream (not shown in FIG. 1E) do not intersect or collide with deflector plates 121a, 121b.

[0051] FIG. 1F illustrates another embodiment of a droplet deflector 125 configured for high-angle deflection of a droplet flow stream in accordance with the present invention. Droplet deflector 125 includes deflector plates 126a, 126b configured to apply a constant deflection force to the deflected droplet flow stream from a plurality of different lateral positions. In particular, deflector plates 126a, 126b are segmented deflector plates. Deflector plate 126a includes proximal ends 126a-p and distal ends 126a-d positioned downstream along a longitudinal axis 198 of the flow stream from proximal ends 126a-p. Deflector plate 126b includes proximal ends 126b-p and distal ends 126b-d positioned downstream along a longitudinal axis 198 of the flow stream from proximal ends 126b-p. Each of the split deflector plates 126a and 126b has six corresponding segments 127a, 127b, 127c, 127d, 127e, and 127f. The segments 127a, 127b, 127c, 127d, 127e, and 127f are electrically isolated from one another so that different voltages can be applied to each segment. That is, segment 127a is separated from segment 127b by an air gap, segment 127b is separated from segment 127c by an air gap, segment 127c is separated from segment 127d by an air gap, segment 127d is separated from segment 127e by an air gap, and segment 127e is separated from segment 127f by an air gap.

[0052] Deflector plates 126a, 126b are angled, i.e., have regions where deflector plates 126a, 126b are parallel to one another and regions where deflector plates are angled away from one another. However, embodiments of split deflector plates are not limited to angled deflector plates, and split deflector plates may have any convenient shape, including, for example, the optimized shape of deflector plates 116a, 116b of droplet deflector 115, or twisted or spiral deflector plates 121a, 121b of droplet deflector 120, or any other desired shape. Segmented electrodes 126a, 126b are segmented such that different voltages may be applied to different segments of segmented electrodes 126a, 126b. The segmented electrodes may be configured such that the voltages applied to different segments vary at different lateral positions. Lateral position refers, for example, to the distance between the segments, e.g., the average distance between the segments. Typically, such distances are measured perpendicular to the longitudinal axis of the droplet flow stream; i.e., the distance from the centerline between the deflector plates may be measured. The voltages applied to different segments of the deflector plates may be selected to compensate for the increasing distance between the deflector plates (as particles move along the longitudinal axis of the flow stream). By increasing the voltages applied to the different segments, the electric field strength between the deflector plates can be substantially maintained, i.e., substantially constant, and therefore the deflection force applied to the deflected droplets can be substantially maintained along the longitudinal axis of the droplet flow stream. Voltages −HV1 and +HV1 are applied to segment 127a, voltages −HV2 and +HV2 are applied to segment 127b, voltages −HV3 and +HV3 are applied to segment 127c, voltages −HV4 and +HV4 are applied to segment 127d, voltages −HV5 and +HV5 are applied to segment 127e, and voltages −HV6 and +HV6 are applied to segment 127f. The voltages applied to the various segments increase along the longitudinal axis of the droplet flow stream, with HV6 being greater than HV5, HV5 being greater than HV4, HV4 being greater than HV3, HV3 being greater than HV2, and HV2 being greater than HV1.

[0053] 1G illustrates another embodiment of a droplet deflector 130 configured for high-angle deflection of a droplet flow stream according to the present invention. Droplet deflector 130 includes split deflector plates 131a and 131b. Droplet deflector 130 further includes an electrical circuit 133. Electrical circuit 133 is a voltage divider. Voltage divider 133 includes a voltage source, a potential source of voltage HV connected to a series resistive element, and a series resistor R. Voltage divider 133 is configured such that different nodes 133a, 133b, 133c, 133d, 133e, and 133f of voltage divider 133 are raised to different potentials, with the absolute value of the voltage applied to the segment of the droplet flow stream furthest from the source along the longitudinal axis of the droplet flow stream being greatest. Voltage divider 133 is configured so that different segments of deflector plates 131a, 131b are electrically connected to different nodes 133a, 133b, 133c, 133d, 133e, and 133f of voltage divider 133, with the voltage at node 133a being greater than the voltage at node 133b, which is greater than the voltage at node 133c, which is greater than the voltage at node 133d, which is greater than the voltage at node 133e, which is greater than the voltage at node 133f. The resistive elements of voltage divider 133 are each identical resistor R. However, in other embodiments, any convenient resistance may be applied at different stages of the voltage divider so that any desired voltage may be applied to different segments of the droplet deflector. In some cases, the resistance of the voltage divider may increase or decrease along the longitudinal axis of the droplet flow stream. In other cases, the resistance of the voltage divider may be varied in other ways.

[0054] The droplet deflector 130 includes multiple segments interconnected by high-resistivity dielectric spacers 134. Any convenient resistive dielectric material may be used, such as a solid dielectric material including, for example, plastic, porcelain, or glass. The high-resistivity dielectric spacers 134 are configured to electrically isolate each deflection plate segment of the droplet deflector 130. The high-resistivity dielectric spacers 134 are further configured to provide structural support for the deflector plates, i.e., structural support not available with a purely air-gap configuration. In other embodiments, a voltage divider may be integrated into the spacers between the deflector segments by selecting and configuring the dielectric material between the deflector segments to function as resistors in a voltage ladder. The electrical resistance provided by each spacer between the segments may be configured based on the material selected for use as the spacer and the shape, e.g., height, length, and width, of each spacer segment.

[0055] flow cytometer Aspects of the present disclosure further include flow cytometers. The flow cytometers of interest include droplet deflectors of the present disclosure. As described in detail herein, the droplet deflectors of interest include droplet deflectors configured for high-angle deflection of a droplet flow stream. Additionally, the flow cytometer includes a light source configured to illuminate particles in the flow stream at an interrogation point within the flow cell.

[0056] Flow cells of interest include cuvettes configured to transport particles in a flow stream. As used herein, the term "flow cell" is used in its conventional sense to refer to an element having a flow path for a liquid flow stream to transport particles in a sheath fluid. Cuvettes of interest include a passageway (i.e., a flow path) extending therethrough. The flow stream may include a liquid sample injected from a sample tube. In some cases, flow cells include optically transparent flow paths. The cuvette may be constructed of, for example, quartz, glass, or clear plastic. In some embodiments, the cuvette is formed from silica, such as fused silica. In some cases, flow cells are configured to be illuminated with light from a light source at one or more interrogation points. The term "interrogation point" used herein refers to an area within the flow cell where particles are illuminated by light from the light source, e.g., for analysis. The size of the interrogation point may vary as desired. For example, if 0 μm represents the axis of light emitted by the light source, the interrogation point may be within a range of -50 μm to 50 μm, e.g., -25 μm to 40 μm, or e.g., -15 μm to 30 μm. Depending on certain considerations (eg, number and placement of lasers), there may be multiple illumination points within the flow cell.

[0057] In some embodiments, the flow cell has or is configured for use with a sample injection port configured to deliver a sample to the flow cell, hi embodiments, the sample injection system is configured to deliver a suitable flow of sample to the internal chamber (i.e., flow path) of the flow cell. Depending on the desired characteristics of the flow stream, the flow rate of the sample delivered by the sample injection port to the chamber of the flow cell may be 1 μL / min or more, such as 2 μL / min or more, for example 3 μL / min or more, such as 5 μL / min or more, for example 10 μL / min or more, such as 15 μL / min or more, for example 25 μL / min or more, for example 50 μL / min or more, for example 100 μL / min or more, and in some cases the flow rate of the sample delivered by the sample injection port to the chamber of the flow cell is 1 μL / sec or more, such as 2 μL / sec or more, for example 3 μL / sec or more, for example 5 μL / sec or more, for example 10 μL / sec or more, for example 15 μL / sec or more, for example 25 μL / sec or more, for example 50 μL / sec or more, for example 100 μL / sec or more.

[0058] The sample injection port may be an orifice in the wall of the internal chamber or a tube located at the proximal end of the internal chamber. When the sample injection port is an orifice in the wall of the internal chamber, the orifice may have any suitable shape. Cross-sectional shapes of interest include, but are not limited to, rectilinear shapes, such as square, rectangular, trapezoidal, triangular, and hexagonal; curved shapes, such as circular and oval; and irregular shapes, such as a parabolic bottom joined to a flat top. In some embodiments, the sample injection port has a circular orifice. The size of the orifice of the sample injection port may vary depending on the shape, and in some cases may have an opening in the range of 0.1 mm to 5.0 mm, e.g., 0.2 to 3.0 mm, e.g., 0.5 mm to 2.5 mm, e.g., 0.75 mm to 2.25 mm, e.g., 1 mm to 2 mm, e.g., 1.25 mm to 1.75 mm, e.g., a 1.5 mm opening.

[0059] In some cases, the sample injection port is a tube located at the proximal end of the internal chamber of the flow cell. For example, the sample injection port may be a tube aligned with the orifice of the flow cell. When the sample injection port is a tube aligned with the orifice of the flow cell, the cross-sectional shape of the sample injection tube may have any suitable shape. Cross-sectional shapes of interest include, but are not limited to, rectilinear cross-sectional shapes, such as square, rectangular, trapezoidal, triangular, and hexagonal; curved cross-sectional shapes, such as circular and oval; and irregular shapes, such as a parabolic bottom joined to a flat top. The orifice of the tube may vary depending on the shape and may in some cases have an opening in the range of 0.1 mm to 5.0 mm, e.g., 0.2 to 3.0 mm, e.g., 0.5 mm to 2.5 mm, e.g., 0.75 mm to 2.25 mm, e.g., 1 mm to 2 mm, e.g., 1.25 mm to 1.75 mm, e.g., 1.5 mm. The shape of the tip of the sample injection port may 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 may have a beveled tip with a bevel angle within a range of 1° to 10°, for example, 2° to 9°, for example, 3° to 8°, for example, 4° to 7°, for example, a bevel angle of 5°.

[0060] In some embodiments, the flow cell further includes a sheath fluid injection port configured to supply sheath fluid to the flow cell. In embodiments, the sheath fluid injection system is configured to supply a flow of sheath fluid, e.g., along with the sample, to the internal chamber of the flow cell to generate a laminated flow stream of sheath fluid surrounding the sample flow stream. Depending on the desired characteristics of the flow stream, the flow rate of the sheath fluid delivered to the chamber of the flow cell may be 25 μL / sec or more, e.g., 50 μL / sec or more, e.g., 75 μL / sec or more, e.g., 100 μL / sec or more, e.g., 250 μL / sec or more, e.g., 500 μL / sec or more, e.g., 750 μL / sec or more, e.g., 1000 μL / sec or more, e.g., 2500 μL / sec or more.

[0061] In some embodiments, the sheath fluid injection port is an orifice in the wall of the internal chamber. The sheath fluid injection port orifice may have any suitable shape, and cross-sectional shapes of interest include, but are not limited to, rectilinear cross-sectional shapes, such as square, rectangular, trapezoidal, triangular, and hexagonal, curved cross-sectional shapes, such as circular and oval, and irregular shapes, such as a parabolic bottom joined to a flat top. The size of the sheath fluid injection port orifice may vary depending on the shape, and in some cases may have an opening in the range of 0.1 mm to 5.0 mm, e.g., 0.2 mm to 3.0 mm, e.g., 0.5 mm to 2.5 mm, e.g., 0.75 mm to 2.25 mm, e.g., 1 mm to 2 mm, e.g., 1.25 mm to 1.75 mm, e.g., a 1.5 mm opening.

[0062] As described above, a flow cytometer includes a light source configured to illuminate particles in the flow stream at an interrogation point within the flow cell. The number of light sources in a flow cytometer may vary. In some embodiments, the flow cytometer includes a single light source. Alternatively, the flow cytometer may include multiple light sources in some cases. In some such cases, the number of light sources is in the range of 2 to 10, e.g., 2 to 5, e.g., 2 to 4. Any convenient light source may be used as the light source described herein. In some embodiments, the light source is a laser. In embodiments, the laser may be any convenient laser, such as a continuous wave laser. For example, the laser may be a diode laser, e.g., an ultraviolet diode laser, a visible diode laser, and a near-infrared diode laser. In other embodiments, the laser may 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 CO laser, a CO laser, an argon fluorine (ArF) excimer laser, a krypton fluorine (KrF) excimer laser, a xenon chlorine (XeCl) excimer laser, or a xenon fluorine (XeF) excimer laser, or a combination thereof. In other cases, the subject flow cytometer is equipped with a dye laser, such as a stilbene laser, a coumarin laser, or a rhodamine laser. In still other cases, 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 still other cases, the subject flow cytometers are equipped with solid-state lasers, such as ruby ​​lasers, Nd:YAG lasers, NdCrYAG lasers, Er:YAG lasers, Nd:YLF lasers, Nd:YVO4 lasers, Nd:YCa4O(BO3)3 lasers, Nd:YCOB lasers, titanium sapphire lasers, thulium YAG lasers, ytterbium YAG lasers, Yb2O3 lasers, or cerium-doped lasers, and combinations thereof.

[0063] In some embodiments, the laser light source may further include one or more optical adjustment components. In some embodiments, the optical adjustment components are disposed between the light source and the flow cell and may include any device capable of changing the spatial width or other characteristics of the illumination from the light source, such as the illumination 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, collimation protocols, and combinations thereof. In some embodiments, the flow cytometer of interest includes one or more focusing lenses. In one example, the focusing lens may be a reduction lens. In yet other embodiments, the flow cytometer of interest includes optical fibers.

[0064] The light source may be positioned at any suitable distance from the flow cell, for example the light source and the flow cell are separated by 0.005 mm or more, such as 0.01 mm or more, for example 0.05 mm or more, for example 0.1 mm or more, such as 0.5 mm or more, for example 1 mm or more, for example 5 mm or more, for example 10 mm or more, for example 25 mm or more, such as 100 mm or more. Additionally, the light source may be positioned at any suitable angle relative to the flow cell, for example between 10 degrees and 90 degrees, for example between 15 degrees and 85 degrees, for example between 20 degrees and 80 degrees, for example between 25 degrees and 75 degrees, for example between 30 degrees and 60 degrees, for example at an angle of 90 degrees.

[0065] In some embodiments, the light source of interest includes multiple lasers, e.g., two or more lasers, e.g., three or more lasers, e.g., four or more lasers, e.g., five or more lasers, e.g., ten or more lasers, e.g., fifteen or more lasers, configured to provide laser light for separate illumination of the flow stream. Depending on the desired wavelength of light for illuminating the flow stream, each laser may have a different specific wavelength within a range of 200 nm to 1500 nm, e.g., 250 nm to 1250 nm, e.g., 300 nm to 1000 nm, e.g., 350 nm to 900 nm, e.g., 400 nm to 800 nm. In certain embodiments, the lasers of interest may include one or more of a 405 nm laser, a 488 nm laser, a 561 nm laser, and a 635 nm laser.

[0066] In some embodiments, the light source is an optical beam generator configured to generate two or more beams of frequency-shifted light. In some cases, the optical beam generator comprises a laser, a radio frequency generator configured to apply a radio frequency drive signal to an acousto-optic device to generate two or more angularly deflected laser beams. In these embodiments, the laser may be a pulsed laser or a continuous wave laser. For example, optical beam generator lasers of interest include those listed above.

[0067] The acousto-optic device may be any convenient acousto-optic protocol configured to frequency-shift laser light using applied acoustic waves. In certain embodiments, the acousto-optic device is an acousto-optic deflector. The acousto-optic device of the subject system is configured to generate an angularly deflected laser beam from light from a laser and an applied high-frequency drive signal. The high-frequency drive signal may be applied to the acousto-optic device using any suitable high-frequency drive signal source, such as a direct digital synthesizer (DDS), an arbitrary waveform generator (AWG), or an electrical pulse generator.

[0068] In an embodiment, the controller is configured to apply high frequency drive signals to the acousto-optic device to generate a desired number of angularly deflected laser beams of the output laser beam, for example configured to apply 3 or more high frequency drive signals, for example 4 or more high frequency drive signals, for example 5 or more high frequency drive signals, for example 6 or more high frequency drive signals, for example 7 or more high frequency drive signals, for example 8 or more high frequency drive signals, for example 9 or more high frequency drive signals, for example 10 or more high frequency drive signals, for example 15 or more high frequency drive signals, for example 25 or more high frequency drive signals, for example 50 or more high frequency drive signals, for example configured to apply 100 or more high frequency drive signals.

[0069] In some cases, to generate an intensity profile of the angularly deflected laser beam of the output laser beam, the controller is configured to apply a high frequency drive signal having a varying amplitude within a range, for example, from about 0.001 V to about 500 V, for example, from about 0.005 V to about 400 V, for example, from about 0.01 V to about 300 V, for example, from about 0.05 V to about 200 V, for example, from about 0.1 V to about 100 V, for example, from about 0.5 V to about 75 V, for example, from about 1 V to about 50 V, for example, from about 2 V to about 40 V, for example, from about 3 V to about 30 V, or for example, from about 5 V to about 25 V. The applied high frequency drive signal in some embodiments has a frequency within the range of about 0.001 MHz to about 500 MHz, for example, about 0.005 MHz to about 400 MHz, for example, about 0.01 MHz to about 300 MHz, for example, about 0.05 MHz to about 200 MHz, for example, about 0.1 MHz to about 100 MHz, for example, about 0.5 MHz to about 90 MHz, for example, about 1 MHz to about 75 MHz, for example, about 2 MHz to about 70 MHz, for example, about 3 MHz to about 65 MHz, for example, about 4 MHz to about 60 MHz, for example, about 5 MHz to about 50 MHz.

[0070] In some embodiments, the controller includes a processor to which a memory is operatively coupled, the memory storing instructions that, when executed by the processor, cause the processor to generate an output laser beam including an angularly deflected laser beam having a desired intensity profile. For example, the memory may include instructions for generating two or more, e.g., three or more, e.g., four or more, e.g., five or more, e.g., ten or more, e.g., twenty-five or more, e.g., fifty or more angularly deflected laser beams of the same intensity, e.g., the memory may include instructions for generating one hundred or more angularly deflected laser beams of the same intensity. In other embodiments, the memory may include instructions for generating two or more, e.g., three or more, e.g., four or more, e.g., five or more, e.g., ten or more, e.g., twenty-five or more, e.g., fifty or more angularly deflected laser beams of different intensities, e.g., the memory may include instructions for generating one hundred or more angularly deflected laser beams of different intensities.

[0071] In some embodiments, the controller has a processor to which the memory is operatively coupled, such that the memory stores instructions that, when executed by the processor, cause the processor to generate an output laser beam that increases in intensity from the center to the edges of the output laser beam along a horizontal axis. In these cases, the intensity of the angularly deflected laser beam at the center of the output beam may be in a range of 0.1% to about 99%, for example, 0.5% to about 95%, for example, 1% to about 90%, for example, about 2% to about 85%, for example, about 3% to about 80%, for example, about 4% to about 75%, for example, about 5% to about 70%, for example, about 6% to about 65%, for example, about 7% to about 60%, for example, about 8% to about 55%, or may be in a range of about 10% to about 50% of the intensity of the angularly deflected laser beam at the edges of the output laser beam along the horizontal axis. In other embodiments, the controller has a processor to which the memory is operatively coupled, such that the memory stores instructions that, when executed by the processor, cause the processor to generate an output laser beam that increases in intensity from the edge to the center of the output laser beam along a horizontal axis. In these cases, the intensity of the angularly deflected laser beam at the edge of the output beam may be in a range of 0.1% to about 99%, for example, 0.5% to about 95%, for example, 1% to about 90%, for example, about 2% to about 85%, for example, about 3% to about 80%, for example, about 4% to about 75%, for example, about 5% to about 70%, for example, about 6% to about 65%, for example, about 7% to about 60%, for example, about 8% to about 55%, or may be in a range of about 10% to about 50% of the intensity of the angularly deflected laser beam at the center of the output laser beam along the horizontal axis. In yet another embodiment, the controller has a processor to which a memory is operatively coupled, such that the memory stores instructions that, when executed by the processor, cause the processor to generate an output laser beam having a Gaussian intensity profile along a horizontal axis.In yet another embodiment, the controller has a processor to which the memory is operatively coupled, such that the memory stores instructions that, when executed by the processor, cause the processor to generate an output laser beam having a top-hat intensity profile along a horizontal axis.

[0072] In embodiments, the optical beam generator of interest may be configured to generate spatially separated angularly deflected laser beams of the output laser beam. Depending on the applied high frequency drive signal and the desired irradiance profile of the output laser beam, the angularly deflected laser beams may be separated by 0.001 μm or more, such as 0.005 μm or more, such as 0.01 μm or more, such as 0.05 μm or more, such as 0.1 μm or more, such as 0.5 μm or more, such as 1 μm or more, such as 5 μm or more, such as 10 μm or more, such as 100 μm or more, such as 500 μm or more, such as 1000 μm or more, such as 5000 μm or more. In some embodiments, the system is configured to generate angularly deflected laser beams of the output laser beam that overlap, for example, adjacent angularly deflected laser beams along a horizontal axis of the output laser beam. The overlap of adjacent angularly deflected laser beams (e.g., overlap of beam spots) may be 0.001 μm or more, for example, 0.005 μm or more, for example, 0.01 μm or more, for example, 0.05 μm or more, for example, 0.1 μm or more, for example, 0.5 μm or more, for example, 1 μm or more, for example, 5 μm or more, for example, 10 μm or more, for example, 100 μm or more.

[0073] In some cases, the light beam generator configured to generate two or more beams of frequency-shifted light may be any of the light beam generators described in U.S. Pat. Nos. 9,423,353, 9,784,661, 9,983,132, 10,006,852, 10,036,699, 10,078,045, 10,222,316, 10,288,546, 10,324,019, 10,408,758, 10,451,538, 10,620,111, 10,684,211, 10,845,295, 10,935,482, U.S. Pat. Nos. 5,623,353, 5,623,353, 5,784,661, 5,983,132, 5,006,852, 5,036,699, 5,078,045, 5,222,316, 5,288,546, 5,324,019, 5,408,758, 5,451,538, 5,620,111, 5,684,211, 5,845,295, 5,935,482 ... The laser pumping module may be one such as those described in U.S. Patent No. 10935485, U.S. Patent No. 11105728, U.S. Patent No. 11280718, U.S. Patent No. 11327016, U.S. Patent No. 11366052, U.S. Patent No. 11371937, U.S. Patent No. 11692926, U.S. Patent No. 11630053, U.S. Patent No. 11774343, U.S. Patent No. 11940369, and U.S. Patent No. 11946851.

[0074] Additionally, the flow cytometer includes a photodetector configured to collect light emitted from the illuminated particles. The photodetector is configured to detect the particle-modulated light transmitted by the fiber optic collection element and generate a signal based on a characteristic (e.g., intensity) of the light. For example, the one or more particle-modulated light detectors may include one or more side scatter detectors for detecting light of a side-scattered wavelength (i.e., light refracted and reflected by the surface and internal structure of the particle). In some embodiments, the flow cytometer includes one side scatter detector. In other embodiments, the flow cytometer includes multiple side scatter detectors, e.g., two or more, e.g., three or more, e.g., four or more, e.g., five or more side scatter detectors.

[0075] Any convenient detector for detecting collected light may be used in the side scatter light detectors described herein. Detectors of interest may include, but are not limited to, optical sensors or photodetectors, such as active pixel sensors (APS), avalanche photodiodes, image sensors, charge-coupled devices (CCDs), intensified charge-coupled devices (ICCDs), light-emitting diodes, photon counters, bolometers, pyroelectric detectors, photoresistors, photocells, photodiodes, photomultiplier tubes (PMTs), phototransistors, quantum dot photoconductors or photodiodes, and combinations thereof, among other detectors. In some embodiments, 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 some embodiments, the detector is a photomultiplier tube, e.g., a 0.01 cm 2 ~10cm 2 , e.g. 0.05cm 2 ~9cm 2 , e.g., 0.1 cm 2 ~8cm 2 , e.g., 0.5 cm 2 ~7cm 2 , e.g. 1 cm 2 ~5cm 2 The photomultiplier tube has an active detection surface area of ​​each region within the range of .times. ...

[0076] In embodiments, the subject flow cytometer further comprises a fluorescence detector configured to detect light at one or more fluorescent wavelengths, hi other embodiments, the flow cytometer comprises a plurality of fluorescence detectors, e.g., 2 or more, e.g., 3 or more, e.g., 4 or more, e.g., 5 or more, e.g., 10 or more, e.g., 15 or more, e.g., 20 or more fluorescence detectors.

[0077] Any convenient detector for detecting collected light may be used in the fluorescence detectors described herein. Detectors of interest may include, but are not limited to, optical sensors or photodetectors, such as active pixel sensors (APS), avalanche photodiodes, image sensors, charge-coupled devices (CCDs), intensified charge-coupled devices (ICCDs), light-emitting diodes, photon counters, bolometers, pyroelectric detectors, photoresistors, photocells, photodiodes, photomultiplier tubes (PMTs), phototransistors, quantum dot photoconductors or quantum dot photodiodes, and combinations thereof, among other detectors. In some embodiments, 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 some embodiments, the detector is a photomultiplier tube, e.g., a 0.01 cm 2 ~10 cm 2 , e.g., 0.05 cm 2 ~9cm 2 , e.g., 0.1 cm 2 ~8cm 2 , e.g., 0.5 cm 2 ~7cm 2 , e.g. 1 cm 2 ~5cm 2 The photomultiplier tube has an active detection surface area of ​​each region within the range of .times. ...

[0078] When a subject flow cytometer includes multiple fluorescence detectors, each fluorescence detector may be identical, or the collection of fluorescence detectors may be a combination of different types of detectors. For example, when a subject flow cytometer includes two fluorescence detectors, in some embodiments, the first fluorescence detector is a CCD-based device and the second fluorescence detector (or image sensor) is a CMOS-based device. In other embodiments, both the first and second fluorescence detectors are CCD-based devices. In still other embodiments, both the first and second fluorescence detectors are CMOS-based devices. In still other embodiments, the first fluorescence detector is a CCD-based device and the second fluorescence detector is a photomultiplier tube (PMT). In still other embodiments, the first fluorescence detector is a CMOS-based device and the second fluorescence detector is a photomultiplier tube. In still other embodiments, both the first and second fluorescence detectors are photomultiplier tubes.

[0079] In embodiments of the present disclosure, a fluorescence detector of interest is configured to measure collected light at one or more wavelengths, e.g., two or more wavelengths, e.g., five or more different wavelengths, e.g., ten or more different wavelengths, e.g., twenty-five or more different wavelengths, e.g., fifty or more different wavelengths, e.g., one hundred or more different wavelengths, e.g., two or more different wavelengths, e.g., three hundred or more different wavelengths, e.g., four hundred or more different wavelengths, e.g., light emitted from a sample in a flow stream. In some embodiments, two or more detectors of a module as described herein are configured to measure collected light of the same wavelength or overlapping wavelengths.

[0080] 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 a spectrum of light over a range of wavelengths. For example, a flow cytometer may include one or more detectors configured to collect a spectrum of light over one or more wavelength ranges from 200 nm to 1000 nm. In yet other embodiments, the detector of interest is configured to measure light emitted from a sample in the flow stream at one or more specific wavelengths. For example, a module may have one or more detectors configured to measure light at one or more of: 450 nm, 518 nm, 519 nm, 561 nm, 578 nm, 605 nm, 607 nm, 625 nm, 650 nm, 660 nm, 667 nm, 670 nm, 668 nm, 695 nm, 710 nm, 723 nm, 780 nm, 785 nm, 647 nm, 617 nm, and any combination thereof. In certain embodiments, the one or more detectors may be configured to pair with a particular fluorophore, such as a fluorophore used with a sample in a fluorescence assay.

[0081] A flow cytometer may have any suitable mechanism or mechanisms for supplying sheath fluid and sample fluid to the sheath fluid input coupler and sample fluid input coupler. For example, the sample fluid input coupler may be fluidly connected to a sample fluid line (e.g., tubing) that is fluidly connected to a sample fluid reservoir. Similarly, the sheath fluid input coupler may be fluidly connected to a sheath fluid line that is fluidly connected to a sheath fluid reservoir. Similarly, a flow cytometer may have any suitable mechanism or mechanisms for managing waste from the flow stream. The fluid output coupler may be fluidly connected to a waste line that is fluidly connected to a waste reservoir. A fluid management system that may be adapted for use in the subject flow cytometer is described in U.S. Patent Application Publication No. 2022 / 0341838, the entire disclosure of which is incorporated herein by reference.

[0082] Suitable flow cytometry systems include those described in Ormerod (ed.), Flow Cytometry: A Practical Approach, Oxford University Press (1997); Jaroszeski et al. (eds.), Flow Cytometry Protocols, Methods in Molecular Biology 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 Thromb 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., the disclosures of which are incorporated herein by reference. 24(3):203-255.In some cases, flow cytometry systems of interest include the BD Biosciences FACSCanto™ flow cytometer, BD Biosciences FACSCanto™ II flow cytometer, BD Accuri™ flow cytometer, BD Accuri™ C6 Plus flow cytometer, BD Biosciences FACSCelesta™ flow cytometer, BD Biosciences FACSLyric™ flow cytometer, BD Biosciences FACSVerse™ flow cytometer, BD Biosciences FACSymphony™ flow cytometer, BD Biosciences LSRFortessa™ flow cytometer, BD Biosciences LSRFortessa™ X-20 flow cytometer, BD Biosciences FACSPresto™ flow cytometer, BD Biosciences FACSVia™ flow cytometer, and the BD Biosciences FACSCalibur™ cell sorter, BD Biosciences FACSCount™ cell sorter, BD Biosciences FACSLyric™ cell sorter, ...elesta™ flow cytometer, BD Biosciences FACSLyric™ flow cytometer, BD Biosciences FACSVerse™ flow cytometer, BD Biosciences FACSymphony™ flow cytometer, BD Biosciences LSRFortessa™ flow cytometer, BD Biosciences LSRFortessa™ X-20 flow cytometer, BD Biosciences FACSPresto™ flow cytometer, BD Biosciences FACSVia™ flow cytometer, and BD Biosciences FACSCalibur™ cell sorter. These include the BD Biosciences Via™ cell sorter, BD Biosciences Influx™ cell sorter, BD Biosciences Jazz™ cell sorter, BD Biosciences Aria™ cell sorter, BD Biosciences FACSAria™ II cell sorter, BD Biosciences FACSAria™ III cell sorter, BD Biosciences FACSAria™ Fusion cell sorter, BD Biosciences FACSMelody™ cell sorter, BD Biosciences FACSymphony™ S6 cell sorter, and BD Biosciences FACSDiscover™ cell sorter.

[0083] In some embodiments, the subject system may be configured to perform a variety of tasks, including but not limited to, the following: U.S. Pat. Nos. 10,663,476, 10,620,111, 10,613,017, 10,605,713, 10,585,031, 10,578,542, 10,578,469, 10,481,074, 10,302,545, 10,145,793, 10,113,967, 10,006,852, 9,952,076, 9,933,341, 9,726,527, 9,453,789, 9,200,334, 9,097,640, 9,095,494, the disclosures of which are incorporated herein by reference in their entireties. Nos. 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, and 4,498,766.

[0084] In some embodiments, the flow cytometer is configured as an imaging flow cytometer. For example, in some cases, the subject system may be configured as described in Diebold, et al. Nature Photonics Vol. 7(10); 806-810 (2013) and U.S. Pat. Nos. 9,423,353 and 9,784,661. Nos., U.S. Patent Nos. 9,983,132, 10,006,852, 10,036,699, 10,078,045, 10,222,316, 10,288,546, 10,324,019, 10,408,758, 10,451,538, 10,620,111, 10,684,211, 10,845,295, 10,935,482, 10,935,485, 11,105,728, and U.S. Patent No. 1,128,071,891, U.S. Patent No. 1,132,016, U.S. Patent No. 1,366,052, U.S. Patent No. 1,137,1937, U.S. Patent No. 1,169,2926, U.S. Patent No. 1,163,0053, U.S. Patent No. 1,177,4343, U.S. Patent No. 1,194,0369, and U.S. Patent No. 1,194,6851 (the disclosures of which are incorporated herein by reference). In some embodiments where the flow cytometer is a particle sorter, the particle sorter is an image-enabled particle sorter. Image-enabled particle sorters are described in U.S. Pat. Nos. 10,324,019, 10,620,111, 11,105,728, and 11,774,343, as well as U.S. patent application Ser. Nos. 18 / 537,103, 18 / 657,618, 18 / 657,623, and 18 / 657,633, the entire disclosures of which are incorporated herein by reference.

[0085] FIG. 2 illustrates a system 200 for flow cytometry according to an exemplary embodiment of the present disclosure. The system 200 includes a laser 201 configured to illuminate particles 211 in a flow stream 214 at an interrogation point 215 within a flow cell 210. While one laser is shown in the example of FIG. 2, it is understood that multiple lasers may also be used. The laser beam from the laser 201 is directed to a focusing lens 202, which focuses the laser beam onto a portion of the fluid stream within the flow cell 210 where the sample particles 211 reside. The flow cell 210 is part of a fluid system that directs particles in the stream, typically one at a time, into the focused laser beam for interrogation. Alternatively, if the flow cytometer is a stream-in-air cytometer, a nozzle top may be used.

[0086] As shown in FIG. 2 , flow cell 210 is fluidly connected to sheath fluid reservoir 203 containing sheath fluid and sample fluid reservoir 204 containing sample fluid. Sheath fluid from sheath fluid reservoir 203 is supplied to at least one sheath fluid injection port 208 via tubing (i.e., sheath fluid line) 207. Additionally, sample fluid containing particles 211 from sample fluid reservoir 204 is supplied to sample injection port 206 via tubing (i.e., sample fluid line) 205. Sample injection port 206 is fluidly connected to sample injector 213 (e.g., a sample injection needle) configured to introduce particles 211 into the interior of flow cell 210. Particles 211 are hydrodynamically focused via sheath fluid flowing from sheath fluid injection port 208 such that flow stream 214 is formed downstream of tapered section 212 of flow cell 210. Particles emitted at the distal end of flow cell 210 may be discarded and / or collected via any suitable protocol. For example, depending on the type of flow cytometry being performed, particles may be collected at the distal end of flow cell 210, for example, via a waste line. Alternatively, particles may be sorted.

[0087] Light from one or more laser beams interacts with particles 211 in the sample through diffraction, refraction, reflection, scattering, and absorption, and is re-emitted at a variety of different wavelengths depending on the particle's characteristics, such as its size, internal structure, and the presence of one or more fluorescent molecules attached to or naturally present on or within the particle. The fluorescent radiation, as well as the diffracted, refracted, reflected, and scattered light, may be sent to one or more detectors. In particular, forward-scattered light (FSC) is sent to a forward-scattered light detector 223. The forward-scattered light detector 223 is positioned slightly off-axis from the direct beam passing through the flow cell 210 and is configured to detect diffracted light, i.e., excitation light traveling primarily in a forward direction through or around the particle. The intensity of the light detected by the forward-scattered light detector 223 depends on the overall size of the particle. The forward-scattered light detector may include, for example, a photodiode. A scattering bar 222 is positioned between the forward-scattered light detector 223 and the optical filter 221a. The optical filter 221a may be configured to remove non-FSC light of at least one wavelength, while the scattering bar 222 may be configured to prevent the incident beam from the laser 201 (i.e., non-scattered light) from being detected by the forward scattered light detector 223.

[0088] Additionally, side-scattered light (SSC) is detected by side-scattered light detector 224. In other words, side-scattered light detector 224 is configured to detect refracted and reflected light from the surface and internal structures of particle 211, which tends to increase as the structural complexity of the particle increases. In the example of FIG. 2, flow cytometer 200 includes dichroic mirror 220a configured to reflect SSC light to side-scattered light detector 224 while passing non-SSC light (e.g., fluorescent light). Optical filter 221b is configured to prevent non-SSC light of at least one wavelength from being detected by side-scattered light detector 224. Fluorescence detectors 225a-225c are also shown, each configured to detect fluorescent light of a different wavelength. For example, dichroic mirror 220b may be configured to reflect fluorescent light (FL) corresponding to a first wavelength (or wavelength range) to fluorescence detector 225a while passing light of other wavelengths. Optical filter 221c may be configured to prevent light of at least one wavelength that does not correspond to the first wavelength (or wavelength range) from being detected by fluorescence detector 225a. Similarly, dichroic mirror 220c is configured to reflect FL light corresponding to the second wavelength (or wavelength range) to fluorescence detector 225b, while passing light of a third wavelength (or wavelength range) for detection by fluorescence detector 225c. Optical filter 221d is configured to prevent light of at least one wavelength that does not correspond to the second wavelength (or wavelength range) from being detected by fluorescence detector 225b. Additionally, optical filter 221e is configured to prevent light of at least one wavelength that does not correspond to the third wavelength (or wavelength range) from being detected by fluorescence detector 225c.

[0089] Those skilled in the art will recognize that flow cytometers according to embodiments of the present disclosure are not limited to the flow cytometer shown in FIG. 2 , but may include any flow cytometer known in the art. For example, a flow cytometer may have any number of lasers, beam splitters, filters, and detectors of various wavelengths and in a variety of different configurations. For example, while three fluorescence detectors are shown in the embodiment of FIG. 2 for illustrative purposes, it will be understood that any suitable number of fluorescence detectors may be used.

[0090] During operation, the operation of the flow cytometer is controlled by the controller / processor 290, and measurement data from the detectors may be stored in the memory 295 and processed by the controller / processor 290. Although not explicitly shown, the controller / processor 290 is coupled to the detectors to receive output signals from the detectors, and may further be coupled to the electrical and electromechanical components of the flow cytometer to control the laser 201, fluid flow parameters, etc. An input / output (I / O) function 297 may also be provided in the system. The memory 295, the controller / processor 290, and the I / O function 297 may be provided entirely as an integral part of the flow cytometer. In such an embodiment, a display may also form part of the I / O function 297 to present experimental data to a user of the flow cytometer 200. Alternatively, some or all of the memory 295 and the controller / processor 290 and the I / O function 297 may be part of one or more external devices, such as a general-purpose computer. In some embodiments, memory 295 and some or all of controller / processor 290 may be in wireless or wired communication with the flow cytometer. Controller / processor 290 in conjunction with memory 295 and I / O functionality 297 may be configured to perform a variety of functions related to the preparation and analysis of flow cytometer experiments.

[0091] The various fluorescent molecules of a panel of fluorescent dyes used in a flow cytometer experiment each emit light in a unique wavelength band. The particular fluorescent labels used in the experiment and their associated fluorescence emission bands may be selected to generally match the filter windows of the detector. I / O function 297 may be configured to receive data regarding a flow cytometer experiment having a panel of fluorescent labels and multiple cell populations with multiple markers, each cell population having a subset of multiple markers. I / O function 297 may be further configured to receive biological data assigning one or more markers to one or more cell populations, marker concentration data, emission spectrum data, data assigning labels to one or more markers, and cytometer configuration data. Flow cytometer experiment data, such as label spectral characteristics and flow cytometer configuration data, may further be stored in memory 295. Controller / processor 290 may be configured to evaluate one or more assignments of labels to markers.

[0092] In some embodiments, the subject system is a particle sorting system configured to sort particles using an enclosed particle sorting module, such as that described in U.S. Patent Application Publication No. 2017 / 0299493, filed March 28, 2017, the disclosure of which is incorporated herein by reference. In certain embodiments, particles (e.g., cells) of a sample are sorted using a sorting determination module having a plurality of sorting determination units, such as that described in U.S. Patent Application Publication No. 2020 / 0256781, filed December 23, 2019, the disclosure of which is incorporated herein by reference. In some embodiments, a system for sorting components of a sample includes a particle sorting module with deflection plates, such as that described in U.S. Patent Application Publication No. 2017 / 0299493, filed March 28, 2017, the disclosure of which is incorporated herein by reference.

[0093] In one embodiment, the system is a fluorescence imaging system using a radio frequency tag emission imaging particle sorter, as shown in FIG. 3. Particle sorter 300 includes an optical illumination unit 300a including a light source 301 (e.g., a 488 nm laser) that generates an output beam of light 301a, which is split into beams 302a and 302b by beam splitter 302. Light beam 302a propagates through an acousto-optic device (e.g., an acousto-optic deflector (AOD)) 303 to generate output beam 303a having one or more angularly deflected beams of light. Optionally, output beam 303a from acousto-optic device 303 includes a local oscillator beam and multiple radio frequency comb beams. Light beam 302b propagates through an acousto-optic device (e.g., an acousto-optic deflector (AOD)) 304 to generate output beam 304a having one or more angularly deflected beams of light. In some cases, output beam 304a from acousto-optic device 304 includes a local oscillator beam and multiple high-frequency comb beams. Output beams 303a and 304a from acousto-optic device 303 and acousto-optic device 304, respectively, are combined in beam combiner 305 to generate output beam 305a, which is transmitted through optics 306 (e.g., an objective lens) to illuminate particles in flow cell 307. In some embodiments, acousto-optic device 303 (AOD) splits a single laser beam into an array of beamlets, each with a different optical frequency and angle. A second AOD 304 adjusts the optical frequency of a reference beam, which is then superimposed with the array of beamlets in beam combiner 305. In some embodiments, the light illumination system having a light source and an acousto-optical device may further include those described in Schraivogel, et al. (“High-speed fluorescence image-enabled cell sorting” Science (2022), 375 (6578): 315-320) and U.S. Patent Application Publication No. 2021 / 0404943, the disclosures of which are incorporated herein by reference.

[0094] Output beam 305a illuminates sample particles 308 propagating through flow cell 307 (e.g., with sheath fluid 309) at illumination region 310. As shown in illumination region 310, multiple beams (e.g., angularly polarized, high-frequency shifted beams of light shown as dots across illumination region 310) overlap with a reference local oscillator beam (shown as a cross-hatched line across illumination region 310). The overlapping beams exhibit beat behavior due to their different optical frequencies, with each beamlet emitting at a different frequency f 1-n transmits sinusoidal modulation.

[0095] Light from the illuminated sample is transmitted to a light detection system 300b having multiple light detectors. The light detection system 300b includes a forward scattered light detector 311 for generating a forward scattered light image 311a and a side scattered light detector 312 for generating a side scattered light image 312a. The light detection system 300b further includes a bright-field light detector 313 for generating a light loss image 313a. In some embodiments, the forward scattered light detector 311 and the side scattered light detector 312 are photodiodes (e.g., avalanche photodiodes (APDs)). In some cases, the bright-field light detector 313 is a photomultiplier tube (PMT). Fluorescence from the illuminated sample is further detected by fluorescence detectors 314-317. In some cases, the light detectors 314-317 are photomultiplier tubes. Light from the illuminated sample is directed via beam splitter 320 to side-scattered light detection channel 312 and fluorescence detection channels 314-317. Light detection system 300b includes bandpass optics 321-324 (e.g., dichroic mirrors) for transmitting light of predetermined wavelengths to photodetectors 314-317, respectively. In some cases, optic 321 is a 534 nm / 40 nm bandpass. In some cases, optic 322 is a 586 nm / 42 nm bandpass. In some cases, optic 323 is a 700 nm / 54 nm bandpass. In some cases, optic 324 is a 783 nm / 56 nm bandpass. The first number represents the center of the spectral band. The second number represents the range of the spectral band. Thus, a 510 / 20 filter extends 10 nm on either side of the center of the spectral band, from 500 nm to 520 nm.

[0096] Data signals generated in response to light detected in the forward scattered light detection channel 311, the side scattered light detection channel 312, the bright-field light detection channel 313, and the fluorescence detection channels 314-317 are processed by real-time digital processing by processors 350 and 351. Images 311a-317a can be generated in each light detection channel based on the data signals generated by processors 350 and 351. Image-based sorting is performed in response to a sorting signal generated by a sorting trigger 352. The sorting unit 300c has a deflector plate 331 for deflecting particles into a sample container 332 or a waste stream 333, i.e., a deflector plate according to an embodiment of the present invention. In some cases, sorting portion 300c is configured to sort particles using an enclosed particle sorting module, such as that described in U.S. Patent Application Publication No. 2017 / 0299493, filed March 28, 2017, the disclosure of which is incorporated herein by reference. In some embodiments, sorting portion 300c includes a sort determination module having multiple sort determination units, such as that described in U.S. Patent Application Publication No. 2020 / 0256781, the disclosure of which is incorporated herein by reference.

[0097] In some embodiments, the system is a particle analyzer, and particle analysis system 401 (FIG. 4) can be used to analyze and characterize particles with or without physical sorting of the particles into a collection vessel. FIG. 4 is a functional block diagram of a particle analysis system for computation-based sample analysis and particle characterization. In some embodiments, particle analysis system 401 is a flow system. Particle analysis system 401 includes a fluid system 402. Fluid system 402 can include or be coupled to a sample tube 405 and a moving fluid column within the sample tube, through which particles 403 (e.g., cells) of the sample move along a common sample path 409.

[0098] The particle analysis system 401 includes a detection system 404 configured to collect a signal from each particle as it passes through one or more detection stations along a common sample path. A detection station 408 generally refers to a monitoring region 407 of the common sample path. Detection, in some embodiments, may involve detecting light or one or more other properties of the particle 403 as it passes through the monitoring region 407. In FIG. 4, one detection station 408 is shown with one monitoring region 407. In some embodiments of the particle analysis system 401, multiple detection stations may be provided. Additionally, some detection stations may monitor more than one region.

[0099] Each signal is assigned a signal value, generating a data point for each particle. As described above, this data may be referred to as event data. The data points may be multidimensional data points that include values ​​for each property measured for the particle. The detection system 404 is configured to collect a series of such data points over a first time interval.

[0100] The particle analysis system 401 may further include a control system 406. The control system 406 may include one or more processors, amplitude control circuitry, and / or frequency control circuitry. The illustrated control system may be operatively associated with the fluid system 402. The control system may be configured to generate a calculated signal frequency for at least a portion of the first time interval based on the Poisson distribution and the number of data points collected by the detection system 404 during the first time interval. The control system 406 may further be configured to generate an experimental signal frequency based on the number of data points in the portion of the first time interval. The control system 406 may further compare the experimental signal frequency to the calculated signal frequency or a predetermined signal frequency.

[0101] 5 is a functional block diagram of an example particle analysis control system, such as an analysis controller (i.e., processor) 500 for analyzing and displaying biological events. Analysis controller 500 can be configured to perform various processes for controlling the graphical display of biological events.

[0102] A particle analyzer or particle sorting system 502 may be configured to acquire the biological event data. For example, a flow cytometer may generate flow cytometry event data. The particle analyzer 502 may be configured to provide the biological event data to the analysis controller 500. A data communication channel may be included between the particle analyzer or particle sorting system 502 and the analysis controller 500. The biological event data may be provided to the analysis controller 500 via the data communication channel.

[0103] The analysis controller 500 may be configured to receive biological event data from a particle analyzer or particle sorting system 502. The biological event data received from the particle analyzer or particle sorting system 502 may include flow cytometry event data. The analysis controller 500 may be configured to provide a graphical display including a first plot of the biological event data on a display device 506. The analysis controller 500 may further be configured to render a region of interest as a gate around a population of the biological event data displayed by the display device 506, e.g., overlaid on the first plot. In some embodiments, the gate may be a logical combination of one or more illustrated regions of interest plotted on a histogram or bivariate plot of a parameter. In some embodiments, the display may be used to display particle parameters or saturation detector data.

[0104] Analysis controller 500 may further be configured to display the in-gate biological event data on display device 506 differently from other events in the outside-gate biological event data. For example, analysis controller 500 may be configured to render the color of the biological event data included within the gate differently from the color of the outside-gate biological event data. Display device 506 may be implemented as a monitor, tablet computer, smartphone, or other electronic device configured to present a graphical interface.

[0105] The analysis controller 500 may be configured to receive a gate selection signal identifying a gate from a first input device. For example, the first input device may be implemented as a mouse 510. The mouse 510 may initiate a gate selection signal to the analysis controller 500 identifying a gate to be displayed or manipulated via the display device 506 (e.g., by clicking on or within the desired gate when the cursor is over the desired gate). In some embodiments, the first device may be implemented as a keyboard 508 or other means for providing input signals to the analysis controller 500, such as a touchscreen, a pen, a photodetector, or a voice recognition system. Some input devices may include multiple input functions. In such embodiments, each input function may be considered an input device. For example, as shown in FIG. 5, the mouse 510 may include a right mouse button and a left mouse button, and the right mouse button and the left mouse button may each generate a trigger event.

[0106] A trigger event can cause the analysis controller 500 to change how the data is displayed, which portions of the data are actually displayed on the display device 506, and / or provide input to further processing, such as selecting a population of interest for particle sorting.

[0107] In some embodiments, the analysis controller 500 may be configured to detect when a gate selection is initiated by the mouse 510. The analysis controller 500 may further be configured to automatically modify the visualization of the plot to facilitate gating. This modification may be made based on a particular distribution of the biological event data received by the analysis controller 500.

[0108] The analysis controller 500 may be connected to a storage device 504. The storage device 504 may be configured to receive and store biological event data from the analysis controller 500. The storage device 504 may be further configured to receive and store flow cytometry event data from the analysis controller 500. The storage device 504 may be further configured to enable retrieval of biological event data, such as flow cytometry event data, by the analysis controller 500.

[0109] A display device 506 may be configured to receive display data from the analysis controller 500. The display data may include plots of the biological event data and gates outlining sections of the plots. The display device 506 may be further configured to modify the information displayed in response to input received from the analysis controller 500 in conjunction with input from the particle analyzer 502, the storage device 504, the keyboard 508, and / or the mouse 510.

[0110] In some embodiments, the analysis controller 500 can generate a user interface for receiving example events for filtering. For example, the user interface can include controls for receiving example events or example images. The example events or images, or example gates, can be provided prior to collection of event data for a sample, or can be provided based on an initial set of events for a portion of the sample.

[0111] FIG. 6A is a schematic diagram illustrating a particle sorting system 600 (e.g., particle analyzer or particle sorting system 502) according to one embodiment presented herein. In some embodiments, the particle sorting system 600 is a cell sorting system. As shown in FIG. 6A, a droplet-forming transducer 602 (e.g., a piezoelectric oscillator) is coupled to a fluid conduit 601, which may be coupled to, include, or be a nozzle 603. Within the fluid conduit 601, a sheath fluid 604 hydrodynamically focuses a sample fluid 606 containing particles 609 into a moving fluid column 608 (e.g., a stream). Within the moving fluid column 608, the particles 609 (e.g., cells) are aligned in a single file and traverse a monitoring region 611 (e.g., where the laser and the stream intersect) that is illuminated by an illumination source 612 (e.g., a laser). Vibration of droplet-forming transducer 602 causes moving fluid column 608 to break up into multiple droplets 610 , some of which contain particles 609 .

[0112] During operation, a detection station 614 (e.g., an event detector) identifies when a particle (or cell) of interest crosses the monitoring region 611. The detection station 614 feeds a timing circuit 628, which in turn feeds a flash charge circuit 630. At a droplet break-off point, signaled by a timed droplet delay (Δt), a flash charge can be applied to the moving fluid column 608 so that the droplet of interest carries a charge. The droplet of interest may contain one or more particles or cells to be sorted. The charged droplets can then be sorted by activating a deflection plate (not shown) to deflect the charged droplets into a receptacle, such as a collection tube or a multi-well or microwell sample plate, where a well or microwell can be specifically associated with the droplet of interest. As shown in FIG. 6A, the droplets can be collected in a drain receptacle 638.

[0113] A detection system 616 (e.g., a droplet boundary detector) serves to automatically determine the phase of the droplet drive signal as a particle of interest passes through the monitoring region 611. An exemplary droplet boundary detector is described in U.S. Pat. No. 7,679,039, the entire contents of which are incorporated herein by reference. The detection system 616 enables the instrument to accurately calculate the position of each detected particle within the droplet. The detection system 616 may provide an amplitude signal 620 and / or a phase signal 618, which are then provided (via an amplifier 622) to an amplitude control circuit 626 and / or a frequency control circuit 624. The amplitude control circuit 626 and / or the frequency control circuit 624 then control the droplet forming transducer 602. The amplitude control circuit 626 and / or the frequency control circuit 624 may be provided within the control system.

[0114] In some embodiments, the 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 on the detected events. The sorting decisions can be included in the event data for the particles. In some embodiments, detection system 616 and detection station 614 can be implemented as a single detection unit or can be communicatively coupled such that event measurements can be collected by either detection system 616 or detection station 614 and provided to a non-collection element.

[0115] FIG. 6B is a schematic diagram illustrating a particle sorting system according to one embodiment presented herein. The particle sorting system 600 shown in FIG. 6B includes deflector plates 652 and 654 according to an embodiment. A charge can be applied via a stream charging wire within the barb, generating a stream of droplets 610 containing particles 609 for analysis. The particles can be illuminated using one or more light sources (e.g., lasers) to generate light scattering and fluorescence information. The information about the particles is analyzed by sorting electronics or other detection systems (not shown in FIG. 6B). Deflector plates 652 and 654 can be independently controlled to attract or repel the charged droplets and direct them toward a desired collection vessel (e.g., one of 672, 674, 676, or 678). 6B, deflector plates 652 and 654 can be controlled to direct particles along a first path 662 toward a container 674 or along a second path 668 toward a container 678. If the particle is not of interest (e.g., does not exhibit scattering or illumination information within a specified sorting range), the deflector plates may allow the particle to continue along path 664. Such uncharged droplets may be directed into a waste container, such as via an aspirator 670.

[0116] Sorting electronics can be included to initiate measurement collection, receive fluorescent signals for the particles, and determine how to adjust the deflection plates to sort the particles. Exemplary implementations of the embodiment shown in Figure 6B include the BD FACSAria™ system of flow cytometers, commercially available from Becton, Dickinson and Company (Franklin Lakes, NJ).

[0117] method Aspects of the present disclosure further include methods for sorting particles of a sample, e.g., cells of a biological sample. In some embodiments, the method includes illuminating a sample containing particles in a flow stream within an interrogation region of a particle sorting module, detecting light (e.g., fluorescence) from the sample, and sorting particles of the sample into two or more sample collection vessels. In some embodiments, the sample is a biological sample, and the method includes sorting and collecting two or more different types of cells.

[0118] In some cases, the sample analyzed in the present method is a biological sample. The term "biological sample" is used in its conventional sense to refer to a whole organism, a plant, a fungus, or a subset of animal tissues, cells, or components, such as may be found in blood, mucus, lymph, synovial fluid, cerebrospinal fluid, saliva, bronchoalveolar lavage fluid, amniotic fluid, amniotic cord blood, urine, vaginal fluid, or semen, as the case may be. Thus, "biological sample" refers to both a naturally occurring organism or a subset of its tissues, and homogenates, lysates, or extracts prepared from an organism or a subset of its tissues, including, but not limited to, plasma, serum, spinal fluid, lymph, skin sections, respiratory tract, gastrointestinal tract, cardiovascular, genitourinary tract, tears, saliva, milk, blood cells, tumors, and organs. Biological samples may be any type of organismal tissue, including both healthy and diseased tissue (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 from venipuncture or finger stick (which may or may not be combined with any reagents, such as preservatives, anticoagulants, etc., prior to assay).

[0119] In some embodiments, the sample source is a "mammal" or "mammalian," which terms are used broadly to describe organisms belonging to the class Mammalia, including the orders Carnivora (e.g., dogs and cats), Rodentia (e.g., mice, guinea pigs, and rats), and Primates (e.g., humans, chimpanzees, and monkeys). In some cases, the subject is a human. The methods may be applied to samples obtained from human subjects of both genders and at any stage of development (i.e., newborn, infant, juvenile, adolescent, adult), and in some embodiments, the human subject is a juvenile, adolescent, or adult. While the present disclosure may be applied to samples from human subjects, it should be understood that the methods may also be performed on samples from other animal subjects (i.e., "non-human subjects"), including, but not limited to, birds, mice, rats, dogs, cats, livestock, and horses.

[0120] Cells of interest may be subject to characterization according to various parameters, such as phenotypic characteristics identified by attaching specific fluorescent labels to the cells of interest. In some embodiments, the system is configured to deflect analysis droplets determined to contain target cells. A variety of cells may be characterized using the subject methods. 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 erythroid cells. Target cells of interest include cells bearing convenient cell surface markers or antigens that may be internalized or labeled by convenient affinity agents or their conjugates. For example, target cells may comprise 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 umbilical cord blood.

[0121] In practicing the subject methods, a volume of an initial fluid sample is injected into a flow cytometer. The volume of sample injected into the particle sorting module may vary, for example, within the range of 0.001 mL to 1000 mL of sample, e.g., 0.005 mL to 900 mL, e.g., 0.01 mL to 800 mL, e.g., 0.05 mL to 700 mL, e.g., 0.1 mL to 600 mL, e.g., 0.5 mL to 500 mL, e.g., 1 mL to 400 mL, e.g., 2 mL to 300 mL, e.g., 5 mL to 100 mL.

[0122] Methods according to embodiments of the present disclosure enumerate and optionally sort labeled particles (e.g., target cells) in a sample. In practicing the subject methods, a fluid sample containing particles is first introduced into a flow nozzle of the system. Upon exiting the flow nozzle, the particles pass substantially one at a time through a sample interrogation region where each particle is illuminated by a light source, and measurements of light scattering parameters, and optionally fluorescence emission measurements (e.g., two or more light scattering parameters and one or more fluorescence emission measurements), are recorded separately for each particle, as desired. Depending on the characteristics of the flow stream being interrogated, the light may illuminate a flow stream of 0.001 mm or greater, e.g., 0.005 mm or greater, e.g., 0.01 mm or greater, e.g., 0.05 mm or greater, e.g., 0.1 mm or greater, e.g., 0.5 mm or greater, e.g., 1 mm or greater. In certain embodiments, the methods illuminate a planar cross-section of the flow stream within the sample interrogation region, e.g., with a laser (as described above). In another embodiment, the method illuminates a predetermined length of the flow stream within the sample interrogation region to correspond to the illumination profile of a diffuse laser beam or lamp.

[0123] In some embodiments, the method irradiates the flow stream at or near the nozzle orifice of the flow cell. For example, the method may irradiate the flow stream at about 0.001 mm or more from the nozzle orifice, such as 0.005 mm or more, such as 0.01 mm or more, such as 0.05 mm or more, such as 0.1 mm or more, such as 0.5 mm or more, for example 1 mm or more from the nozzle orifice. In some embodiments, the method irradiates the flow stream directly adjacent to the nozzle orifice of the flow cell.

[0124] In embodiments of this method, detectors such as photomultiplier tubes (PMTs) are used to record the light passing through each particle (sometimes referred to as forward scattered light), the light reflected perpendicular to the direction of particle flow through the detection region (sometimes referred to as orthogonal or side scattered light), and, if the particles are labeled with one or more fluorescent markers, the fluorescence emitted by the particles as they pass through the detection region and are illuminated by an energy source. Forward scattered light (FSC), side scattered light (SSC), and fluorescent emission each have separate parameters per particle (or "event"). Thus, for example, two, three, or four parameters may be collected (and recorded) from particles labeled with two different fluorescent markers. The data recorded for each particle may be analyzed in real time or stored in a data storage and analysis means, such as a computer, as desired.

[0125] In some embodiments, particles are detected and uniquely identified by exposing them to excitation light and measuring the fluorescence of each particle in one or more detection channels, as desired. The fluorescence emitted in the detection channels used to identify particles and their associated binding complexes may be measured after excitation by a single light source, or may be measured separately after excitation by different light sources. When separate excitation light sources are used to excite particle labels, the particle labels may be selected such that all particle labels are excitable by each of the excitation light sources used.

[0126] In some embodiments, the method further includes data collection, analysis, and recording, e.g., using a computer, where multiple data channels record data from each detector regarding light scattering and fluorescence emitted by each particle as it passes through the sample interrogation region of the particle sorting module. In these embodiments, particles are classified and counted during analysis, with each particle present as a set of digitized parameter values. The subject system may be configured to trigger on selected parameters to distinguish particles of interest from background and noise. "Trigger" refers to a preset threshold for parameter detection and may be used as a means for detecting when a particle has passed through a light source. Detection of an event exceeding the selected parameter threshold triggers the collection of light scattering and fluorescence data for the particle. Data related to particles or other components in the analysis medium that cause a response below the threshold is not acquired. The trigger parameter may be detection of forward scattered light resulting from a particle passing through a light beam. In this manner, the flow cytometer detects and collects light scattering and fluorescence data for particles.

[0127] Specific subpopulations of interest are then further analyzed by "gating" based on the data collected for the entire population. To select an appropriate gate, the data is plotted to separate the subpopulations as best as possible. This procedure may be performed by plotting forward scatter (FSC) versus side (i.e., orthogonal) scatter (SSC) on a two-dimensional dot plot. A subpopulation of particles (i.e., cells within the gate) is then selected, and particles not within the gate are excluded. If desired, a gate may be selected by drawing a line around the desired subpopulation using a cursor on the computer screen. Only those particles within the gate are then further analyzed by plotting other parameters of these particles, such as fluorescence. If desired, the above analysis may be configured to calculate the number of particles of interest in the sample.

[0128] To select droplets of interest containing particles of interest, the analyzed flow stream is subjected to a deflection force by a droplet deflector, i.e., an embodiment of a droplet deflector as described herein, that includes deflection plates configured for high-angle deflection of the droplet flow stream. A voltage is applied to the metal plates (deflection plates) to accelerate and deflect (i.e., high-angle deflect) droplets flowing past the deflection plates based on the charge and charge polarity of the droplets. The voltage applied to the deflection plates to deflect the charged particles may be 10 mV or more, e.g., 25 mV or more, e.g., 50 mV or more, e.g., 100 mV or more, e.g., 250 mV or more, e.g., 500 mV or more, e.g., 750 mV or more, e.g., 1000 mV or more, e.g., 2500 mV or more, e.g., 5000 mV or more, e.g., 10,000 V or more, e.g., 15,000 V or more, e.g., 25,000 V or more, e.g., 50,000 V or more, e.g., 100,000 V or more. In one embodiment, the voltage applied to the metal plates is in the range of 0.5 kV to 15 kV, e.g., 1 kV to 15 kV, e.g., 1.5 kV to 12.5 kV, e.g., 2 kV to 10 kV. Therefore, the electric field strength between the deflection plates is in the range of 0.1 V / m to 1×10 7 V / m, e.g., 0.5 V / m ~5×10 6 V / m, e.g., 1V / m ~1×10 6 V / m, e.g., 5V / m ~5×10 5 V / m, e.g., 10 V / m~1×10 5 V / m, e.g., 50 V / m~5×10 4 V / m, e.g., 1×10 5 V / m~2×10 6 V / m range.

[0129] Where the droplet deflector comprises a deflection plate having two or more segments, the voltage applied to each segment of the deflection plate may be different, and the voltage applied to each segment of the deflection plate may differ by 0.01 mV or more, such as 0.05 mV or more, for example 0.1 mV or more, such as 0.5 mV or more, for example 1 mV or more, such as 5 mV or more, for example 10 mV or more, such as 25 mV or more, for example 50 mV or more, such as 75 mV or more, for example 100 mV or more, such as 250 mV or more, for example 500 mV or more, such as 750 mV or more, for example 1 V or more, such as 2.5 V or more, for example 5 V or more, such as 10 V or more, for example 25 V or more, such as 50 V or more, for example 100 V or more. Thus, the electric field strength between each segment of the deflection plates may be substantially the same or may differ by 0.001 V / m or more, such as 0.01 V / m or more, for example 0.1 V / m or more, such as 0.5 V / m or more, for example 1 V / m or more, such as 2 V / m or more, for example 5 V / m or more, such as 10 V / m or more, for example 25 V / m or more.

[0130] In some embodiments, the system operates to determine a time slot in which one or more sample collection containers are aligned to receive deflected droplets. In some cases, the deflection signal includes a first deflection sub-signal and a last deflection sub-signal, and the system operates to generate the deflection signal by transmitting a first deflection sub-signal at the beginning of the time slot that configures the deflector to deflect analyzed droplets, if present. In some cases, the method transmits a last deflection sub-signal at the end of the time slot that configures the droplet deflector not to deflect analyzed droplets. In some embodiments, the method transmits a last deflection sub-signal at the end of the time slot that configures the droplet deflector not to deflect analyzed droplets after one analyzed droplet is deflected during the time slot.

[0131] Featured methods may also use the particles in research, clinical trials, or treatment. In some embodiments, the subject methods obtain individual cells prepared from a target fluid or tissue biological sample. For example, the subject methods obtain cells from a fluid or tissue sample used as a research or diagnostic specimen for a disease such as cancer. Similarly, the subject methods obtain cells from a fluid or tissue sample used for treatment. Cell therapy protocols are protocols in which viable cellular material, including, for example, cells and tissue, may be prepared and introduced into a subject as a therapeutic treatment. Conditions that may be treated by administration of flow cytometry-sorted samples include, but are not limited to, blood disorders, immune system disorders, organ damage, and the like.

[0132] A typical cell therapy protocol may include the steps of sample collection, cell isolation, genetic modification, culture, in vitro expansion, cell harvesting, sample volume reduction, sample washing, biopreservation, storage, and cell introduction into a subject. A protocol may begin by collecting viable cells and tissue from a tissue source in a subject to generate a cell and / or tissue sample. The sample may be collected by any suitable procedure, including, for example, administering a cell mobilizing agent to the subject, drawing blood from the subject, or removing bone marrow from the subject. After sample collection, cell enrichment may be performed by multiple methods, including, for example, centrifugation-based methods, filter-based methods, elutriation, magnetic separation, fluorescence-activated cell sorting (FACS), and the like. In some cases, the enriched cells may be genetically modified by any convenient method, such as nuclease-mediated gene editing. Genetically modified cells may be cultured, activated, and expanded in vitro. In some cases, the cells are preserved, e.g., cryopreserved, and stored for future use. At the time of use, the cells are thawed and then administered to a patient, e.g., the cells may be infused into a patient.

[0133] Aspects of the present disclosure further include a method for determining an optimized shape of a deflection plate configured for high-angle deflection of a droplet flow stream. The disclosed method applies the numerical techniques described herein for simulating the path of a droplet flow stream under the influence of an electric field imposed by a deflection plate to iteratively modify the shape of the deflection plate in one or two dimensions. Such a method may be applied to arrive at a deflection plate configured for high-angle deflection that minimizes the distance between the deflected droplets and the deflection plate, subject to certain constraints.

[0134] Computer Control System Aspects of the present disclosure further include computer control systems for carrying out the subject methods, the systems further comprising one or more computers for fully or partially automating the systems for carrying out the methods described herein. In some embodiments, the systems comprise a computer having a computer-readable storage medium having stored thereon a computer program, which, when loaded into the computer, comprises instructions for illuminating a sample in a flow stream within a sample interrogation region, an algorithm for detecting light from the sample and measuring the detected light at one or more wavelengths, and an algorithm for sorting particles in the sample by applying droplet deflection for high-angle deflection of droplets in the flow stream into two or more sample collection vessels.

[0135] 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 can access a memory in which instructions for executing the steps of the subject method are stored. The processing module may include an operating system, a graphical user interface (GUI) controller, system memory, memory storage devices, an input / output controller, 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 or become available. The processor executes an operating system that interfaces with firmware and hardware in a well-known manner to facilitate the processor's coordination and execution of functions of various computer programs, which may be written 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 functions of the 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 in accordance with known techniques. In some embodiments, the processor includes analog electronics that provide feedback control, such as negative feedback control.

[0136] The system memory may be any of a variety of known or future memory storage devices. Examples include any commonly available random access memory (RAM), magnetic media such as a resident hard disk or tape, optical media such as a read-write compact disk, flash memory device, or other memory storage device. The memory storage device may be any of a variety of known or future devices, including a compact disk drive, tape drive, or diskette drive. These types of memory storage devices typically read from and / or write to a program storage medium (not shown), such as a compact disk. Any of these program storage media, or other program storage media now in use or that may be developed in the future, may be considered a computer program product. As will be appreciated, these program storage media typically store computer software programs and / or data. Computer software programs, also referred to as computer control logic, are typically stored in the system memory and / or on program storage devices used in conjunction with the memory storage devices.

[0137] In some embodiments, a computer program product is described that includes a computer-usable medium having stored thereon control logic (a computer software program including program code). The control logic, when executed by a processor, a computer, causes the processor to perform the functions described herein. In other embodiments, some functions are implemented primarily in hardware, for example, using hardware state machines. Implementation of hardware state machines to perform the functions described herein will be apparent to one skilled in the relevant art.

[0138] The memory may be any suitable device from which the processor can store and retrieve data, such as a magnetic, optical, or solid-state storage device (including a magnetic or optical disk, tape, RAM, or any other suitable device, fixed or portable). The processor may include a general-purpose digital microprocessor appropriately programmed from a computer-readable medium storing the necessary program code. The program may be provided to the processor remotely via a communication channel or pre-recorded on a computer program product, such as a memory, or on other portable or fixed computer-readable storage media using one of these devices connected to the memory. For example, a magnetic or optical disk may store the program and be read by a disk writer / reader. The system of the present disclosure may further include a program, e.g., in the form of a computer program product, an algorithm for use in implementing the method as described above. The program according to the present disclosure may be recorded on a computer-readable medium, e.g., 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 tape; 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.

[0139] The processor may also access a communication channel to communicate with a user at a remote location, meaning that the user does not have direct contact with the system but instead relays input information to the input manager from an external device, such as a computer connected to a wide area network ("WAN"), telephone network, satellite network, or any other suitable communication channel, including a mobile phone (i.e., a smartphone).

[0140] In some embodiments, a system according to the present disclosure may be configured to include a communications interface. In some embodiments, the communications interface includes a receiver and / or a transmitter for communicating with a network and / or another device. The communications interface may be configured for wired or wireless communications, including, but not limited to, radio frequency (RF) communications such as radio frequency identification (RFID), ZigBee communications protocol, Wi-Fi, infrared, wireless universal serial bus (USB), ultra-wideband (UWB), Bluetooth® communications protocol, and cellular communications such as code division multiple access (CDMA) or global system for mobile communications (GSM).

[0141] In one embodiment, the communication interface is configured to include one or more communication ports, e.g., 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 computer terminals (e.g., in a clinic or hospital environment) configured for similar complementary data communication.

[0142] In one embodiment, the communication interface is configured for infrared communication, Bluetooth® communication, or any other suitable wireless communication protocol, allowing the subject system to communicate with other devices, such as computer terminals and / or networks, communication-enabled mobile phones, personal digital assistants, or any other communication devices that a user may use in conjunction with the device.

[0143] In one embodiment, the communication interface is configured to provide connectivity for data transfer using Internet Protocol (IP) via a cellular 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.

[0144] In one embodiment, the subject system is configured to communicate wirelessly with a server device via a communications interface using a common standard, such as 802.11, Bluetooth® RF protocol, or IrDA infrared protocol. The server device may be another portable device, such as a smartphone, personal digital assistant (PDA), or notebook computer; or a larger device, such as a desktop computer, 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 touchscreen.

[0145] In some embodiments, the communications interface is configured to automatically or semi-automatically communicate data stored in the subject system, e.g., any data storage unit, with a network or server device using one or more of the communications protocols and / or mechanisms described above.

[0146] 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 may 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 / 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 using a network or other type of remote communication in alternative embodiments. The output manager may also provide information generated by the processing module to a user at a remote location, for example, via the Internet, telephone, or satellite network, according to known techniques. Presentation of data by the output manager may be performed according to various known techniques. In some examples, the data may include SQL, HTML, or XML documents, emails or other files, or other forms of data. The data may include Internet URL addresses so that the user can obtain additional SQL, HTML, XML, or other documents or data from remote sources. The platform or platforms present in the subject system are typically a class of computers commonly referred to as servers, but may be any type of computer platform now known or later developed. However, the platforms may also be mainframe computers, workstations, or other computer types. The platforms may be networked or not, and may be connected via any type of cabling, now known or later, or other communication systems, including wireless systems. The platforms may be co-located or physically separated.Various operating systems may be used on any of the computer platforms, depending in some cases on the type and / or configuration of the computer platform selected. Suitable operating systems include Windows NT, Windows XP, Windows 7, Windows 8, Windows 10, iOS, macOS, Linux, Ubuntu, Fedora, OS / 400, i5 / OS, IBM i, Android, SGI IRIX, Oracle Solaris, and the like.

[0147] FIG. 7 illustrates a general configuration of an exemplary computing device 700 according to one embodiment. The general configuration of computing device 700 illustrated in FIG. 7 includes an arrangement of computer hardware and software components. However, not all of these typical conventional elements need be shown to provide a useful disclosure. As illustrated, computing device 700 includes a processing unit 710, a network interface 720, a computer-readable medium drive 730, an input / output device interface 740, a display 750, and input devices 760, all of which may communicate with each other via a communications bus. Network interface 720 may provide connectivity to one or more networks or computing systems. Thus, processing unit 710 may receive information and instructions from other computing systems or services via a network. Processing unit 710 may further communicate with memory 770 and may further provide output information for an optional display 750 via input / output device interface 740. For example, analysis software (e.g., data analysis software or program, e.g., FlowJo®) stored as executable instructions in non-transitory memory of the analysis system can display flow cytometry event data to a user. The input / output device interface 740 may further accept input from any input device 760, such as a keyboard, mouse, digital pen, microphone, touch screen, gesture recognition system, voice recognition system, gamepad, accelerometer, gyroscope, or other input device.

[0148] Memory 770 may include computer program instructions (grouped in some embodiments as modules or components) that processing unit 710 executes to implement one or more embodiments. Memory 770 typically includes RAM, ROM, and / or other persistent, secondary, or non-transitory computer-readable media. Memory 770 may store an operating system 772 that provides computer program instructions for use by processing unit 710 in the general management and operation of computing device 700. Data may be stored in data storage device 790. Memory 770 may further include computer program instructions and other information for implementing aspects of the present disclosure.

[0149] kit Aspects of the present disclosure further include kits, which include one or more of the droplet deflectors described herein. In some embodiments, the kits include deflection plates configured for high-angle deflection of the droplet flow stream of the subject droplet deflectors, e.g., deflection plates having a shape corresponding to the path of the deflected droplet flow stream, deflection plates configured to apply a deflection force to the flow stream at multiple different angles, twisted deflection plates, and droplet deflector deflection plates configured to apply a constant deflection force to the deflected droplet flow stream from multiple different lateral positions, along with instructions for assembling the droplet deflector and / or its deflection plates. In some cases, the kits may include one or more assay components (e.g., labeled reagents, buffers, etc., as described above). In some cases, the kits may further include a sample collection device, such as a lancet or needle configured to pierce the skin to obtain a whole blood sample, a pipette, etc., as desired.

[0150] The various assay components of the kit may be present in separate containers, or some or all of the assay components may be premixed. For example, in some cases, one or more components of the kit, e.g., each plate of a pair of droplet deflection plates, is present within a sealed pouch, e.g., a sterile foil pouch or package.

[0151] In addition to the above components, the subject kits may (in some embodiments) further comprise instructions for use. These instructions may be present in the subject kits in a variety of forms, and one or more of these instructions may be present in the kit. One form in which these instructions may be provided is as information printed on a suitable medium or substrate, such as one or more pieces of paper on which the information is printed, kit packaging, a package insert, etc. Another form in which these instructions may be provided is as a computer-readable medium on which the information is recorded, such as a diskette, a compact disc (CD), a portable flash drive, etc. Another form in which these instructions may be present is a website address that may be used via the Internet to access the information at a remote location.

[0152] usefulness The subject droplet deflectors, particle sorting modules, particle sorting systems, methods, and computer systems are used in a variety of applications where it is desirable to analyze and sort particle components in a sample within a fluid medium, such as a biological sample. Embodiments of the present invention are used where it is desirable to provide a flow cytometer with increased cell sorting accuracy, improved particle collection, particle charging efficiency, more accurate particle charging, and improved particle deflection during cell sorting.

[0153] Embodiments of the present disclosure find use in applications where cells prepared from a biological sample may be desirable for use in research, clinical trials, or therapy. In some embodiments, the subject methods and devices may facilitate obtaining and / or analyzing individual cells prepared from a targeted fluid or tissue biological sample. For example, the subject methods and systems facilitate obtaining cells from fluid or tissue samples used as research or diagnostic specimens for diseases such as cancer. Similarly, the subject methods and systems may facilitate obtaining cells from fluid or tissue samples used for therapy.

[0154] The following are given by way of example and not limitation.

[0155] experiment In droplet deflector embodiments including deflector plates configured for high-angle deflection of a droplet flow stream, the droplet deflector may have a deflector plate shape that is the result of iterative numerical calculations. A process for iteratively determining such a deflector plate shape is illustrated in FIGS. 8A-8B. This process begins by assuming a flat deflector plate 801 (having a proximal end 801p and a distal end 801d located downstream from the proximal end 801p along the longitudinal axis of the flow stream) among parallel deflector plates 801, 805. The length of each deflector plate is limited to a fixed length, and the initial spacing between the deflector plates is constant along the length of the deflector plates 801, 805. For modeling purposes, the distance from the droplet at the center of the deflector plate to the electrode 801 is calculated over approximately n=1000 points [d0, d1, . . . d i . . . d n ]. An initial value is chosen, in this case, for all d i is 3 mm. At each of these points, the electric field, acceleration, velocity, and position of the droplet (assuming ejection from a 100 micron nozzle) are calculated. At each position x of the deflected droplet, the distance between the droplet and electrode 801 is compared. Based on the results of these comparisons, (d i -x)<ε (ε is a design constraint that is the selected buffer distance between the deflection droplet and the deflector plate 801), then d i . . . d n increases, for example, d i . . . d n d i plus an arbitrary small number. The process is then repeated using the new distance between the deflector plates until numerical simulations show that the droplets fall beyond the length of the deflector plates 801, i.e., off the droplet deflector.

[0156] The iterative calculation process is illustrated in Figure 8B, which shows a relatively enlarged cross section of deflector plate 801. The initial shape of deflector plate 801 is shown on the left and has an initial shape that is a flat, parallel plate as shown in Figure 8A (i.e., a plate parallel to deflector plate 805 and parallel to the longitudinal axis of the droplet flow stream), i.e., d0...d n Each d i is 3mm.

[0157] After the first iteration of the numerical calculation process, the shape of the deflector plate 801 becomes a step 802a (i.e., the deflected droplet falls within a distance of less than ε from the deflector plate 801), as shown in the center of FIG. 8B. a The deflector plate 801 is modified to include a step corresponding to the position of d0. . . d (a-1) is 3 mm, and d a . . . d n has been modified to be 3 mm plus any increment, i.e., to include the step, i.e., the step away from the longitudinal axis of the droplet flow stream.

[0158] After the second iteration of the numerical calculation process, the shape of the deflector plate 801 becomes a step 802b (i.e., the deflected droplet falls within a distance of less than ε from the deflector plate 801), as shown on the right side of FIG. 8B. b The deflector plate 801 is further modified to include a step corresponding to the second position of d0. . . d (a-1) is 3 mm, and d a . . . d (b-1) is 3 mm plus any incremental step, and d b . .. d n has been changed to be 3 mm plus any two incremental steps.

[0159] 8B shows a relatively enlarged cross section of deflector plate 801, the iterative process continues until the shape of deflector plate 801 is configured such that deflected droplet 810 can be deflected along the entire length of deflector plate 801 without intersecting or colliding with deflector plate 801, at which point the iterative process terminates. The final shape of the corresponding deflector plate 805 can be considered a mirror image of the final shape of deflector plate 801 (e.g., with steps corresponding to steps 802a, 802b).

[0160] An embodiment of a numerical solution technique for determining the geometry of deflector plates configured for high-angle deflection of a droplet flow stream is shown in flowchart 900 of Figure 9. Flowchart 900 begins with step 910, which specifies an initial condition for the deflector plates, in this case, flat, parallel deflector plates spaced 6 mm apart from each other, as the initial shape to be adjusted by proceeding through flowchart 900. After completing step 910, flowchart 900 proceeds to step 920.

[0161] In step 920, various properties of the simulated deflected droplets are calculated at longitudinal positions along the longitudinal axis of the droplet flow stream (corresponding to the time points when the deflected droplets fall). In this case, the electric field between the deflector plates is calculated based on the distance between the deflector plates, and the velocity and lateral position of the deflected droplets are calculated. The initial longitudinal position at which these properties are calculated corresponds to the longitudinal position of the droplets when they first enter the droplet deflector, which corresponds to the initial time (i.e., t=0). After completing step 920, flowchart 900 proceeds to step 930.

[0162] In step 930, the simulation progresses over time so that the deflected droplets advance to more distal positions along the longitudinal axis of the droplet flow stream. After completing step 930, flowchart 900 proceeds to step 940.

[0163] In step 940, the distance between the droplet and the deflector plate is compared, in each case, with (i) time and (ii) the corresponding longitudinal position of the deflected droplet, as determined in step 930. If the distance between the deflected droplet and the deflector plate is greater than or equal to a threshold, flowchart 900 returns to step 930 and advances time so that the deflected droplet moves to a more distal longitudinal position along the longitudinal axis of the droplet flow stream. If the distance between the deflected droplet and the deflector plate is less than the threshold, flowchart 900 instead proceeds to step 950.

[0164] In step 950, the shape of the deflection plate (i.e., electrode) is modified to prevent the deflected droplet from moving within a specified threshold distance from the deflection plate. In particular, a step is introduced into the deflection plate so that the distance between the deflected droplet and the deflection plate increases by a specified distance at the longitudinal position assigned in step 930 and at all subsequent longitudinal positions along the deflection plate. After completing step 950, flowchart 900 proceeds to step 960.

[0165] In step 960, the updated shape of the deflector blades is recorded, for example, by writing or storing in computer memory a new file representing the shape of the deflector blades. After completing step 960, flowchart 900 returns to step 920.

[0166] Flowchart 900 progresses through time and ends when the deflected droplets move to a longitudinal position such that they exit the droplet deflector. That is, flowchart 900 ends when the deflected droplets fall a distance along the longitudinal axis that is greater than the longitudinal length of the deflector plate. Upon completing the steps of flowchart 900, the final shape of the droplet deflector is the shape recorded in the last plate file written the last time flowchart 900 executed step 960.

[0167] FIG. 10A shows simulated results of deflected droplet paths under the influence of a deflector plate configured in a conventional, i.e., angled, shape. Plot 1000a shows x- and y-axes corresponding to the x- and y-positions of droplets in the deflected droplet flow stream. In plot 1000a, y-axis 1001 corresponds to the position along the longitudinal axis of the droplet flow stream. In plot 1000a, x-axis 1002 corresponds to the lateral position of the droplet flow stream, i.e., the deflection position. Deflected droplet flow stream path 1005a shows a drop along the y-axis and a further deflection along the x-axis. The position and shape of deflector plate 1010a are shown in plot 1000a. Deflected droplet flow stream path 1005a is deflected under the influence of deflector plate 1010a. Deflector plate 1010a has proximal ends 1010a-p and distal ends 1010a-d located downstream along the longitudinal axis of the flow stream from proximal ends 1010a-p. Although deflection path 1005a does not collide with deflector plate 1010a, the shape of deflector plate 1010a is further away from the path 1005a of the deflected droplet flow stream than necessary to prevent collision (and thus droplet deflector 1010a is further away from the opposing deflector, reducing the deflection force applied to the droplets).

[0168] FIG. 10B shows the results 1000b of a numerical simulation to determine the shape of a deflector plate 1010b for high-angle deflection according to an embodiment and the path 1005b of deflected droplets under the influence of the deflector plate 1010b, calculated using an embodiment of the method described in connection with FIGS. 8A, 8B, and 9. The deflector plate 1010b has a proximal end 1010b-p and a distal end 1010b-d located downstream along the longitudinal axis of the flow stream from the proximal end 1010b-p. The deflector plate 1010b is shaped to avoid collisions between the deflected droplet flow path 1005b and the deflector plate 1010b. However, in contrast to the deflector plate 1010a, the deflector plate 1010b closely mimics the path 1005b of the deflected droplet flow stream. In particular, deflector plate 1010b is configured to minimize the distance to deflected droplet flow path 1005b along its length along the longitudinal axis such that the distance between deflector plate 1010b and its opposing deflector plate (not shown) is also minimized, thereby maximizing the deflection force applicable to droplet flow stream 1005b.

[0169] In FIG. 10B, deflector plate 1010b is shown for deflected droplet flow stream 1005b, which is the path of 3 nl charged droplets. Deflector plate 1010b (and its corresponding plate (not shown)) are initially straight and parallel (i.e., in the proximal region) because bringing the deflector plates closer together would result in electrical breakdown. Once the droplets in deflected droplet flow stream 1005b reach within 0.1 mm of deflector plate 1010b, deflector plate 1010b and its corresponding plate (not shown) are changed to 0.0175 mm apart, and the simulation of the deflected droplets is repeated. After many iterations, the simulation is able to reach the bottom of deflector plate 1010b (i.e., distal end 1010b-d), satisfying the 0.1 mm boundary condition around deflector plate 1010b. If desired, deflection plate 1010b and its opposing plate (not shown) generate an electric field that deflects the deflected droplet flow stream into a shape that closely mimics the shape of deflection plate 1010b, i.e., deflection plate 1010b is configured for high-angle deflection of the droplet flow stream according to embodiments of the present invention. This optimization of the shape for high-angle deflection of the droplet flow stream results in approximately 10% more deflection than conventional electrodes such as electrode 1010a.

[0170] As described herein, one-dimensional deflection plates can have the limitation that deflected droplets will eventually collide, intersect, or strike the surface of the deflection plate. Adjusting the shape of the deflection plate by moving the plate surface away from the deflected droplets can prevent droplets from striking the surface, but can also reduce the electric field (and thus the deflection force) and the acceleration of the deflected droplets. To address this issue, some embodiments use relatively short, straight plates, where the plate terminates at the point where the droplet intersects the plate; instead, particles are accelerated in an orthogonal direction by an orthogonally positioned deflection plate. In such embodiments, droplets are deflected in a straight line, which, for equal electrodes, is at a 45-degree angle to the plate. Other embodiments address this issue by using helical or twisted deflection plates, as described herein.

[0171] The numerical method embodiments described in connection with FIGS. 8A, 8B, and 9, and the results shown in FIG. 10B, relate to optimizing deflector plate shapes in one dimension. Such methods can be extended to optimizing deflector plate shapes in two dimensions, such as optimizing deflector plates having a helical configuration, e.g., deflector plates 121a and 121b of droplet deflector 120. To arrive at a two-dimensional optimized deflector plate shape, the numerical optimization is performed in a manner similar to the one-dimensional optimization, e.g., as shown in FIGS. 8A, 8B, and 9. However, to extend such techniques to two dimensions, the two-dimensional techniques further require keeping track of the deflector plate angle (i.e., the angle corresponding to the twist angle or the angle about the longitudinal axis of the droplet flow stream) to calculate acceleration, velocity, and position in both the x and y directions (i.e., extending the calculations of step 920 of flowchart 900 to two dimensions in both the x and y directions). If the position of the deflected droplet is determined to be within a threshold distance of, for example, 0.1 mm from the deflection plate, the shape of the distal portion of the deflection plate is shifted or spaced a nominal amount (i.e., similar to steps 940 and 950 of flowchart 900) and the simulation is restarted. The numerical calculation process is repeated until the final shape of the deflected droplet is achieved as it exits the droplet deflector (i.e., the simulation reaches a shape where the deflected droplet travels the longitudinal length of the deflection plate without colliding with the deflection plate).

[0172] FIG. 10C illustrates the application of an embodiment of the numerical computation technique to arrive at a two-dimensional shape for deflector plate 1010c. Deflector plate 1010c has proximal end 1010c-p and distal end 1010c-d located downstream along the longitudinal axis of the flow stream from proximal end 1010c-p. Displacements in both the x and y directions are shown in the same plot 1000c. A one-dimensional representation of the shape of the final electrode 1010c is shown for x-axis deflected droplet displacement 1006c (i.e., droplet displacement along the x-axis, orthogonal to the droplet flow stream) and y-axis deflected droplet displacement 1007c (i.e., droplet displacement along the y-axis, orthogonal to the droplet flow stream and also orthogonal to the x-axis). Total droplet displacement 1005c is also shown in plot 1000c, representing the total distance of the deflected droplet flow stream from the longitudinal axis of the droplet flow stream. The result shown in plot 1000c is that the net effect of twisting the deflector plates in two dimensions according to the embodiment is that the deflector plates remain straight for a longer period of time (i.e., the straight portion at the top (i.e., proximal region) of the deflector plate 1010c's shape is longer than the straight portion at the top (i.e., proximal region) of the deflector plate 1010b), resulting in a higher exit velocity and displacement position of particles deflected by deflector plate 1010c relative to particles deflected by deflector plate 1010a or deflector plate 1010b. The net effect is an increased amount of deflection 1005c of the deflected droplet flow stream. Additionally, because the velocities of the deflected droplets in both the x and y directions are equal, the deflection is at a 45-degree angle from the initial orientation of the electrodes. These simulation results have been confirmed experimentally using a prototype deflector plate as described herein.

[0173] FIG. 11 illustrates an embodiment of twisted deflection plates 1101, 1102 according to an embodiment of the present invention. The deflection plates 1101, 1102 are twisted deflection plates for high-angle deflection of droplets. The shapes of the deflection plates 1101, 1102 were obtained using a two-dimensional deflection plate numerical calculation as described in connection with FIG. 10C. That is, the amount and degree of "twist" of the deflection plates 1101, 1102, as well as the distance between the deflection plates 1101, 1102, were calculated as the distance the deflection plates should be spaced apart to avoid deflected droplets colliding with the deflection plates. The shape of one deflection plate 1101 can be simulated using the techniques described herein, for example, in connection with FIG. 10C, and the shape of the opposing deflection plate 1102 is determined to be a mirror image of the calculated shape of the deflection plate 1101.

[0174] FIG. 12A shows an experimental setup for evaluating the deflection plates of a droplet deflector according to an embodiment of the present invention against conventional angled deflector plates. Experimental setup 1210 includes aspects of a flow cytometry particle sorter equipped with deflection plates 1220a and 1220b. Experimental setup 1210 allows for the replacement of conventional angled deflection plates, such as deflection plate 105, with deflection plates according to an embodiment of the present invention configured for high-angle deflection of droplets. Experimental setup 1210 was used to baseline two-way, four-way, and six-way deflections with a 100 micron nozzle at three different deflection plate voltages (i.e., three different voltages applied to deflection plates 1220a and 1220b). Due to limitations of experimental setup 1210 (e.g., the possibility that deflected droplets of an embodiment of the present invention may overshoot available receiving wells), the droplet charge of the deflected droplets was used as a proxy for the total deflection distance. In this manner, experimental setup 1210 was used to compare the performance of conventional deflection plates against deflection plates according to embodiments of the present invention optimized in one dimension and deflection plates according to embodiments of the present invention optimized in two dimensions. Figure 12B is another view of experimental setup 1210 with conventional angled deflection plates 1220a, 1220b attached.

[0175] FIG. 12C shows experimental droplet deflection results comparing conventional angled deflection plates (e.g., deflection plates 111a and 111b) ("S8") with one-dimensional high-angle deflection plates (e.g., deflection plate 1005b) ("AD") according to embodiments of the present invention. As can be seen, across all deflection positions (LLL, LL, L, R, RR, RRR), the droplet charge required to strike a different displacement location (i.e., the droplet charge required to displace a droplet into a tube or well of a multiwell plate) is reduced for each of the three voltages applied to the deflection plates ("S8 50" and "AD 50"; "S8 70" and "AD 70"; "S8 90" and "AD 90"). The effect is an approximately 5-10% reduction in droplet charge. These results demonstrate the improved performance of the one-dimensionally optimized prototype embodiment of the present invention relative to conventional angled deflection plates.

[0176] Figure 12D is another view of experimental setup 1210 with deflection plates 1230a, 1230b according to an embodiment of the invention, in this case with a twisted shape optimized in two dimensions. Figure 12E is another view of experimental setup 1210 with deflection plates 1230a, 1230b according to an embodiment of the invention, with a twisted shape optimized in two dimensions. Figure 12F is another view of experimental setup 1210 with deflection plates 1230a, 1230b according to an embodiment of the invention, with a twisted shape optimized in two dimensions. Figure 12G shows an exemplary multiwell plate 1240 used in conjunction with experimental setup 1210 for receiving deflected droplets at different locations.

[0177] FIG. 12H shows experimental droplet deflection results comparing conventional angled deflection plates (e.g., deflection plates 111a and 111b) ("S8") with a twisted, two-dimensional high-angle deflection plate (e.g., deflection plate 1005c) ("Twister" or "TW") according to embodiments of the present invention. As can be seen from the results, across deflection positions (R, RR), the droplet charge required to strike a different displacement location (i.e., the droplet charge required to displace a droplet into a tube or well of a multiwell plate) decreases for each of the three voltages applied to the deflection plate ("S8 50" and "TW 50"; "S8 70" and "TW 70"; "S8 90" and "TW 90"). These results demonstrate the continued improved performance of a prototype embodiment of a two-dimensionally optimized twisted high-angle deflection plate according to embodiments of the present invention compared to conventional angled deflection plates.

[0178] Regardless of the scope of the appended claims, the present invention may be further defined by the following notes.

[0179] Appendix 1. A droplet deflector comprising a deflection plate configured for high angle deflection of a droplet flow stream.

[0180] Clause 2. The droplet deflector of clause 1, wherein the deflection plate has a shape corresponding to a path of the deflected droplet flow stream.

[0181] Clause 3. The droplet deflector of clause 1 or 2, wherein the deflection plate has a shape that minimizes the distance between the deflected droplet flow stream and the deflection plate of the droplet deflector over the length of the deflection plate.

[0182] Appendix 4. A droplet deflector according to any one of appendices 1 to 3, wherein the deflection plate has a shape that maintains a constant buffer region between the deflected droplet flow stream and the deflection plate of the droplet deflector over the length of the deflection plate.

[0183] Clause 5. The droplet deflector of any one of clauses 1-4, wherein the deflection plate is configured to maximize the deflection force applied to the droplet flow stream at a plurality of different downstream locations.

[0184] Clause 6. The droplet deflector of any one of clauses 1 to 5, wherein the deflection plate has a shape configured to prevent the deflected droplet stream from colliding with the deflection plate.

[0185] Clause 7. The droplet deflector of any one of clauses 1-6, wherein the distance between the deflection plates is configured to minimally increase at each of a plurality of downstream positions.

[0186] Appendix 8. The droplet deflector of any one of appendices 1 to 7, wherein the deflection plate has a non-linear surface.

[0187] Appendix 9. A droplet deflector according to any one of appendices 1 to 8, wherein the deflection plate has a splined shape.

[0188] Clause 10. The droplet deflector of any one of clauses 1-9, wherein the deflection plate is configured to apply a deflection force to the flow stream at a plurality of different angles.

[0189] Clause 11. The droplet deflector of clause 10, wherein the deflection plates are twisted.

[0190] Clause 12. The droplet deflector of clause 11, wherein the twist angle is 5 degrees or greater.

[0191] Clause 13. The droplet deflector of clause 11, wherein the twist angle is 30 degrees or greater.

[0192] Clause 14. The droplet deflector of clause 11, wherein the twist angle is 60 degrees or greater.

[0193] Clause 15. The droplet deflector of any one of clauses 1-14, configured to apply a constant deflection force to the deflected droplet flow stream from a plurality of different lateral positions.

[0194] Clause 16. The droplet deflector of clause 15, wherein the different lateral positions are different distances from the centerline of the deflector plate.

[0195] Clause 17. The droplet deflector of clause 15 or 16, wherein the distance between the deflector plates increases at different downstream positions.

[0196] Clause 18. The droplet deflector of any one of clauses 15 to 17, configured so that different voltage potentials are applied to the deflector plates at different downstream locations.

[0197] Clause 19. The droplet deflector of any one of clauses 15-18, wherein each deflector plate has two or more segments.

[0198] Clause 20. The droplet deflector of clause 19, wherein the segmented deflector plates have aligned corresponding segments.

[0199] Clause 21. The droplet deflector of clause 19 or 20, wherein each segment is electrically isolated from the other segments.

[0200] Clause 22. The droplet deflector of any one of clauses 19-21, wherein each segment of the segmented deflector plate is configured to receive a different electrical potential.

[0201] Addendum 23. The droplet deflector of any one of Addendums 19 to 22, wherein each segment is separated from the other segments by an electrical insulator.

[0202] Clause 24. The droplet deflector of any one of clauses 19 to 23, further comprising a plurality of resistors connected in series.

[0203] Clause 25. The droplet deflector of clause 24, wherein each resistor of the plurality of resistors is electrically connected to a segment of the segmented deflector plate.

[0204] Clause 26. The droplet deflector of any one of clauses 19 to 25, wherein each segment is separated from the other segments by a resistive dielectric.

[0205] Clause 27. The droplet deflector of any one of clauses 19-26, wherein each segment of the segmented deflector plate is configured to receive a different magnitude of electrical potential based on the distance between corresponding segments of the electrode.

[0206] Clause 28. The droplet deflector of any one of clauses 19-27, further comprising a voltage source operably connected to the deflector plates.

[0207] Clause 29. The droplet deflector of clause 28, further comprising a voltage divider having a plurality of resistors connected in series.

[0208] Clause 30. The droplet deflector of clause 29, wherein each resistor of the plurality of resistors is electrically connected to a segment of the segmented deflector plate.

[0209] Clause 31. The droplet deflector of any one of clauses 1-30, configured to apply a deflection force sufficient to deflect particles by 5 mm or more.

[0210] Clause 32. The droplet deflector of any one of clauses 1-31, configured to apply a deflection force sufficient to deflect particles by 15 mm or more.

[0211] Clause 33. The droplet deflector of any one of clauses 1-32, configured to apply a deflection force sufficient to deflect particles by 30 mm or more.

[0212] Clause 34. The droplet deflector of any one of clauses 1 to 33, configured to apply a deflection force sufficient to deflect particles within a range of 5 mm to 100 mm.

[0213] Clause 35. The droplet deflector of any one of clauses 1-34, configured to apply a deflection force sufficient to deflect particles by 30 degrees or more.

[0214] Clause 36. The droplet deflector of any one of clauses 1-35, configured to deposit droplets of the deflected droplet flow stream into each well in a column of a 96-well plate.

[0215] Appendix 37. The droplet deflector of any one of appendices 1 to 36, wherein the deflection plate comprises a metal plate.

[0216] Clause 38. A droplet deflector according to any one of clauses 1 to 37, wherein the deflector plates have a width in the range of 0.5 mm to 10 mm.

[0217] Clause 39. The droplet deflector of any one of clauses 1 to 38, wherein the deflector plate has a length in the range of 1 mm to 25 mm.

[0218] Clause 40. The droplet deflector of any one of clauses 1 to 39, wherein the deflector plates are spaced at least 1 mm apart.

[0219] Clause 41. The droplet deflector of any one of clauses 1 to 40, wherein the deflector plates are spaced at least 3 mm apart.

[0220] Clause 42. The droplet deflector of any one of clauses 1 to 41, wherein the electrodes are spaced apart at intervals in the range of 1 mm to 10 mm.

[0221] Clause 43. The droplet deflector of any one of clauses 1 to 42, wherein the deflector plate is rectangular.

[0222] Clause 44: A flow cell nozzle having a nozzle orifice configured to direct a flow stream through the flow cell nozzle; 44. A droplet deflector having a deflection plate configured for high-angle deflection of a droplet flow stream according to any one of claims 1 to 43. a particle sorting module comprising:

[0223] Clause 45. The particle sorting module of clause 44, further comprising a sample interrogation region in fluid communication with the orifice of the flow cell nozzle.

[0224] Clause 46. The particle sorting module of clause 45, further comprising a cuvette disposed in the sample interrogation region.

[0225] Clause 47. The particle sorting module of any one of clauses 44-46, further comprising a sample inlet in fluid communication with the flow cell nozzle.

[0226] Clause 48. The particle sorting module of any one of clauses 44-47, further comprising a sheath fluid inlet in fluid communication with the flow cell nozzle.

[0227] Clause 49. The particle sorting module of any one of clauses 44-48, further comprising a sample collection vessel of clause 2 or more.

[0228] Appendix 50. Light source and a flow cell nozzle having a nozzle orifice configured to direct a flow stream through the flow cell nozzle; a detector for measuring light of one or more wavelengths; 44. A droplet deflector having a deflection plate configured for high-angle deflection of a droplet flow stream according to any one of claims 1 to 43. A system comprising:

[0229] Clause 51. The system of clause 50, further comprising a sample interrogation region in fluid communication with the orifice of the flow cell nozzle.

[0230] Clause 52. The system of clause 50 or 51, further comprising a cuvette disposed in the sample interrogation region.

[0231] Clause 53. The system of any one of clauses 50-52, further comprising a sample inlet in fluid communication with the flow cell nozzle.

[0232] Clause 54. The system of any one of clauses 50-53, further comprising a sheath fluid inlet in fluid communication with the flow cell nozzle.

[0233] Clause 55.2 or more of the system of any one of clauses 50-54, further comprising a sample collection container.

[0234] Addendum 56. The system of any one of Addendums 50 to 55, wherein the light source is a laser.

[0235] Clause 57. Illuminating a sample containing particles in a flow stream using a light source; detecting light of one or more wavelengths; 44. A method of sorting particles in a sample into two or more sample collection vessels using a droplet deflector having deflection plates configured for high-angle deflection of a droplet flow stream according to any one of claims 1 to 43.

[0236] Clause 58. The method of clause 57, wherein the deflection plate comprises a split deflection plate.

[0237] Clause 59. The method of clause 58, wherein different segments of the split deflector plate are provided with different voltage potentials.

[0238] Item 60. The method of any one of items 57-59, wherein the flow stream is irradiated with a laser.

[0239] Although the foregoing disclosure has been described in some detail by way of illustration and example for ease of understanding, it will be readily apparent to those skilled in the art, in view of the teachings of the present disclosure, that certain changes and modifications may be made without departing from the spirit and scope of the appended claims.

[0240] Thus, the foregoing merely illustrates the essence of the present disclosure. It is clear that those skilled in the art will be able to devise various configurations that embody the essence of the present disclosure and are within the spirit and scope of the present disclosure, even though not explicitly described or shown herein. Furthermore, all examples and conditional language set forth herein are intended essentially to aid the reader in understanding the essence of the disclosure and the concepts provided by the inventors to advance the art, and should not be construed as limiting the examples and conditions specifically set forth. Furthermore, all statements herein that describe the essence, aspects, and embodiments of the present disclosure, as well as specific examples of the present disclosure, are intended to encompass both structural and functional equivalents of the present disclosure. Additionally, such equivalents are intended to include both currently known equivalents and future-developed equivalents, i.e., all elements developed that perform the same function, regardless of structure. Furthermore, the descriptions disclosed herein are not intended to be publicly disclosed, regardless of whether such disclosure is explicitly recited in the claims.

[0241] Accordingly, it is not intended that the scope of the present disclosure be limited to the exemplary embodiments shown and described herein. Rather, the scope and spirit of the present disclosure are embodied by the appended claims. With respect to claims, 35 U.S.C. 112(f) or 35 U.S.C. 112(6) are expressly provided to be invoked with respect to a limitation in a claim only when the precise phrase "means for" or "step for" appears at the beginning of such limitation in the claim; if such precise phrase is not used in a claim limitation, 35 U.S.C. 112(f) or 35 U.S.C. 112(6) is not invoked.

[0242] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority under 35 U.S.C. §119(e) of the filing date of U.S. Provisional Patent Application No. 63 / 658,746, filed June 11, 2024, the entire disclosure of which is incorporated herein by reference.

Claims

1. A droplet deflector comprising a deflection plate configured for high angle deflection of a droplet flow stream.

2. The droplet deflector of claim 1 , wherein the deflection plate has a shape corresponding to a path of a deflected droplet flow stream.

3. 3. A droplet deflector as claimed in claim 1 or 2, wherein the deflection plates have a shape that minimizes the distance between the deflected droplet flow stream and the deflection plates of the droplet deflector over the length of the deflection plates.

4. 4. A droplet deflector as described in any one of claims 1 to 3, wherein the deflection plate has a shape that maintains a constant buffer area between the deflected droplet flow stream and the deflection plate of the droplet deflector over the length of the deflection plate.

5. The droplet deflector of any one of claims 1 to 4, wherein the deflection plates are configured to maximize the deflection force applied to the droplet flow stream at a plurality of different downstream locations.

6. A droplet deflector according to any one of claims 1 to 5, wherein the deflection plate has a shape configured to prevent a deflected droplet flow stream from colliding with the deflection plate.

7. A droplet deflector according to any preceding claim, wherein the distance between the deflection plates is configured to increase minimally at each of a plurality of downstream positions.

8. A droplet deflector according to any one of claims 1 to 7, wherein the deflection plate has a non-linear surface.

9. A droplet deflector according to any one of claims 1 to 8, wherein the deflection plates have a splined shape.

10. A droplet deflector according to any one of claims 1 to 9, wherein the deflector plate is configured to apply a deflection force to the flow stream at a plurality of different angles.

11. A droplet deflector according to any one of claims 1 to 10, configured to apply a constant deflection force to the deflected droplet flow stream from a plurality of different lateral positions.

12. The droplet deflector of claim 11 , wherein different lateral positions are different distances from a centerline of the deflector plates.

13. 13. A droplet deflector according to claim 11 or 12, wherein each deflection plate has two or more segments.

14. A light source and a flow cell nozzle having a nozzle orifice configured to direct a flow stream through the flow cell nozzle; a detector for measuring light of one or more wavelengths; A droplet deflector according to any one of claims 1 to 13, comprising deflection plates configured for high-angle deflection of a droplet flow stream; A system comprising:

15. illuminating a sample containing particles in the flow stream with a light source; detecting light of one or more wavelengths; 14. A method for sorting particles in a sample into two or more sample collection vessels using a droplet deflector having deflection plates configured for high-angle deflection of a droplet flow stream according to any one of claims 1 to 13.

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