Flow cytometer with adjustable position offset sort deflection plate and methods of use thereof

Abstract

Aspects of the present disclosure include particle sorters having droplet deflectors configured to apply a known offset deflection force to a stream of droplets. Particle sorters according to certain embodiments include a flow cell, a light source (e.g., a laser) for illuminating an interrogation point of the flow cell, a detector for detecting light from the interrogation point, a droplet generator for generating a stream of droplets from a fluid flowing out of the flow cell, and a droplet deflector configured to apply a known offset deflection force to the stream of droplets. In certain cases, the droplet deflector includes a first plate and a second plate configured to be offset from one another. Methods and particle sorting particle modules for applying a known offset deflection force are also provided.

Description

[0001] Cross-reference to related applications

[0002] Pursuant to 35 U.S.SC §119(e), this application claims priority to the filing date of U.S. Provisional Patent Application Serial No. 63 / 041,597, filed on June 19, 2020, the disclosure of which is incorporated herein by reference in its entirety. Background Technology

[0003] Flow-type particle sorting systems, such as sorting flow cytometers, are used to sort particles in a fluid sample based on at least one measurement characteristic of the particles. In a flow-type particle sorting system, particles (e.g., molecules, analyte-bound beads, or single cells) in a fluid suspension pass through a detection region in which a sensor detects particles contained in the flow of a type to be sorted. Upon detection of particles of the type to be sorted, the sensor triggers a sorting mechanism that selectively separates particles of interest. The sorted particles of interest are separated into partitions, such as sample containers, test tubes, or wells in a multi-well plate.

[0004] Particle sensing is typically performed by flowing fluid through a detection area where particles are exposed to light from one or more lasers, and the light scattering and fluorescence properties of the particles are measured. Particles or their components can be labeled with fluorescent dyes for easy detection, and by using fluorescent dyes with different spectra to label different particles or components, multiple different particles or components can be detected simultaneously. Detection is performed using one or more photoelectric sensors to independently measure the fluorescence of each different fluorescent dye.

[0005] To sort particles in a sample, a droplet charging mechanism charges droplets containing the types of particles to be sorted in the flow at the breakpoint of the flow. The droplets are deflected into one or more zones, such as sample collection containers, by an electrostatic field and based on the polarity and size of the charge on the droplets. Uncharged droplets are not deflected by the electrostatic field and are collected by the container along the longitudinal axis of the flow. Summary of the Invention

[0006] Aspects of this disclosure include a particle sorter with a droplet deflector configured to apply a known deflection force to a droplet stream. A particle sorter according to some embodiments includes a flow cell, a light source (e.g., a laser) for illuminating an interrogation point of the flow cell, a detector for detecting light from the interrogation point, a droplet generator for generating a droplet stream from fluid flowing out of the flow cell, and a droplet deflector configured to apply a known deflection force to the droplet stream.

[0007] The particle sorter according to some embodiments includes a droplet deflector having a first plate and a second plate configured to be offset from each other. In embodiments, the first and second plates of the droplet deflector are configured to be adjustablely offset from each other. In some embodiments, the first and second plates are configured to be adjustablely offset from each other relative to a horizontal plane. In such embodiments, the horizontal plane may be perpendicular to the axis of the droplet flow. In some cases, the first plate includes an extension portion configured to allow the first plate to be adjustablely offset relative to the horizontal plane relative to the second plate. In some cases, the extension portion of the first plate includes a positioning screw configured to allow the first plate to be adjustablely offset relative to the horizontal plane relative to the second plate.

[0008] In some examples, the first and second plates are configured to be adjustablely offset from each other by more than 0 mm to 5 mm or more. In other examples, the first and second plates are configured to be adjustablely offset from each other in increments determined by the thread pitch of the locating screws used to adjust the offset.

[0009] In some embodiments, the known deflection force is sufficient to offset the droplet deposition location by 2 mm or more (e.g., when such offset is measured at a distance of 140 mm below the lowest point of the first deflection plate). In other cases, the known deflection force is sufficient to offset the droplet deposition location by one droplet diameter or less (e.g., when such offset is measured at a distance of 140 mm below the lowest point of the first deflection plate).

[0010] In embodiments, the particle sorter may further include multiple partitions configured to receive droplets deflected by droplet deflectors. In some embodiments, the partitions include collection containers. In an example, the collection container is a multi-well plate. In some cases, the multi-well plate contains 1536 or fewer wells. In some cases, the partitions include collection tubes. In an example, each partition has a diameter of 1.8 mm or less.

[0011] In one embodiment, the first plate and the second plate are configured to be parallel to each other. In another embodiment, the first plate and the second plate are configured to be adjustable to face each other.

[0012] In some embodiments, the second plate includes an elongated portion configured to allow the second plate to be adjustably offset relative to the horizontal plane relative to the first plate. In some cases, the elongated portion of the second plate includes a locating screw configured to allow the second plate to be adjustably offset relative to the horizontal plane relative to the first plate.

[0013] In embodiments, the droplet deflector may include an actuator, such as a motor, configured to adjust the offset between a first plate and a second plate. In some embodiments, the actuator (e.g., a motor) is operatively connected to a feedback subsystem. In an example, the feedback subsystem includes a controller operatively connected to the actuator (e.g., the motor) and to a detector configured to detect whether droplets in the droplet stream have deviated by a distance. In some cases, the feedback subsystem is configured to iteratively adjust the offset between the first plate and the second plate.

[0014] In some embodiments, the first and second plates are metal. In one example, the metal plates are spaced 1 mm or more apart. In other examples, the metal plates are spaced 3 mm or more apart. In some cases, the first and second plates are rectangular.

[0015] A method for deflecting droplets using a known offset deflection force is also provided. The method according to some embodiments includes illuminating an interrogation point of a flow cell with a light source, detecting the light from the interrogation point with a detector, generating a droplet stream from fluid flowing out of the flow cell using a droplet generator, and deflecting the droplets of the droplet stream with a droplet deflector configured to apply a known offset deflection force to the droplet stream.

[0016] This disclosure also includes a particle sorting module configured to apply a known offset deflection force to the droplet stream. According to some embodiments, the particle sorting module includes a droplet deflector configured to apply a known offset deflection force to the droplet stream. In some embodiments, the droplet deflector includes a first plate and a second plate configured to be offset from each other.

[0017] Embodiments of the present invention address the problem of insufficient ability in current flow chamber sorters to finely adjust the droplet deposition position (near or far from the flow position) in the horizontal plane. Embodiments of the present invention provide droplet positioning for microplates of 1536 wells, and positioning adjustment for small collection tubes or other containers. Embodiments of the present invention address the problem of sorting and collecting devices that are rectangular and not located at a precise right angle to the horizontal plane of the sorting deflection flow. Attached Figure Description

[0018] The invention will be best understood from the following detailed description when read in conjunction with the accompanying drawings. The drawings include the following figures:

[0019] Figure 1 A schematic droplet deflector of a particle sorter according to the present disclosure is depicted.

[0020] Figure 2 An embodiment of the first plate according to the present disclosure is depicted, wherein the first plate is configured to be adjustablely offset relative to the second plate (the offset relative to the second plate is not shown).

[0021] Figure 3 A particle sorter according to an embodiment of the present disclosure is depicted, comprising an offset between a first plate and a second plate on a front-to-back axis in a horizontal plane.

[0022] Figure 4A A schematic diagram of a particle sorter according to certain embodiments is depicted.

[0023] Figure 4B A schematic diagram of a particle sorter according to certain embodiments is depicted.

[0024] Figure 5 The effect of applying a known offset deflection force to the droplets of a droplet stream is depicted in an embodiment of the particle sorter according to the present disclosure, by showing the droplet deposition location when a deflection force is applied with a known offset of varying degrees.

[0025] Figure 6 A flow cytometer according to certain embodiments is described. Detailed Implementation

[0026] This disclosure includes a particle sorter with a droplet deflector configured to apply a known deflection force to a droplet stream. According to some embodiments, the particle sorter includes a flow cell, a light source (e.g., a laser) for illuminating an interrogation point of the flow cell, a detector for detecting light from the interrogation point, a droplet generator for generating a droplet stream from fluid flowing out of the flow cell, and a droplet deflector configured to apply a known deflection force to the droplet stream. Methods for applying the known deflection force and particle sorting modules are also provided.

[0027] Before describing the invention in more detail, it should be understood that the invention is not limited to the specific embodiments described, as these can certainly be varied. It should also be understood that, because the scope of the invention will be limited only by the appended claims, the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0028] Where numerical ranges are provided, it should be understood that each intermediate value to one-tenth of the lower limit unit, unless the context explicitly specifies otherwise, is included in this invention as the range between the upper and lower limits of that range and any other specified or intermediate value. The upper and lower limits of these smaller ranges may be independently included within the smaller ranges and also within this invention, but are subject to any specific exclusions within the ranges. Where the range includes one or both limitations, the range excluding one or both of those included limitations is also included in this invention.

[0029] Certain ranges presented herein are preceded by the term "approximately". The term "approximately" is used herein to provide literal support for the exact number that follows it, as well as to indicate that it is close to or approximate to the number following the term. In determining whether a number is close to or approximate to a specifically cited number, a number that is close to or approximate to an uncited number can be a number that provides a substantial equivalent of the specifically cited number in the context in which it appears.

[0030] 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 invention pertains. Although any methods and materials similar to or equivalent to those described herein may also be used in the practice or testing of this invention, representative illustrative methods and materials are described hereafter.

[0031] All publications and patents referenced in this specification are incorporated herein by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference, and are incorporated by reference to disclose and describe the methods and / or materials relating to the referenced publications. Reference to any publication is for publication prior to the filing date and should not be construed as an admission that the present invention is not entitled to prior publication of such publications based on prior art. Furthermore, the publication dates provided may differ from the actual publication dates and may require independent verification.

[0032] It should be noted that, unless the context clearly specifies otherwise, as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural indicators. It should also be noted that claims can be drafted to exclude any optional elements. Therefore, this statement is intended to serve as a preliminary basis for the use of exclusive terms such as “unique,” ​​“only,” etc., when referencing claim elements or using the “negative” limitation.

[0033] As will be apparent to those skilled in the art upon reading this disclosure, each individual embodiment described and illustrated herein has discrete components and features that can be readily separated from or combined with features of any of the other several embodiments without departing from the scope or spirit of the invention. Any enumerated methods may be performed in the order of the enumerated events or in any other logically possible order.

[0034] Although the device and method have been or will be described for grammatical fluency and functional interpretation, it should be clearly understood that, unless expressly stated in accordance with 35 U.SC §112, the claims should not be construed as necessarily being limited in any way by the interpretation of “means” or “steps,” but should be given the full scope of meaning and equivalents provided by the definition provided by the claims under the principle of equivalence, and, where the claims are expressly stated, will acquire the full statutory equivalence provided in 35 U.SC §112.

[0035] As described above, this disclosure provides a particle sorter including a droplet deflector configured to apply a known offset deflection force to a droplet stream. In embodiments further described in this disclosure, the particle sorter has a first and second plate configured offset from each other, adjustable plates, partitions configured to accommodate deflected droplets and adjusted by an actuator (e.g., a motor), and a feedback subsystem is described first in more detail. Next, a method for deflecting droplets using a known offset deflection force is described. A particle sorting module is also described.

[0036] Particle sorter

[0037] Aspects of this disclosure include a particle sorter with a droplet deflector configured to apply a known deflection force to a droplet stream. Specifically, the particle sorter according to certain embodiments includes a flow cell, a light source (e.g., a laser) for illuminating an interrogation point of the flow cell, a detector for detecting light from the interrogation point, a droplet generator for generating a droplet stream from fluid flowing out of the flow cell, and a droplet deflector configured to apply a known deflection force to the droplet stream.

[0038] The term “deflection” is used in its conventional sense in this article, referring to the force applied that causes a droplet in a flow to deviate from its normal trajectory (i.e., in the absence of a deflection force) to a different trajectory.

[0039] Applying an "offset deflection force" means applying a deflection force to a droplet in a droplet stream that deviates from the standard side-to-side orientation (i.e., in a direction different from the direction in which the deflection force is applied without offset). In other words, the direction of the deflection force relative to the direction of the deflection force before the offset may be tilted. For example, the orientation of the droplet to which the deflection force is applied may be offset in a horizontal plane orthogonal to the longitudinal axis of the droplet stream. Therefore, without offset, the deflection force will be applied to the droplet stream only in the "side-to-side" direction in the horizontal plane. With offset, the deflection force can then include both "side-to-side" and "front-to-back" components. That is, if an xyz coordinate system is superimposed on the droplet deflector, a standard droplet deflector will apply a deflection force only along the x-axis of the coordinate system. Conversely, a droplet deflector configured to apply a known offset deflection force will apply a deflection force with directional components in both the x and y axes. In some cases, the deflection force is deflected by applying the deflection force after the droplet flow has been oriented by rotating around the longitudinal axis of the droplet flow.

[0040] Applying a "known offset deflection force" means applying a deflection force to the droplets in the droplet stream that deflects them by a designed or predetermined amount. That is, in some cases, a "known offset deflection force" is a deflection force that deflects the expected amount.

[0041] As described in more detail herein, the target particle sorter according to certain embodiments provides droplet deflectors including a first plate and a second plate configured to be offset from each other. In other embodiments, the target particle sorter according to certain embodiments provides a first plate and a second plate configured to be adjustablely offset from each other. When sorting samples, sorting efficiency is improved by using a target particle sorter configured to apply a known offset deflection force to the droplet flow, thereby reducing the waste of sample particles (e.g., by carefully deflecting droplets containing target particles, such as cells, to unintended locations, such as locations outside the intended wells of a multi-well plate). In some cases, sorting efficiency can be improved such that when using a target particle sorter and method, the sorting efficiency can be increased to allow the collection and sorting of more particle variants or more quantities of particles corresponding to each type of sorted particle. When used as part of flow cytometry for sample sorting, the target method can improve the yield of particle sorting.

[0042] In embodiments of the particle sorter according to this disclosure, droplets in a droplet stream can be deflected by a known offset deflection force along the longitudinal axis of the droplet stream from their normal trajectory by a distance of 0.001 mm or more, as measured radially through a plane orthogonal to the longitudinal axis of the droplet stream (such that such radial measurement reflects the known offset—i.e., composed of both the x-axis component and the y-axis component relative to the overlapping xyz plane), for example 0.005 mm or more, for example 0.01 mm or more, for example 0.05 mm or more, for example 0.1 mm or more, for example 0.5 mm or more, for example 1 mm or more, for example 2 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, for example 30 mm or more, for example 35 mm or more, and including 50 mm or more. For example, droplets in a droplet stream can travel distances from 0.001 mm to 100 mm, such as 0.005 mm to 95 mm, 0.001 mm to 90 mm, 0.05 mm to 85 mm, 0.01 mm to 80 mm, 0.05 mm to 75 mm, 0.1 mm to 70 mm, 0.5 mm to 65 mm, 1 mm to 60 mm, 5 mm to 55 mm, and including 10 mm to 50 mm.

[0043] The particle sorter according to embodiments of this disclosure can be configured to sort particles in a sample, such as cells in a biological sample. In these embodiments, the droplet deflector of the particle sorter is configured to apply a known deflection force sufficient to deflect droplets into one or more sample collection containers. Thus, the droplet deflector can be configured to apply a known deflection force such that droplets are deflected into sample collection containers at a distance of 0.001 mm or more from the longitudinal axis of the droplet flow, for example, 0.005 mm or more, 0.01 mm or more, 0.05 mm or more, 0.1 mm or more, 0.5 mm or more, 1 mm or more, 2 mm or more, 5 mm or more, 10 mm or more, 15 mm or more, 20 mm or more, 25 mm or more, 30 mm or more, 35 mm or more, and including 50 mm or more. For example, a droplet deflector can be configured to deflect a droplet into a sample collection container, which is moved from a distance of 0.001 mm to 100 mm from the longitudinal axis of the flow, for example, 0.005 mm to 95 mm, 0.001 mm to 90 mm, 0.05 mm to 85 mm, 0.01 mm to 80 mm, 0.05 mm to 75 mm, 0.1 mm to 70 mm, 0.5 mm to 65 mm, 1 mm to 60 mm, 5 mm to 55 mm, and including 10 mm to 50 mm.

[0044] As described above, the particle sorter according to the embodiments includes a droplet deflector configured to deflect droplets in a flow stream by applying a known offset deflection force to the droplet stream. In some cases, the different offset values ​​of this disclosure can be described based on the angle formed between the direction of the applied deflection force and a line representing the intersection of the horizontal plane and the droplet deflector plate. This angle describing the offset deflection force can vary depending on the structural configuration of the target droplet deflector of the particle sorter, as described in more detail below, and can vary 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°, and including 30° to 50°. In other cases, the different known offset deflection forces of this disclosure can be described based on the degree of orientation of the deflection force rotating about the longitudinal axis of the droplet stream before the application of the known offset deflection force. The rotation angle describing the known deflection force can vary depending on the structural configuration of the target droplet deflector of the particle sorter and can range from 0.01° to 360°, for example 0.05° to 355°, for example 0.1° to 350°, for example 0.5° to 300°, for example 10° to 270°, for example 15° to 135°, for example 20° to 90°, for example 25° to 75° and including 30° to 50°.

[0045] In embodiments described in this disclosure, the droplet deflector includes a first plate and a second plate configured to be offset from each other. By offsetting the first plate and the second plate from each other, in some cases, this means that when the two plates are offset, they no longer face each other directly and immediately, and the droplet flow remains in the middle position between the two plates. In other words, the faces of the two plates are no longer horizontally opposite each other in the Y-axis plane. In some cases, as described above, the two plates are offset by moving one plate along the front-back axis (i.e., the Y-axis). In some cases, the different offset values ​​of the first plate and the second plate described in this disclosure can be described based on the distance the plates are offset along the front-back axis (i.e., the Y-axis). This distance describing the offset between the plates can vary depending on the structural configuration of the target droplet deflector of the particle sorter and can range from 0.01 mm to 10 mm or greater, for example, 0.05 mm to 9.9 mm, 0.1 mm to 9 mm, 0.5 mm to 7.5 mm, 0.1 mm to 6 mm, 1.5 mm to 5 mm, 2 mm to 4 mm, and including 2.5 mm to 3.5 mm. In some cases, the different offsets of the first and second plates described in this disclosure can be described by the angle formed between the line connecting the midpoint of the first plate and the midpoint of the second plate when the plates face each other directly and without gaps (i.e., before offsetting the first and second plates) and the line formed between the midpoints of the first and second plates after offsetting the plates. The angle of offset between the descriptors can vary depending on the structural configuration of the target droplet deflector of the particle sorter, as described in more detail below, and can vary 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°, and includes 30° to 50°.

[0046] In some cases, the first and second plates are configured to be adjustablely offset from each other. Adjustable offset from each other means that the amount of offset between the first and second plates is dynamically configurable. In this case, the first and second plates can be adjusted to increase or decrease the desired offset between them. In this scenario, the offset can be repeatedly adjusted iteratively within a range of offset values, and in doing so, converge empirically to the desired offset value.

[0047] As described above, in some embodiments, the first and second plates are configured to be adjustablely offset from each other relative to a horizontal plane. The horizontal plane may be parallel to the plane containing the collection container for the droplets—that is, the plane of the multi-well plate. In some cases, the horizontal plane is perpendicular to the longitudinal axis of the droplet flow. Perpendicular to the longitudinal axis of the droplet flow means that the horizontal plane is orthogonal to the longitudinal axis of the droplet flow. The longitudinal axis of the droplet flow is the axis along which the droplets flow when not affected by deflection forces.

[0048] Figure 1 A schematic droplet deflector of a particle sorter according to the present disclosure is depicted. The droplet deflector 100 includes a first plate 100a and a second plate 100b. The first plate 100a and the second plate 100b of the droplet deflector 100 are configured to be adjustablely offset from each other. In the illustrated embodiment, the offset is indicated by the available offset position of the first plate 100a along dashed line 105. The first plate 100a and the second plate 100b are configured to be adjustablely offset from each other relative to a horizontal plane 110 shown below the first plate 100a and the second plate 100b. The horizontal plane 110 is traversed by a "side-to-side" axis (i.e., the x-axis) 120 and a "front-to-back" axis (i.e., the y-axis) 130. In some embodiments, the droplet deposition position along the "side-to-side" axis 120 can be determined, for example, by a charge applied to the droplet and a voltage applied to the first plate 100a and the second plate 100b. In an embodiment, the droplet deposition position along the front-to-back axis 130 (i.e., the degree of droplet deposition position offset) can be determined by, for example, the amount of available offset position offset shown by the dashed line 105 along the first plate 100a. In some cases, velocity can be used to adjust the degree of deflection. For example, at lower flow rates, the droplet spends a longer time period in the deflection field of the plate, thereby increasing the influence of the field and increasing the deflection. In this case, a lower velocity can be used to achieve a greater deflection with a given plate configuration.

[0049] In an embodiment, the first plate may include an elongated portion configured to allow the first plate to be adjustablely offset relative to a horizontal plane relative to a second plate. The elongated portion can be any convenient construction of the first plate that allows for adjustable offset relative to the second plate. In an embodiment, the elongated portion may refer to a portion that extends along the length of an available offset position of the first plate. That is, as described above, the elongated portion may extend along a front-to-back axis (i.e., the y-axis). In some cases, the elongated portion may extend across the length of the available offset position of the first plate and may restrict movement of the first plate such that the position and orientation of the first plate can only be adjusted along the front-to-back axis. In an embodiment, the elongated portion may be a keyed opening designed to match the relative fixing device of the droplet deflector. This keyed opening may extend laterally along the available offset position, such that the first plate is offset by translating it along the length of the keyed opening.

[0050] In this embodiment, the extended portion of the first plate may include a positioning screw configured to allow the first plate to be adjustably offset relative to the second plate relative to a horizontal plane. The positioning screw can be any convenient screw and can be positioned in the first plate as needed to adjust its position, thereby offsetting the first plate relative to the second plate. In embodiments, the first plate may include a threaded hole through which the positioning screw is added, such that the end of the positioning screw protrudes through the threaded hole in the first plate. In some examples, the positioning screw may be positioned such that rotating the positioning screw in the first plate causes the first plate to offset in a forward or backward direction along a front-back axis (i.e., the y-axis). In examples, the positioning screw may be positioned such that rotating the positioning screw causes the end of the positioning screw to apply a force to the fixing device of the droplet deflector, such that, as a result of the applied force, the first plate also offsets along the front-back axis. Furthermore, both adjustable plates can have positioning screws that can be adjusted completely "inward" or completely "outward," allowing both plates to move forward or backward without offset to finely adjust the entire electrostatic field through which the droplet will pass if necessary. Locating screws can have any convenient length, diameter, and thread pitch. In some cases, locating screws may have fine threads for better, more precise adjustment of the offset position of the first plate.

[0051] Figure 2 A first plate 200 configured for adjustable offset according to this disclosure is depicted. The first plate 200 configured for adjustable offset according to this disclosure is shown by comparison with a standard plate 250 of a droplet deflector not configured for adjustable offset. As described above, the first plate 200 includes an extension 210 configured to allow the first plate 200 to be adjustablely offset relative to a second plate with respect to a horizontal plane. As described above, the extension 210 of the first plate 200 also includes a positioning screw 220 configured to allow the first plate to be adjustablely offset relative to a second plate with respect to a horizontal plane.

[0052] In embodiments of the particle sorter disclosed herein, the first plate and the second plate are configured to be adjustablely offset from each other by a factor greater than 0 mm to 5 mm or more, for example, 0.01 mm to 4.99 mm, 0.05 mm to 4 mm, 0.5 mm to 3.5 mm, 1 mm to 3 mm, and including 1.5 mm to 2.5 mm. In this embodiment, the first plate and the second plate are configured to be adjustablely offset from each other in increments determined by the thread pitch of a positioning screw used to adjust the offset between the first plate and the second plate. In instances where the positioning screw includes a fine thread pitch, the adjustment of the first plate and the second plate can be finer than the adjustment increments available when the positioning screw includes a less fine thread pitch. As described above, the first plate and the second plate can be adjustablely offset from each other relative to a horizontal plane in different increments. In some cases, the first plate may include an elongated portion and a positioning screw configured to adjustably offset the first plate relative to the second plate by the offset amounts and increments described above.

[0053] Figure 3 A particle sorter according to an embodiment of the present disclosure is depicted, as described above, including an offset between a first plate and a second plate in the horizontal plane along a front-to-back axis (i.e., the y-axis). The particle sorter 300 according to an embodiment of the present disclosure includes a first deflector plate 300a, which includes an extension portion 310 and a positioning screw 320, and is configured to be adjustablely offset relative to a second plate 300b, which is a standard deflector plate (i.e., it is not configured to be adjustablely offset according to the present disclosure). The front-to-back offset between the first plate 300a and the second plate 300b is depicted as the horizontal space between dashed lines 330. The horizontal offset 330 between the first and second plates illustrates how the first plate is offset in the rearward direction in the horizontal plane such that the resulting deflection force is a known offset deflection force, i.e., the deflection force includes a known offset in the front-to-back direction.

[0054] In some embodiments, the known deflection force is sufficient to offset the droplet deposition location by 2 mm or more. For example, when measured at a distance (e.g., 140 mm below the lowest point of the first plate), the droplet may be offset by 2 mm or more on the front-back axis. That is, when measured at a distance of 140 mm below the lowest point of the first plate of the droplet deflector, the droplet deflector can be configured to apply a known deflection force sufficient to deflect the droplets of the droplet stream by 2 mm or more on the front-back axis in the horizontal plane. Therefore, when measured at 140 mm below the lowest point of the first plate, the resulting offset of the droplet deposition location can be 2 mm or more. In other embodiments, the known deflection force is sufficient to offset the droplet deposition location by one droplet diameter or less. In other words, when measured at a distance below the lowest point of the first plate of the droplet deflector, the droplet deflector can be configured to apply a known deflection force to deflect the droplet flow along the front-back axis of the horizontal plane by only one droplet diameter or less. Therefore, when measured at a distance below the lowest point of the first plate, the resulting offset of the droplet deposition position can be only one droplet diameter or less.

[0055] In embodiments of the particle sorter according to this disclosure, the droplet deflector of the particle sorter is configured such that the first and second plates are metallic. The metal plates of the target particle sorter can be formed of any suitable metal capable of generating an electric field and may include, but are not limited to, 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-manganese-platinum alloys, cobalt alloys, cobalt-chromium alloys, cobalt-tungsten alloys, cobalt-molybdenum-carbon alloys, cobalt-nickel-molybdenum-iron-tungsten alloys, copper alloys, copper-arsenic alloys, copper-beryllium alloys, copper-silver alloys, copper-zinc alloys (e.g., brass), copper-tin alloys (e.g., bronze), copper-nickel alloys, copper-tungsten alloys, copper-gold-silver alloys, copper-nickel-iron alloys, copper-manganese-tin alloys, copper-aluminum-zinc-tin alloys, copper-gold alloys, gold alloys, gold-silver alloys, indium alloys, indium-tin alloys, indium-tin oxide alloys, iron alloys, and 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, titanium-nickel alloys, silver alloys, silver-copper alloys (e.g., pure silver), silver-cobalt-germanium alloys (e.g., pure silver), silver-gold alloys, silver-copper-gold alloys, silver-platinum alloys, tin alloys, tin-copper-antimony alloys, tin-lead-antimony alloys, tin-lead-antimony alloys, titanium alloys, titanium-vanadium-chromium alloys, titanium-aluminum alloys, titanium-aluminum-vanadium alloys, zinc alloys, zinc-copper alloys, zinc-aluminum-magnesium-copper alloys, zirconium alloys, zirconium-tin alloys, or combinations thereof.

[0056] In the embodiments described in this disclosure, a known deflection force is applied to the droplets in the droplet stream by applying a voltage to the first and second metal plates of the droplet deflector, resulting in an electric field between the first and second metal plates. This electric field between the first and second plates accelerates and diverts the trajectory of the charged target droplet from the longitudinal axis of the droplet stream. In this embodiment, the known deflection force generated by the electric field can accelerate and divert the trajectory of the target droplet from the longitudinal axis of the droplet stream to one or more sample collection containers. As described above, the voltage applied to the first and second plates to transfer the charged droplets can be 10 mV or higher, for example, 25 mV or higher, 50 mV or higher, 100 mV or higher, 250 mV or higher, 500 mV or higher, 750 mV or higher, 1000 mV or higher, 2500 mV or higher, 5000 mV or higher, 10000 V or higher, 15000 V or higher, 25000 V or higher, 50000 V or higher, or higher, and includes 100000 V or higher. In some embodiments, the voltage applied to the first and second metal plates is from 0.5 kV to 15 kV, for example, 1 kV to 15 kV, 1.5 kV to 12.5 kV, and includes 2 kV to 10 kV. In some embodiments, the voltage applied to the first and second metal plates is from 0.5 kV to 15 kV, for example, from 1 kV to 15 kV, from 1.5 kV to 12.5 kV, and includes 2 kV to 10 kV. Depending on the voltage applied to the first and second metal plates, the electric field strength between the metal plates can vary, ranging from 0.001 V / m to 1 × 10⁻⁶. 7 V / m, for example, 0.01V / m to 5×10 6 V / m, for example 0.1V / m to 1×10 6 V / m, for example 0.5V / m to 5×10 5 For example, 1V / m to 1×10 5 V / m, for example, 5V / m to 5×10 4 V / m, for example, 10V / m to 1×10 4 V / m and includes 50V / m up to 5×10 3 V / m, for example 1×10 5 ·V / m up to 2×10 6 V / m.

[0057] The first and second metal plates are spaced apart from each other by a distance sufficient to generate an electric field therebetween. For example, the first and second metal plates may be spaced 0.01 mm or more, such as 0.05 mm or more, 0.1 mm or more, 0.5 mm or more, 1 mm or more, 1.5 mm or more, 2 mm or more, 2.5 mm or more, 3 mm or more, 3.5 mm or more, 4 mm or more, 4.5 mm or more, 5 mm or more, 10 mm or more, 15 mm or more, 20 mm or more, and including 25 mm or more. In some cases, the distance between the first and second metal plates ranges from 0.01 mm to 50 mm, such as 0.05 mm to 45 mm, 0.1 mm to 40 mm, 0.5 mm to 35 mm, 1 mm to 30 mm, 1.5 mm to 25 mm, 2 mm to 20 mm, and including 3 mm to 15 mm.

[0058] In some embodiments of the particle sorter, the first and second plates of the droplet deflector are configured to be parallel to each other. That is, even when the first and second plates are offset from each other, the plane of the first plate is parallel to the plane of the second plate. In some instances, the first and second plates are configured to be adjustablely rotated to face each other. That is, in instances where the offset between the first and second plates can be adjusted, the orientation of the first and second plates can also be adjusted so that the first and second plates remain parallel to each other with different degrees of offset from each other. In some cases, the first plate or the second plate, or both plates, are rotated about their respective longitudinal axes (in some cases parallel to the longitudinal axis of the droplet flow) to be oriented to face each other.

[0059] In one embodiment, the second plate of the droplet deflector of the particle sorter includes an elongated portion configured to allow the second plate to be adjustablely offset relative to the first plate with respect to a horizontal plane. The elongated portion can be any convenient configuration of the second plate that allows for adjustable offset relative to the first plate. In one embodiment, the elongated portion can refer to a portion that extends along the length of an available offset position of the second plate. That is, as described above, the elongated portion can extend along a front-to-back axis. In some cases, the elongated portion can extend across the length of the available offset position of the second plate and can restrict the movement of the second plate such that the position and orientation of the second plate can only be adjusted along the front-to-back axis. In one embodiment, the elongated portion can be a keyed opening designed to match the relative fixing device of the droplet deflector of the particle sorter. This keyed opening can extend along a lateral range of the available offset position, such that the second plate is offset by translating it along the length of the keyed opening. In this embodiment, the elongated portion of the second plate can include a positioning screw configured to allow adjustable offset of the second plate relative to the first plate with respect to a horizontal plane. The positioning screw can be any convenient positioning screw and can be positioned in the second plate as needed to adjust the position of the second plate to offset it relative to the first plate in the horizontal plane. In embodiments, the second plate may include a threaded hole through which the positioning screw is added, such that the end of the positioning screw protrudes through the threaded hole in the second plate. In some examples, the positioning screw can be positioned such that rotating the positioning screw in the second plate causes the second plate to offset in a forward or backward direction along a front-back axis. In examples, the positioning screw can be positioned such that rotating the positioning screw causes the end of the positioning screw to apply a force to the stationary fixture of the droplet deflector, such that, as a result of the applied force, the second plate also offsets along a front-back axis. The positioning screw can have any convenient length, diameter, and thread pitch. In some cases, the positioning screw may have fine threads for better fine adjustment of the offset position of the second plate.

[0060] In some instances, the particle sorter according to this disclosure may be configured to further include an actuator (e.g., a motor) configured to adjust the offset between a first plate and a second plate. When the actuator is a motor, the motor can be integrated into the droplet deflector of the particle sorter in any convenient manner, enabling the motor to automatically adjust the offset between the first and second plates. In some cases, the motor may be attached directly or indirectly to a positioning screw via, for example, a gearing mechanism, such that rotation of the motor causes the positioning screw to rotate, thereby adjusting the offset between the first and second plates. Any convenient displacement protocol can be used as the motor configured to adjust the offset between the first and second plates. In some cases, the motor may be configured with an actuated translation stage, a lead screw translation assembly, or a gear transmission. The motor may include a stepper motor, a servo motor, a brushless motor, a brushed DC motor, a microstepping drive motor, a high-resolution stepper motor, and other types of motors.

[0061] In one embodiment, an actuator (e.g., a motor) is operatively connected to a feedback subsystem. The feedback subsystem can be any convenient system for automatically controlling the amount of adjustable offset between the first and second plates. In this embodiment, the feedback subsystem may include a controller operatively connected to the actuator (e.g., the motor) and to a detector configured to detect the distance of droplet displacement in the droplet flow. For example, as described above, the detector may be configured to detect the distance of droplet displacement, including the distance the droplet is offset by a particle sorter along the front-to-back axis in the horizontal plane. In this example, the detector may include any convenient camera system (e.g., a camera) configured to capture images of the droplet deposition location, and the controller may be any convenient controller (e.g., a microcontroller or microprocessor) configured to evaluate the droplet displacement based on the images received from the camera and adjust the offset between the first and second plates as needed to refine the offset of the droplet deposition location. That is, in some cases, the controller is configured by instructions stored in a memory operatively connected to the controller, which, when executed, cause the controller to adjust the offset between the first and second plates. In some examples, the feedback subsystem is configured to iteratively adjust the offset between the first and second plates. That is, the feedback subsystem can be configured to adjust the offset between the first and second plates multiple times, such that a known offset deflection force is iteratively adjusted, and correspondingly, the offset of the droplet deposition position is iteratively adjusted. Therefore, in some instances, the feedback subsystem can provide additional precision regarding the specific offset used to achieve the droplet deposition position. In this embodiment, the feedback subsystem can also be configured to use calibration particles (e.g., beads) added to the droplet stream to detect and measure the droplet offset. Such beads can include, for example, Accudrop beads, such as BD FACS. TM Accudrop beads.

[0062] The first and second plates of the droplet deflector in the target particle sorter can be any suitable shape, such as circular, elliptical, semi-circular, crescent-shaped, star-shaped, square, triangular, rhomboid, pentagonal, hexagonal, heptagonal, octagonal, rectangular, or other suitable polygons. In some embodiments, the first and second plates are rectangular.

[0063] In embodiments, the shape and size of the first plate may be the same as or different from the shape and size of the second plate. In some embodiments, the shape of the first plate is the same as the shape of the second plate (e.g., both are rectangular). In other embodiments, the shape of the first plate is different from the shape of the second plate (e.g., the first plate is a square, and the second plate is a rectangle). In some instances, the dimensions of the first plate are the same as the dimensions of the second plate. In one example, the width of the first plate is the same as the width of the second plate. In other instances, the length of the first plate is the same as the length of the second plate. In still other instances, the width and length of the first plate are the same as the width and length of the second plate. In some examples, the dimensions of the first plate are different from the dimensions of the second plate. In one example, the width of the first plate is different from the width of the second plate. In another example, the length of the first plate is different from the length of the second plate. In yet another example, both the width and length of the first plate are different from the width and length of the second plate.

[0064] The dimensions of the first and second plates can vary depending on their shapes. In some embodiments, the width of each of the first and second plates ranges from 0.5 mm to 10 mm, for example, 1 mm to 9.5 mm, 1.5 mm to 9 mm, 2 mm to 8.5 mm, 2.5 mm to 8 mm, 3 mm to 7.5 mm, 3.5 mm to 7 mm, 4 mm to 6.5 mm, and includes a width ranging from 4.5 mm to 6 mm. In some cases, the widths of the first and second plates are the same, while in others, the widths of the first and second plates are different. The lengths of the first and second plates vary from 10 mm to 500 mm, for example, 15 mm to 450 mm, 20 mm to 400 mm, 25 mm to 350 mm, 30 mm to 300 mm, 35 mm to 250 mm, 40 mm to 200 mm, 45 mm to 150 mm, and includes a length ranging from 50 mm to 100 mm. In some cases, the first and second plates are of the same length, while in others, they are of different lengths. In some embodiments, the first and second plates are asymmetrical polygons, with the width of the first end being smaller than the width of the second end. The width of each end can range from 0.01 mm to 10 mm, for example, 0.05 mm to 9.5 mm, 0.1 mm to 9 mm, 0.5 mm to 8.5 mm, 1 mm to 8 mm, 2 mm to 8 mm, 2.5 mm to 7.5 mm, and includes 3 mm to 6 mm. In some embodiments, the first and second plates are asymmetrical polygons, with the width of the first end being 1 to 10 mm and the width of the second end being 2 to 5 mm. For example, the first and second plates can be asymmetrical polygons with the width of the first end being 5 mm and the width of the second end being 10 mm. In embodiments, the surface area of ​​the first and second plates can vary as needed and can range from 0.25 to 15 cm².2 Within a range, for example, 0.5 to 14 cm 2 For example, 0.75 to 13 cm 2 For example, 1 to 12 cm 2 For example, 1.5 to 11 cm 2 And including 2 to 10 cm 2 .

[0065] In some embodiments, the particle sorter of interest may include one or more sorting decision modules configured to generate sorting decisions for particles (e.g., cells) based on identifying the cell phenotype. A droplet deflector may be configured to sort particles (e.g., cells) from a flow stream based on the sorting decisions generated by the sorting decision modules. As described above, the term "sorting" is used herein in its conventional sense to refer to separating components of a sample (e.g., cells, non-cellular particles, such as biomolecules) and, in some instances, conveying the separated components to one or more partitions, such as a sample collection container. For example, a target particle sorter may be configured to sort samples having two or more components, such as three or more components, four or more components, five or more components, ten or more components, fifteen or more components, and includes sorting samples having 25 or more components. One or more sample components may be isolated from a sample and transferred to a sample collection container, such as two or more sample components, three or more sample components, four or more sample components, five or more sample components, ten or more sample components, and up to fifteen or more sample components may be isolated from a sample and transferred to a sample collection container. In some cases, the phrase "sample component" refers to cells with different cell phenotypes.

[0066] In some embodiments, the particle sorting system of interest is configured to sort particles (e.g., cells) using a closed particle sorting module, such as the particle sorting module described in U.S. Patent Publication No. 2017 / 0299493, filed March 28, 2017, the disclosure of which is incorporated herein by reference. In some embodiments, a sorting decision module having multiple sorting decision units is used to sort particles (e.g., cells) of a sample, such as as described in U.S. Provisional Patent Application No. 16 / 725,756, filed December 23, 2019, the disclosure of which is incorporated herein by reference.

[0067] Figure 4A This is a schematic diagram of a particle sorter 400 according to one embodiment presented herein. In some embodiments, the particle sorter 400 is a cell sorter system. Figure 4AAs shown, a droplet-forming transducer 402 (e.g., a piezoelectric oscillator) is coupled to a fluid conduit 401, which may be coupled to, include, or be a nozzle 403. Within the fluid conduit 401, a sheath fluid 404 hydrodynamically focuses a sample fluid 406 containing particles 409 into a moving fluid column 408 (e.g., a stream). Within the moving fluid column 408, particles 409 (e.g., cells) align in a single file to pass through a monitoring area 411 (e.g., a laser flow intersection, an interrogation point) irradiated by an illumination source 412 (e.g., a laser stream). Vibration of the droplet-forming transducer 402 causes the moving fluid column 408 to break into multiple droplets 410 (droplet streams), some of which contain particles 409.

[0068] In operation, a detection station 414 (e.g., an event detector) identifies when a particle (or cell) of interest crosses a monitoring area 411. The detection station 414 is fed into a timing circuit 428, which in turn feeds into a flash charge circuit 430. At the droplet break point, notified by a timing droplet delay (Δt), a flash charge can be applied to a moving fluid column 408, causing the droplet of interest to carry a charge. The droplet of interest may include one or more particles or cells to be sorted. The charged droplet can then be sorted by activating a deflection plate (not shown) to deflect it into a partition (e.g., a vessel, such as a collection tube or a multi-well or micro-well sample plate), where the partition or well or micro-well can be associated with a droplet of particular interest. Figure 4A As shown, the droplets can be collected in the discharge container 438.

[0069] A detection system 416 (e.g., a droplet boundary detector) is used to automatically determine the phase of the droplet drive signal as a particle of interest passes through a monitoring region 411. An exemplary droplet boundary detector is described in U.S. Patent No. 7,679,039, the entire contents of which are incorporated herein by reference. The detection system 416 allows the instrument to accurately calculate the position of each detected particle within the droplet. The detection system 416 may be fed an amplitude signal 420 and / or a phase signal 418, which in turn are fed (via amplifier 422) an amplitude control circuit 426 and / or a frequency control circuit 424. The amplitude control circuit 426 and / or the frequency control circuit 424 then control the droplet forming transducer 402. The amplitude control circuit 426 and / or the frequency control circuit 424 may be included in a control system.

[0070] In some embodiments, sorting electronics (e.g., detection system 416, detection station 414, and processor 440) may be coupled to a memory configured to store detected events and sorting decisions based thereon. The sorting decisions may be included in event data for particles. In some embodiments, detection system 416 and detection station 414 may be implemented as a single detection unit or communicationally coupled such that event measurements can be collected by one of detection system 416 or detection station 414 and provided to non-collecting elements.

[0071] Figure 4B This is a schematic diagram of a particle sorter according to one embodiment presented herein. Figure 4B The particle sorter 400 shown includes a first deflector plate 452 and a second deflector plate 454 as described in this disclosure. Charge can be applied via charging lines in a barb. This generates a droplet stream 410 containing particles 410 for analysis. The particles can be illuminated with one or more light sources (e.g., lasers) to generate light scattering and fluorescence information. This can be achieved, for example, through sorting electronics or other detection systems. Figure 4B (Not shown) Information about the analyzed particles. The first deflector plate 452 and the second deflector plate 454 can be independently controlled to attract or repel charged droplets to guide them to a target collection container (e.g., one of 472, 474, 476, or 478), such as a partition. Figure 4B As shown, the first deflector plate 452 and the second deflector plate 454 can be controlled to guide particles along a first path 462 toward container 474 or along a second path 468 toward container 478. The first deflector plate 452 and the second deflector plate 454 can be offset from each other (e.g., by adjusting the position of the first deflector plate 452 up or down outside the plane of the figure) so that they apply a known deflection force. In some cases, this known deflection force can be applied to more precisely align the deflected droplets with collection containers 472, 474, 476, and 478. If the droplets are of no interest (do not exhibit scattering or illumination information within a specific sorting range), the deflector plates can allow the particles to continue along the flow path 464. Such uncharged droplets can be fed into the waste container via a suction device such as a suction device 470.

[0072] It may include sorting electronics to initiate the collection of measurements, receive the fluorescence signal of the particles, and determine how to adjust the deflection plate to induce particle sorting. Figure 4B The example implementations of the embodiments shown include the BD FACSAria™ series flow cytometers commercially available from Becton, Dickinson and the Company (Franklin Lakes, NJ).

[0073] In embodiments, the particle sorter according to this disclosure further includes a droplet generator. The droplet generator can be any convenient device suitable for generating a droplet flow from fluid exiting a flow cell. In embodiments, the fluid exiting the flow cell is continuous and interconnected, and the droplet generator causes the continuous and interconnected fluid exiting the flow cell to form separate and discrete droplets. In some examples, the droplet generator is an oscillating transducer. For example, in some cases, the oscillating transducer is a piezoelectric oscillator. Vibration of the droplet generator causes the fluid moving therein to break into multiple droplets in the droplet flow. The amplitude and frequency of the droplet generator's vibration affect the characteristics of the droplets and droplet flow formed by the droplet generator.

[0074] In examples, the particle sorter also includes multiple partitions configured to contain droplets generated by a droplet generator and deflected by a droplet deflector. A partition refers to any convenient container capable of receiving one or more droplets from a droplet stream (e.g., droplets containing sorted particles (e.g., cells) sorted by the particle sorter) and keeping the contents of the partition separate from other material not sorted into the partition. Embodiments include more than one partition, such as two partitions, four partitions, 96 partitions, or 1536 or more partitions. A partition can be any convenient size capable of containing and holding droplets from a droplet stream (e.g., droplets containing sorted particles (e.g., cells) separated from the droplet stream). In some cases, the partition size is designed to contain more than one droplet, such as 10 droplets, 100 droplets, 1000 droplets, 10000 droplets, or more. In some embodiments, the partition includes a collection container. In an example, the collection container is a multi-well plate. The wells of the multi-well plate can be of any convenient shape. In some cases, the cross-sectional shape of the wells is circular; in others, it is rectangular or square. Wells can be of any size, sufficient to accommodate droplets, such as those containing sorted particles (e.g., cells), as needed. For example, the volume of a well can be 0.001 ml or more, such as 0.005 ml, 0.015 ml, 0.1 ml, 2 ml, or 5 ml or more. A multi-well plate can include any number of wells. In examples, a multi-well plate can include six, 12, 24, 48, 96, 384, 1536, 3456, or 9600 or more wells. In some examples, a multi-well plate has 1536 or fewer wells. The wells of a multi-well plate can be arranged in any convenient manner. In some examples, the wells are arranged in a rectangular shape with an aspect ratio of approximately 2:3. In some examples, the multi-well plate described herein conforms to recognized standards, such as those developed by the Society for Biomolecular Sciences according to ANSI standards. The multi-well plate can be composed of any convenient material. In some cases, multi-well plates may be composed of polypropylene, polystyrene, or polycarbonate. In other instances, the compartments may include collection tubes. Wells can be of any size and shape with sufficient capacity to accommodate droplets as needed, such as droplets containing sorted particles (e.g., cells). In some cases, each collection tube has a circular cross-sectional shape with a diameter of 1.8 mm or less.

[0075] As described in detail above, the particle sorter according to embodiments of this disclosure can be configured to sort particles in a sample, such as cells in a biological sample. In these embodiments, the droplet deflector of the particle sorter is configured to apply a known deflection force sufficient to deflect particles flowing in the droplet stream into one or more sample collection containers. Figure 5Panel AC illustrates the effect of applying a known offset deflection force to the droplets in a particle sorter according to this disclosure by showing the droplet deposition locations when a known offset deflection force with different offsets is applied. The effect of the known offset deflection force can be seen with reference to the wells of a multi-well plate configured to accommodate droplets deflected by the droplet deflectors of the particle sorter, whose positions and orientations remain substantially constant between panels AC. As described above, the multi-well plate is located on a horizontal plane orthogonal to the longitudinal axis of the droplet flow and includes a row of wells extending only in the "side-to-side" direction of the horizontal plane (i.e., the x-axis of the overlapping xyz coordinate system), as described above.

[0076] Figure 5 Panel A shows the resulting droplet deposition locations (i.e., the locations of droplet deposition) with a standard deflection force, i.e., a deflection force excluding any known offset (in other words, a deflection force with a known offset of zero). In panel A, the multi-well plate 510 includes a row of wells 520 extending only along the "side-to-side" axis and the horizontal plane of the multi-well plate 510. Droplet deposition locations 530 are generated by applying a deflection force to the droplets of the droplet flow without any known offset component (i.e., a known offset of zero). Because the known offset of the deflection force is zero, the droplet deposition locations 530 extend only along the "side-to-side" axis of the horizontal plane. As mentioned above, because the deflection force applied to these droplets does not include a known offset component, the droplet deposition locations 530 do not extend along the "front-to-back" axis (i.e., the y-axis of the overlapping xyz coordinate system).

[0077] Figure 5 Panel B illustrates the droplet deposition location resulting from a known offset deflection force, which includes a known offset guiding the droplet toward the "backward" direction of the "front-back" axis (i.e., the y-axis of the overlapping xyz coordinate system). In panel B, the multi-well plate 540 includes a row of wells 550 extending only along the "side-to-side" axis and horizontal plane of the multi-well plate 540. The droplet deposition location obtained by applying a known offset deflection force to the droplets in the droplet flow, using a known offset component guided toward the "backward" direction of the "front-back" axis of the multi-well plate, is shown in panel B. As seen on the left side of the multi-well plate 540 in panel B, because the known offset of the deflection force is guided "backward," the droplet deposition location 560 extends along the "side-to-side" axis and also toward the rear of the "front-back" axis. Because the deflection force applied to the droplets includes a known offset component that guides some of the droplets toward the "backward" direction, the droplet deposition location 560 extends partially along the "front-back" axis of the multi-well plate. That is, the droplet deposition location shown in panel B, obtained from the application of the known offset deflection force, is considered to extend along the x-axis and the y-axis of the overlapping xyz plane.

[0078] Figure 5Panel C shows the droplet deposition location obtained by the known offset deflection force, which includes a known offset guiding the droplets toward the "forward" direction of the "front-back" axis (i.e., the y-axis of the overlapping xyz coordinate system). In panel C, the multi-well plate 570 includes a row of wells 580 extending only along the "side-to-side" axis and the horizontal plane of the multi-well plate 570. Using the known offset component guiding the "forward" direction toward the "front-back" axis of the multi-well plate 570 in panel C, the droplet deposition location 590 is obtained by the droplets to which the deflection force is applied to the droplet flow. As seen on the left side of the multi-well plate 570 in panel C, because the deflection force applied to the droplets includes a known offset component guiding some droplets toward the "forward" direction, the droplet deposition location 590 extends along the "side-to-side" axis in the horizontal plane and also in the forward direction along the "front-back" axis. Because the deflection force applied to the droplet includes a known offset component that guides the droplet toward the "forward" direction, the droplet deposition location 590 extends partially along the "front-back" axis of the multi-well plate. That is, the droplet deposition location shown in panel C, obtained based on the application of the known offset deflection force, is considered to extend along the x-axis and the y-axis of the overlapping xyz plane.

[0079] The particle sorter described in this disclosure includes a flow cell. The flow cell of the target particle sorter includes an interrogation point. The interrogation point of the flow cell is configured to be illuminated by a light source. In embodiments, the flow cell of the target particle sorting system may also include a flow cell nozzle with a nozzle orifice configured to allow flow through the flow cell nozzle. In some instances, the target particle sorter includes a flow cell with a flow cell nozzle having an orifice for propagating a fluid sample to the sample interrogation point, wherein in some embodiments, the flow cell nozzle includes a proximal cylindrical portion defining a longitudinal axis and a distal truncated conical portion terminating on a flat surface, having a nozzle orifice transverse to the longitudinal axis. The length (measured along the longitudinal axis) of such a proximal cylindrical portion can vary from 1 mm to 15 mm, for example, 1.5 mm to 12.5 mm, for example, 2 mm to 10 mm, for example, 3 mm to 9 mm, and includes 4 mm to 8 mm. The length (measured along the longitudinal axis) of this distal truncated conical portion can also vary, ranging from 1 mm to 10 mm, for example 2 mm to 9 mm, for example 3 mm to 8 mm, and including 4 mm to 7 mm. In some embodiments, the diameter of the flow pool nozzle chamber can vary, ranging from 1 mm to 10 mm, for example 2 mm to 9 mm, for example 3 mm to 8 mm, and including 4 mm to 7 mm.

[0080] In some instances, the nozzle chamber does not include a cylindrical portion and the entire flow cell nozzle chamber is truncated conical. In these embodiments, the length of the truncated conical nozzle chamber (measured along the longitudinal axis transverse to the nozzle orifice) can range from 1 mm to 15 mm, for example, 1.5 mm to 12.5 mm, for example, 2 mm to 10 mm, for example, 3 mm to 9 mm, and includes 4 mm to 8 mm. The diameter of the proximal portion of the truncated conical nozzle chamber can range from 1 mm to 10 mm, for example, 2 mm to 9 mm, for example, 3 mm to 8 mm, and includes 4 mm to 7 mm.

[0081] In an embodiment, the sample flow may exit from an orifice at the distal end of the flow cell nozzle. Depending on the desired characteristics of this flow, the flow cell nozzle orifice can be of any suitable shape, with the cross-sectional shape of interest including, but not limited to: linear cross-sectional shapes, such as squares, rectangles, trapezoids, triangles, hexagons, etc.; curved cross-sectional shapes, such as circles, ellipses; and irregular shapes, such as the bottom of a parabola coupled to the top of a plane. In some embodiments, the flow cell nozzle of interest has a circular orifice. The nozzle orifice size can vary, ranging from 1 μm to 20,000 μm in some embodiments, such as 2 μm to 17,500 μm, 5 μm to 15,000 μm, 10 μm to 12,500 μm, 15 μm to 10,000 μm, 25 μm to 7,500 μm, 50 μm to 5,000 μm, 75 μm to 1,000 μm, 100 μm to 750 μm, and including 150 μm to 500 μm. In some embodiments, the nozzle orifice is 100 μm.

[0082] In some embodiments, the flow cell nozzle includes a sample injection port configured to provide a sample to the flow cell nozzle. In embodiments, the sample injection system is configured to provide a suitable sample flow to the flow cell nozzle chamber. Depending on the desired characteristics of the flow flow, the sample rate delivered to the flow cell nozzle chamber through the sample injection port can be 1 μL / sec or higher, for example, 2 μL / sec or higher, for example, 3 μL / sec or higher, for example, 5 μL / sec or higher, for example, 10 μL / sec or higher, for example, 15 μL / sec or higher, for example, 25 μL / sec or higher, for example, 50 μL / sec or higher, for example, 100 μL / sec or higher, for example, 150 μL / sec or higher, for example, 200 μL / sec or higher, for example, 250 μL / sec or higher, for example, 300 μL / sec or higher, for example, 350 μL / sec or higher, for example, 400 μL / sec or higher, for example, 450 μL / sec or higher, and includes 500 μL / sec or higher. For example, the sample flow rate can range from 1 μL / sec to about 500 μL / sec, such as 2 μL / sec to about 450 μL / sec, such as 3 μL / sec to about 400 μL / sec, such as 4 μL / sec to about 350 μL / sec, such as 5 μL / sec to about 300 μL / sec, such as 6 μL / sec to about 250 μL / sec, such as 7 μL / sec to about 200 μL / sec, such as 8 μL / sec to about 150 μL / sec, such as 9 μL / sec to about 125 μL / sec, and includes 10 μL / sec to about 100 μL / sec.

[0083] The sample injection port can be an orifice located in the nozzle chamber wall or a conduit located near the proximal end of the nozzle chamber. When the sample injection port is an orifice located in the nozzle chamber wall, the orifice can be of any suitable shape, with cross-sectional shapes of interest including but not limited to: straight cross-sectional shapes, such as squares, rectangles, trapezoids, triangles, hexagons, etc.; curved cross-sectional shapes, such as circles, ellipses, etc.; and irregular shapes, such as the bottom of a parabola coupled to the top of a plane. In some embodiments, the sample injection port has a circular orifice. The size of the sample injection port orifice may vary depending on the shape, and in some instances, its opening ranges from 0.1 mm to 5.0 mm, for example 0.2 to 3.0 mm, for example 0.5 mm to 2.5 mm, for example 0.75 mm to 2.25 mm, for example 1 mm to 2 mm, and includes 1.25 mm to 1.75 mm, for example 1.5 mm.

[0084] In some instances, the sample injection port is a conduit located proximal to the flow cell nozzle chamber of the flow cell. For example, the sample injection port may be a conduit positioned with an orifice aligned with the flow cell nozzle orifice. When the sample injection port is a conduit aligned with the flow cell nozzle orifice, the cross-sectional shape of the sample injection tube can be any suitable shape, including but not limited to: straight-line cross-sectional shapes such as squares, rectangles, trapezoids, triangles, hexagons, etc.; curved cross-sectional shapes such as circles, ellipses; and irregular shapes, such as the bottom of a parabola coupled to the top of a plane. The orifice of the conduit can vary in shape, and in some cases has an opening ranging from 0.1 mm to 5.0 mm, such as 0.2 to 3.0 mm, 0.5 mm to 2.5 mm, 0.75 mm to 2.25 mm, 1 mm to 2 mm, and including 1.25 mm to 1.75 mm, such as 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 sample injection port may include a beveled tip with a bevel angle ranging from 1° to 10°, such as 2° to 9°, such as 3° to 8°, such as 4° to 7°, and including bevel angles of 5°.

[0085] In some embodiments, the flow cell nozzle further includes a sheath fluid injection port configured to supply sheath fluid to the flow cell nozzle. In embodiments, the sheath fluid injection system is configured to supply a sheath fluid flow to the flow cell nozzle chamber, for example, in combination with a sample to generate a laminated flow of sheath fluid surrounding a sample flow. Depending on the desired flow characteristics, the rate at which the sheath fluid is delivered to the flow cell nozzle chamber can be 25 μL / sec or higher, for example 50 μL / sec or higher, for example 75 μL / sec or higher, for example 100 μL / sec or higher, for example 250 μL / sec or higher, for example 500 μL / sec or higher, for example 750 μL / sec or higher, for example 1000 μL / sec or higher, and includes 2500 μL / sec or higher. For example, the range of sheath fluid flow rate can be from 1 μL / sec to about 500 μL / sec, such as 2 μL / sec to about 450 μL / sec, such as 3 μL / sec to about 400 μL / sec, such as 4 μL / sec to about 350 μL / sec, such as 5 μL / sec to about 300 μL / sec, such as 6 μL / sec to about 250 μL / sec, such as 7 μL / sec to about 200 μL / sec, such as 8 μL / sec to about 150 μL / sec, such as 9 μL / sec to about 125 μL / sec, and includes 10 μL / sec to about 100 μL / sec.

[0086] In some embodiments, the sheath fluid inlet is an orifice located in the wall of the nozzle chamber of the flow cell. The sheath fluid inlet orifice can be of any suitable shape, with the cross-sectional shape of interest including, but not limited to: straight cross-sectional shapes, such as squares, rectangles, trapezoids, triangles, hexagons, etc.; curved cross-sectional shapes, such as circles, ellipses; and irregular shapes, such as the bottom of a parabola coupled to the top of a plane. The size of the sample inlet orifice can vary depending on its shape, and in some cases has an opening ranging from 0.1 mm to 5.0 mm, for example 0.2 to 3.0 mm, for example 0.5 mm to 2.5 mm, for example 0.75 mm to 2.25 mm, for example 1 mm to 2 mm, and including 1.25 mm to 1.75 mm, for example 1.5 mm.

[0087] The particle sorter also includes an interrogation point (i.e., a sample interrogation point). In some cases, the sample interrogation point is in fluid communication with the flow cell nozzle orifice. As described in more detail below, the sample flow can exit from an orifice at the distal end of the flow cell nozzle, and particles in the flow can be illuminated by a light source at the sample interrogation point in the flow cell of the particle sorter. The size of the interrogation point of the particle sorter can vary depending on the characteristics of the flow nozzle, such as the size of the nozzle orifice and the size of the sample inlet. In embodiments, the width of the interrogation point can be 0.01 mm or greater, for example 0.05 mm or greater, for example 0.1 mm or greater, for example 0.5 mm or greater, for example 1 mm or greater, for example 2 mm or more, for example 3 mm or greater, for example 5 mm or greater, and includes 10 mm or greater. The length of the inquiry point can also vary, and in some instances it ranges from 0.01 mm or greater, such as 0.1 mm or greater, such as 0.5 mm or greater, such as 1 mm or greater, such as 1.5 mm or greater, such as 2 mm or greater, such as 3 mm or greater, such as 5 mm or greater, such as 10 mm or greater, such as 15 mm or greater, such as 20 mm or greater, such as 25 mm or greater, including particle sorters of 50 mm or more.

[0088] The interrogation point of the flow cell in the particle sorter can be configured to facilitate illumination of a planar cross-section of the emission flow or to facilitate illumination of a diffuse field of a predetermined length (e.g., using a diffuse laser or lamp). In some embodiments, the interrogation point of the flow cell includes a transparent window that facilitates illumination of a predetermined length of the emission flow, such as 1 mm or longer, 2 mm or longer, 3 mm or longer, 4 mm or longer, 5 mm or longer, and including 10 mm or longer. Depending on the light source used to illuminate the emission flow (described below), the interrogation region of the particle sorting module can be configured to allow light in the range of 100 nm to 1500 nm, such as 150 nm to 1400 nm, 200 nm to 1300 nm, 250 nm to 1200 nm, 300 nm to 1100 nm, 350 nm to 1000 nm, 400 nm to 900 nm, and including 500 nm to 800 nm.Therefore, the flow cell of the particle sorter at the inquiry point can be formed of any transparent material that spans the desired wavelength range, including but not limited to optical glass, borosilicate glass, Pyrex glass, ultraviolet quartz, infrared quartz, sapphire, and plastics such as polycarbonate, polyvinyl chloride (PVC), polyurethane, polyether, polyamide, polyimide, or copolymers of these thermoplastics, such as PETG (ethylene glycol-modified polyethylene terephthalate). The polyester of interest may include, but is not limited to, poly(alkylene terephthalate), such as polyethylene terephthalate (PET), bottle-grade PET (based on monobutylene terephthalate), etc. ethylene glycol, terephthalic acid and other comonomers such as isophthalic acid, cyclohexenedimethyl alcohol, etc., copolymers, poly(butylene terephthalate) (PBT) and poly(hexamethylene terephthalate); polyalkylene adipate, such as polyethylene adipate, polybutylene adipate 1,4- and polyhexamethylene adipate; poly(alkylene octanoate) such as poly(ethylene octanoate); poly(alkylene sebacate), such as poly(ethylene sebacate); poly(ε-caprolactone) and poly(β-propiolactone); poly(alkylene isophthalate), such as poly(ethylene isophthalate); poly(2, 6-Naphthalene dicarboxylate), for example, poly(2,6-naphthalene dicarboxylate); poly(sulfonyl-4,4'-dibenzoylene), for example, poly(sulfonyl-4,4'-dibenzoylene); poly(p-phenylene alkyl dicarboxylate), for example, poly(p-phenylene ethyl dicarboxylate); poly(trans-1,4-cyclohexanedialkyl dicarboxylate), for example, poly(trans-1,4-cyclohexanediethylene ethyl dicarboxylate); poly(1,4-cyclohexane-dimethylene alkyl dicarboxylate), for example, poly(1,4-cyclohexane-dimethylene ethyl dicarboxylate; poly([2.2.2] -Bicyclooctane-1,4-dimethylenealkylene dicarboxylate), such as poly([2.2.2]-bicyclooctane-1,4-dimethylene ethylenedicarboxylate); lactic acid polymers and copolymers, such as (S)-polylactide, (R,S)-polylactide, poly(tetramethylglycolic acid) and poly(lactide-co-glycolic acid); and polycarbonates of bisphenol A, 3,3'-dimethylbisphenol A, 3,3',5,5'-tetrachlorobisphenol A, and 3,3',5,5'-tetramethylbisphenol A; polyamides such as poly(p-phenylene terephthalamide); polyesters, such as polyethylene terephthalate, such as Mylar. TMPolyethylene terephthalate; etc. In some embodiments, the flow cell of interest comprises a test tube positioned at the sample interrogation point. In embodiments, the test tube may be able to transmit light in the range of 100 nm to 1500 nm, such as 150 nm to 1400 nm, such as 200 nm to 1300 nm, such as 250 nm to 1200 nm, such as 300 nm to 1100 nm, such as 350 nm to 1000 nm, such as 400 nm to 900 nm, and including 500 nm to 800 nm.

[0089] In some embodiments, the sample interrogation point includes one or more optical adjustment components. "Optical adjustment" refers to the modification of light incident on the sample interrogation point or light collected from an irradiated flow as needed. In some embodiments, the sample interrogation point includes optical adjustment components for adjusting the light incident on the sample interrogation point by a light source. In other embodiments, the sample interrogation point includes optical adjustment components for adjusting light emitted from an irradiated flow before being transmitted to a detector for measurement. In still other embodiments, the sample interrogation point includes optical adjustment components for adjusting the light incident on the sample interrogation point by a light source and the light emitted by the flow before being transmitted to a detector for measurement. For example, optical adjustment can be increasing the size of the light, the focal point of the light, or collimating the light. In some instances, optical adjustment is a magnification protocol to increase the size of the light (e.g., beam point), such as increasing the size by 5% or more, for example 10% or more, for example 25% or more, for example 50% or more, and includes increasing the size by 75% or more. In other embodiments, optical adjustment includes focusing the collected light to reduce the light size, for example, by 5% or more, 10% or more, 25% or more, 50% or more, and includes reducing the size of the beam spot by 75% or more. In some embodiments, optical adjustment includes collimating the light. The term "collimating," in its conventional sense, refers to optical adjustment of collinearity of light propagation or reduction of divergence from a common propagation axis. In some instances, collimation includes narrowing the spatial cross-section of the beam.

[0090] The optical adjustment element can be any convenient device or structure that provides the desired variation of the collected light, and can include, but is not limited to, lenses, mirrors, pinholes, slits, gratings, light refractors, and any combination thereof. The particle sorter may include one or more optical adjustment elements at the sample inquiry point as needed, such as two or more, three or more, four or more, and even five or more optical adjustment elements.

[0091] In some embodiments, the optical adjustment element is a focusing lens with a magnification ratio of 0.1 to 0.95, such as 0.2 to 0.9, 0.3 to 0.85, 0.35 to 0.8, 0.5 to 0.75, and including 0.55 to 0.7, such as 0.6. For example, in some instances, the focusing lens is a double achromatic de-magnifying lens with a magnification ratio of about 0.6. The focal length of the focusing lens can vary, ranging from 5 mm to 20 mm, such as 6 mm to 19 mm, 7 mm to 18 mm, 8 mm to 17 mm, 9 mm to 16 mm, and including focal lengths of 10 mm to 15 mm. In some embodiments, the focusing lens has a focal length of about 13 mm.

[0092] In other embodiments, the optical adjustment component is a collimator. The collimator can be any convenient collimation protocol, such as one or more mirrors or curved lenses, or combinations thereof. For example, in some instances, the collimator is a single collimating lens. In other instances, the collimator is a collimating mirror. In still other instances, the collimator includes two lenses. In still other instances, the collimator includes a mirror and a lens. When the collimator includes one or more lenses, the focal length of the collimating lens can vary from 5 mm to 40 mm, for example, 6 mm to 37.5 mm, for example, 7 mm to 35 mm, for example, 8 mm to 32.5 mm, for example, 9 mm to 30 mm, for example, 10 mm to 27.5 mm, for example, 12.5 mm to 25 mm, and includes focal lengths ranging from 15 mm to 20 mm.

[0093] In some embodiments, the optical adjustment component is a wavelength splitter. The term "wavelength splitter," used herein in its conventional sense, refers to an optical protocol for separating multicolor light into its constituent wavelengths. According to some embodiments, wavelength separation may include selectively passing through or blocking specific wavelengths or wavelength ranges of multicolor light. The wavelength separation protocol of interest may be part of or in combination with the flow cell nozzle discussed above, including but not limited to colored glass, bandpass filters, interference filters, dichroic mirrors, diffraction gratings, monochromators, combinations thereof, and other wavelength separation protocols. In some embodiments, the wavelength splitter is an optical filter. For example, the optical filter may be a bandpass filter with a minimum bandwidth range of 2 nm to 100 nm, such as 3 nm to 95 nm, such as 5 nm to 95 nm, such as 10 nm to 90 nm, such as 12 nm to 85 nm, such as 15 nm to 80 nm, and includes bandpass filters with a minimum bandwidth range of 20 nm to 50 nm.

[0094] In embodiments, the light source of the particle sorter can be any suitable broadband or narrowband light source. In some cases, depending on the sample composition at the flow cell interrogation point (e.g., cells, calibration particles such as beads, non-cellular particles, etc.), the light source can be configured to emit light with varying wavelengths, ranging from 200 nm to 1500 nm (e.g., 250 nm to 1250 nm), 300 nm to 1000 nm, 350 nm to 900 nm, and including 400 nm to 800 nm. For example, the light source can include a broadband light source emitting light with wavelengths from 200 nm to 900 nm. In other instances, the light source includes a narrowband light source emitting light with wavelengths ranging from 200 nm to 900 nm. For example, the light source can be a narrowband LED (1 nm–25 nm) emitting light with wavelengths ranging from 200 nm to 900 nm. In some embodiments, the light source is a laser. In some instances, the target particle sorter includes gas lasers, such as helium-neon lasers, argon lasers, krypton lasers, xenon lasers, nitrogen lasers, CO2 lasers, CO lasers, argon-fluorine (ArF) excimer lasers, krypton-fluorine (KrF) excimer lasers, xenon chloride (XeCl) excimer lasers, or xenon-fluorine (XeF) excimer lasers, or combinations thereof. In other instances, the target particle sorter includes dye lasers, such as stilbene, coumarin, or rhodamine lasers. In still other instances, the lasers of interest include metal vapor lasers, such as helium-cadmium (HeCd) lasers, helium-mercury (HeHg) lasers, helium-selenium (HeSe) lasers, helium-silver (HeAg) lasers, strontium lasers, neon-copper (NeCu) lasers, copper lasers, or gold lasers, or combinations thereof. In other instances, the target particle sorter includes 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, titania-sapphire lasers, thulium YAG lasers, ytterbium YAG lasers, ytterbium oxide lasers, or cerium-doped lasers and combinations thereof.

[0095] In other embodiments, the light source is a non-laser light source, such as a lamp, including but not limited to halogen lamps, deuterium arc lamps, xenon arc lamps, and light-emitting diodes (LEDs), such as broadband LEDs with a continuous spectrum, superluminescent LEDs, semiconductor LEDs, broadband LED white light sources, and multi-LED integrated light sources. In some instances, the non-laser light source is a stable fiber-coupled broadband light source, a white light source, and other light sources or any combination thereof.

[0096] The light source can be at any suitable distance from the sample at the query point of the flow cell (e.g., the flow stream in a flow cytometer), such as 0.001 mm or more, 0.005 mm or more, 0.01 mm or more, 0.05 mm or more, 0.1 mm or more, 0.5 mm or more, 1 mm or more, 5 mm or more, 10 mm or more, 25 mm or more, and including distances of 100 mm or more. Furthermore, the light source can illuminate the sample at the query point of the flow cell at any suitable angle (e.g., relative to the vertical axis of the flow stream), such as angles ranging from 10° to 90°, 15° to 85°, 20° to 80°, 25° to 75°, and including angles from 30° to 60°, such as 90°.

[0097] A light source can be configured to continuously or discretely illuminate a sample at an interrogation point in a flow cell. In some instances, the particle sorter includes a light source configured to continuously illuminate the sample, such as a continuous-wave laser that continuously illuminates the flow stream at an interrogation point, for example, in a flow cytometer. In other instances, the particle sorter of interest includes a light source configured to illuminate the sample at discrete intervals, such as every 0.001 ms, every 0.01 ms, every 0.1 ms, every 1 ms, every 10 ms, every 100 ms, and including every 1000 ms, or some other interval. When the light source is configured to illuminate the sample at discrete intervals, the particle sorter may include one or more additional components to provide intermittent illumination of the sample with the light source. For example, the target particle sorter in these embodiments may include one or more laser beam choppers, manually or computer-controlled beam stoppers, for blocking and exposing the sample to the light source.

[0098] In some embodiments, the light source is a laser. The laser of interest may include pulsed lasers or continuous-wave lasers. For example, the laser may be a gas laser, such as a helium-neon laser, an argon laser, a krypton laser, a xenon laser, a nitrogen laser, a CO2 laser, a CO laser, an argon-fluorine (ArF) excimer laser, a krypton-fluorine (KrF) excimer laser, a xenon chloride (XeCl) excimer laser, or a xenon-fluorine (XeF) excimer laser or a combination thereof; a dye laser, such as a stilbene, coumarin, or rhodamine laser; or 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, or a neon-copper laser. (NeCu) lasers, copper lasers, or gold lasers and combinations thereof; 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, Ti:sapphire lasers, thulium YAG lasers, ytterbium YAG lasers, ytterbium oxide lasers, or cerium-doped lasers and combinations thereof; semiconductor diode lasers, optically pumped semiconductor lasers (OPSL), or second or third harmonics of any of the above lasers.

[0099] In some embodiments, the light source is a beam generator configured to generate two or more beams of frequency-shifted light. In some instances, the beam generator includes a laser, or an RF generator configured to apply an RF drive signal to an acousto-optic device to generate two or more angle-deflected laser beams. In these embodiments, for example as described above, the laser may be a pulsed laser or a continuous-wave laser.

[0100] The acousto-optic device can be any convenient acousto-optic protocol configured to use frequency-shifted laser light from an applied acoustic wave. In some embodiments, the acousto-optic device is an acousto-optic deflector. The acousto-optic device in the target system is configured to generate an angle-deflected laser beam from light from a laser and an applied radio frequency (RF) drive signal. The RF drive signal can be applied to the acousto-optic device using any suitable RF drive signal source, such as a direct digital synthesizer (DDS), an arbitrary waveform generator (AWG), or an electrical pulse generator.

[0101] In an embodiment, the controller is configured to apply radio frequency drive signals to the acousto-optic device to generate a desired number of angularly deflected laser beams in the output laser beam, for example, to apply 3 or more radio frequency drive signals, such as 4 or more radio frequency drive signals, such as 5 or more radio frequency drive signals, such as 6 or more radio frequency drive signals, such as 7 or more radio frequency drive signals, such as 8 or more radio frequency drive signals, such as 9 or more radio frequency drive signals, such as 10 or more radio frequency drive signals, such as 15 or more radio frequency drive signals, such as 25 or more radio frequency drive signals, such as 50 or more radio frequency drive signals, and includes being configured to apply 100 or more radio frequency drive signals.

[0102] In some instances, in order to generate an intensity distribution of an angularly deflected laser beam in the output laser beam, the controller is configured to apply an RF drive signal having an amplitude that varies, for example, from about 0.001V to about 500V, from about 0.005V to about 400V, from about 0.01V to about 300V, from about 0.05V to about 200V, from about 0.1V to about 100V, from about 0.5V to about 75V, from about 1V to 50V, from about 2V to 40V, from about 3V to about 30V, and including from about 5V to about 25V. In some embodiments, each applied radio frequency drive signal has a frequency 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, and includes about 5 MHz to about 50 MHz.

[0103] In some embodiments, the controller has a processor, and a memory is operatively coupled to the processor such that the memory includes instructions stored thereon that, when executed by the processor, cause the processor to generate an output laser beam with an angle-deflected laser beam having a desired intensity distribution. For example, the memory may include instructions for generating two or more angle-deflected laser beams with the same intensity, such as three or more, four or more, five or more, ten or more, 25 or more, or 50 or more, and may include instructions for generating 100 or more angle-deflected laser beams with the same intensity. In other embodiments, the memory may include instructions for generating two or more angle-deflected laser beams with different intensities, such as three or more, four or more, five or more, ten or more, 25 or more, or 50 or more, and may include instructions for generating 100 or more angle-deflected laser beams with different intensities.

[0104] In some embodiments, the controller has a processor, and a memory is operatively coupled to the processor such that the memory includes instructions stored thereon that, when executed by the processor, cause the processor to generate an output laser beam having an intensity that increases from the edge to the center along a horizontal axis. In these examples, the intensity of the angle-deflected laser beam at the center of the output laser beam can vary from 0.1% to 99% of the intensity of the angle-deflected laser beam at the edge of the output laser beam along the horizontal axis, for example, from 0.5% to 95%, from 1% to 90%, from about 2% to 85%, from about 3% to 80%, from about 4% to 75%, from about 5% to 70%, from about 6% to 65%, from about 7% to 60%, from about 8% to 55%, and includes from about 10% to 50% of the intensity of the angle-deflected laser beam at the edge of the output laser beam along the horizontal axis. In other embodiments, the controller has a processor, and a memory is operatively coupled to the processor such that the memory includes instructions stored thereon that, when executed by the processor, cause the processor to generate an output laser beam with an intensity increasing from the edge to the center along a horizontal axis. In these examples, the intensity of the angle-deflected laser beam at the edge of the output laser beam can vary from 0.1% to 99% of the intensity of the angle-deflected laser beam at the center of the output laser beam along the horizontal axis, for example, from 0.5% to 95%, from 1% to 90%, from about 2% to 85%, from about 3% to 80%, from about 4% to 75%, from about 5% to 70%, from about 6% to 65%, from about 7% to 60%, from about 8% to 55%, and includes from about 10% to 50% of the intensity of the angle-deflected laser beam at the center of the output laser beam along the horizontal axis. In another embodiment, the controller has a processor, and a memory is operatively coupled to the processor such that the memory includes instructions stored thereon, which, when executed by the processor, cause the processor to generate an output laser beam with a Gaussian intensity distribution along a horizontal axis. In still other embodiments, the controller has a processor, and a memory is operatively coupled to the processor such that the memory includes instructions stored thereon, which, when executed by the processor, cause the processor to generate an output laser beam with a top-hat intensity distribution along a horizontal axis.

[0105] In embodiments, the beam generator of interest can be configured to generate an angularly deflected laser beam within spatially separated output laser beams. Depending on the applied radio frequency drive signal and the desired illumination distribution of the output laser beams, the angularly deflected laser beams can be separated by 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, for example, 500 μm or more, for example, 1000 μm or more, and including 5000 μm or more. In some embodiments, the system is configured to generate an angularly deflected laser beam within overlapping output laser beams, for example, overlapping with an angularly deflected laser beam along a horizontal axis of an adjacent output laser beam. The overlap between adjacent angle-deflected laser beams (e.g., beam spot overlap) can be 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, and includes 100 μm or more.

[0106] In some instances, beam generators configured to generate two or more frequency-shifted beams include laser excitation modules, such as U.S. Patent Nos. 9,423,353; 9,784,661 and 10,006,852 and U.S. Patent Publications Nos. 2017 / 0133857 and 2017 / 0350803, the disclosures of which are incorporated herein by reference.

[0107] In an embodiment, the particle sorter includes a detector for detecting light from a query point in a flow cell that includes a light detection system. The light detection system of interest may include one or more photodetectors. The photodetectors of interest may include, but are not limited to, optical sensors such as active pixel sensors (APSs), avalanche photodiodes, image sensors, charge-coupled devices (CCDs), enhancement charge-coupled devices (ICCDs), light-emitting diodes, photon counters, calorimeters, pyroelectric detectors, photoresistors, photovoltaic cells, photodiodes, photomultiplier tubes, phototransistors, quantum dot photoconductors, or combinations thereof, and other photodetectors. In some embodiments, the light from the query point is emitted using a charge-coupled device (CCD), a semiconductor charge-coupled device (CCD), an active pixel sensor (APSs), a complementary metal-oxide-semiconductor (CMOS) image sensor, or an N-type metal-oxide-semiconductor (NMOS) image sensor.

[0108] In some embodiments, the photodetector of interest includes multiple photodetectors. In some instances, the photodetector includes multiple solid-state detectors, such as photodiodes. In some instances, the photodetector includes a photodetector array, such as a photodiode array. In these embodiments, the photodetector array may include four or more photodetectors, such as 10 or more, 25 or more, 50 or more, 100 or more, 250 or more, 500 or more, 750 or more, and may include 1000 or more photodetectors. For example, the detector may be a photodiode array having four or more photodiodes, such as 10 or more, 25 or more, 50 or more, 100 or more, 250 or more, 500 or more, 750 or more, and may include 1000 or more photodiodes.

[0109] The photodetectors can be arranged in any geometric configuration as needed, including but not limited to square, rectangular, trapezoidal, triangular, hexagonal, heptagonal, octagonal, nonagonal, decagonal, dodecagonal, circular, elliptical, and irregular pattern configurations. The photodetectors in the photodetector array can be oriented at angles ranging from 10° to 180° relative to other photodetectors (e.g., referenced in the XZ plane), such as 15° to 170°, 20° to 160°, 25° to 150°, 30° to 120°, and including 45° to 90°. The photodetector array can be any suitable shape and can be linear, such as square, rectangular, trapezoidal, triangular, hexagonal, etc.; curved, such as circular, elliptical; and irregular, such as a parabolic base coupled to the top of a plane. In some embodiments, the photodetector array has a rectangular active surface.

[0110] Each photodetector (e.g., photodiode) in the array may have an active surface with a width ranging from 5 μm to 250 μm, such as 10 μm to 225 μm, 15 μm to 200 μm, 20 μm to 175 μm, 25 μm to 150 μm, or 30 μm to 125 μm, and includes an active surface of 50 μm to 100 μm and a length ranging from 5 μm to 250 μm, such as 10 μm to 225 μm, 15 μm to 200 μm, 20 μm to 175 μm, 25 μm to 150 μm, or 30 μm to 125 μm, and includes an active surface of 50 μm to 100 μm, wherein the surface area of ​​each photodetector (e.g., photodiode) is in the range of 25 μm. 2 Up to 10000μm 2 Arrays, such as 50μm 2 Up to 9000μm 2 For example, 75μm 2 Up to 8000μm 2 For example, 100μm 2 Up to 7000μm 2 For example, 150μm 2 Up to 6000μm 2 And including 200μm 2 Up to 5000μm 2 .

[0111] The size of the photodetector array can vary depending on the amount and intensity of light, the number of photodetectors, and the desired sensitivity. The photodetector array can have a length ranging from 0.01 mm to 100 mm, for example, 0.05 mm to 90 mm, 0.1 mm to 80 mm, 0.5 mm to 70 mm, 1 mm to 60 mm, 2 mm to 50 mm, 3 mm to 40 mm, 4 mm to 30 mm, and including 5 mm to 25 mm. The width of the photodetector array can also vary within the range of 0.01 mm to 100 mm, for example, 0.05 mm to 90 mm, 0.1 mm to 80 mm, 0.5 mm to 70 mm, 1 mm to 60 mm, 2 mm to 50 mm, 3 mm to 40 mm, 4 mm to 30 mm, and including 5 mm to 25 mm. Therefore, the active surface area of ​​the photodetector array can range from 0.1 mm. 2 Up to 10000mm 2 For example, 0.5mm 2 Up to 5000mm 2 For example, 1mm 2 Up to 1000mm 2 For example, 5mm 2 Up to 500mm2 And including 10mm 2 Up to 100mm 2 .

[0112] The photodetector of interest is configured to measure light collected at one or more wavelengths, such as two or more wavelengths, five or more different wavelengths, ten or more different wavelengths, 25 or more different wavelengths, 50 or more different wavelengths, 100 or more different wavelengths, 200 or more different wavelengths, 300 or more different wavelengths, and includes measuring light emitted by the sample in the flow at 400 or more different wavelengths.

[0113] In some embodiments, the photodetector is configured to measure light collected within a wavelength range (e.g., 200 nm–1000 nm). In some embodiments, the photodetector of interest is configured to collect the spectrum of light within the wavelength range. For example, the system may include one or more detectors configured to collect the spectrum of one or more lights within the wavelength range of 200 nm–1000 nm. In other embodiments, the detector of interest is configured to measure one or more specific wavelengths of light from a sample in a flowing stream. For example, the system may include one or more detectors configured to measure one or more of the following wavelengths: 450 nm, 518 nm, 519 nm, 561 nm, 578 nm, 605 nm, 607 nm, 625 nm, 650 nm, 660 nm, 667 nm, 670 nm, 668 nm, 695 nm, 710 nm, 723 nm, 780 nm, 785 nm, 647 nm, 617 nm, and any combination thereof. In some embodiments, the photodetector may be configured to pair with a specific fluorophore (e.g., a fluorophore used with the sample in a fluorescence assay). In some embodiments, the photodetector is configured to measure the light collected across the entire fluorescence spectrum of each fluorophore in the sample.

[0114] The detector is configured to measure light continuously or at discrete intervals. In some instances, the detector of interest is configured to continuously measure the collected light. In other instances, the photodetector is configured to take measurements at discrete intervals, such as every 0.001 milliseconds, every 0.01 milliseconds, every 0.1 milliseconds, every 1 millisecond, every 10 milliseconds, every 100 milliseconds, and including every 1000 milliseconds or some other interval.

[0115] In some instances, the photodetector also includes optical adjustment components. In some instances, optical adjustment is an amplification protocol configured to increase the size of the light field captured by the detector, for example, by 5% or more, for example, 10% or more, for example, 25% or more, for example, 50% or more, and including increasing the light field captured by the detector by 75% or more. In other instances, optical adjustment is a reduction protocol configured to reduce the light field captured by the detector, for example, by 5% or more, for example, 10% or more, for example, 25% or more, for example, reducing by 50% or more, and including reducing the light field captured by the detector by 75% or more. In some embodiments, optical adjustment is a focusing protocol configured to focus the light collected by the detector, for example, focusing the collected beam by 5% or more, for example, 10% or more, for example, 25% or more, for example, 50% or more, and including focusing the collected beam by 75% or more.

[0116] The optical adjustment components can be any convenient device or structure that provides the desired variation of the collected beam and can include, but are not limited to, lenses, mirrors, pinholes, slits, gratings, light refractors, and any combination thereof. The detector may include one or more optical adjustment components as needed, such as two or more, three or more, four or more, and even five or more. In some embodiments, the detector includes a focusing lens. The focusing lens may, for example, be a reducing lens. In other instances, the focusing lens is a magnifying lens. In other embodiments, the detector includes a collimator.

[0117] In some embodiments, the particle sorter includes a combination of different optical adjustment components, such as a combination of pinhole, lens, mirror, slit, etc. For example, in some embodiments, the particle sorter includes a focusing lens and a collimating lens. In other embodiments, the particle sorter includes a collimating lens and a focusing lens. In still other embodiments, the particle sorter includes a focusing lens and a pinhole structure. In yet another embodiment, the particle sorter includes a collimating lens and a pinhole structure. In still still embodiments, the particle sorter includes a collimating lens and a slit structure.

[0118] In some embodiments, the detector and the optical adjustment component communicate optically but do not physically contact each other. Depending on the size of the detector, the optical adjustment component may be positioned at a distance of 0.05 mm or more, 0.1 mm or more, such as 0.5 mm or more, such as 1 mm or more, such as 10 mm or more, such as 25 mm or more, such as 50 mm or more, such as 100 mm or more, such as 250 mm or more, including 500 mm or more. In other embodiments, the optical adjustment component is physically coupled to the detector, for example using an adhesive, and co-molded or integrated together in a housing having the optical adjustment component positioned adjacent to the detector. Thus, the optical adjustment component and the detector can be integrated into a single unit.

[0119] In some embodiments, the optical adjustment component is a focusing lens with a magnification ratio of 0.1 to 0.95, such as 0.2 to 0.9, 0.3 to 0.85, 0.35 to 0.8, 0.5 to 0.75, and including 0.55 to 0.7, such as 0.6. For example, in some instances, the focusing lens is a biachromatic reducing lens with a magnification ratio of approximately 0.6. Depending on the distance between the detector and the lens, and the surface area of ​​the detector's active surface, the focal length of the focusing lens can vary within a range of 5 mm to 20 mm, such as 6 mm to 19 mm, 7 mm to 18 mm, 8 mm to 17 mm, 9 mm to 16 mm, and including focal lengths ranging from 10 mm to 15 mm. In some embodiments, the focusing lens has a focal length of approximately 13 mm.

[0120] In some embodiments, the optical conditioning components include one or more optical fibers configured to relay light from the interrogation point of the flow cell to the detector. Suitable optical fibers for propagating light from the interrogation point of the flow cell to the active surface of the detector include, but are not limited to, flow cytometry fiber optic systems, such as those described in U.S. Patent No. 6,809,804, the disclosure of which is incorporated herein by reference.

[0121] In other embodiments, the detector of interest is coupled to a collimator. The collimator can be any convenient collimation protocol, such as one or more mirrors or curved lenses, or combinations thereof. For example, in some instances, the collimator is a single collimating lens. In other instances, the collimator is a collimating mirror. In still other instances, the collimator includes a series of two or more lenses, such as three or more lenses, and includes four or more lenses. In still other instances, the collimator includes both mirrors and lenses. When the collimator includes one or more lenses, the focal length of the collimating lens can vary within the range of 5 mm to 40 mm, for example, 6 mm to 37.5 mm, for example, 7 mm to 35 mm, for example, 8 mm to 32.5 mm, for example, 9 mm to 30 mm, for example, 10 mm to 27.5 mm, for example, 12.5 mm to 25 mm, and includes focal lengths ranging from 15 mm to 20 mm.

[0122] In some embodiments, the optical adjustment component is a wavelength splitter. As described above, the wavelength splitter of interest refers to an optical protocol for separating multicolor light into its component wavelengths for detection. According to some embodiments, wavelength separation may include selectively passing through or blocking specific wavelengths or wavelength ranges of multicolor light. To separate the wavelengths of light, light emitted from a sample in the flow stream can be passed through any convenient wavelength separation protocol, including but not limited to colored glass, bandpass filters, interference filters, dichroic mirrors, diffraction gratings, monochromators, combinations thereof, and other wavelength separation protocols. The particle sorter may include one or more wavelength splitters, such as two or more, three or more, four or more, five or more, and including ten or more wavelength splitters. In one example, the detector includes a bandpass filter. In another example, the detector includes two or more bandpass filters. In yet another example, the detector includes two or more bandpass filters and a diffraction grating. In still another example, the detector includes a monochromator. In some embodiments, the detector includes multiple bandpass filters and diffraction gratings configured as a filter wheel setup. When the detector includes two or more wavelength splitters, the wavelength splitters can be used individually or in series to separate multicolor light into constituent wavelengths. In some embodiments, the wavelength splitters are arranged in series. In other embodiments, the wavelength splitters are arranged individually, such that each wavelength splitter is used for one or more measurements.

[0123] In some embodiments, the detector includes one or more optical filters, such as one or more bandpass filters. For example, the optical filters of interest may include bandpass filters with a minimum bandwidth range of 2 nm to 100 nm, such as 3 nm to 95 nm, 5 nm to 95 nm, 10 nm to 90 nm, 12 nm to 85 nm, 15 nm to 80 nm, and bandpass filters with a minimum bandwidth range of 20 nm to 50 nm. In other embodiments, the wavelength separator is a diffraction grating. The diffraction grating may include, but is not limited to, transmission, dispersive, or reflection diffraction gratings. The appropriate spacing of the diffraction grating may vary depending on the construction of the flow cell nozzle chamber, the detector, and other available optical conditioning protocols (e.g., focusing lenses), ranging from 0.01 μm to 10 μm, such as 0.025 μm to 7.5 μm, 0.5 μm to 5 μm, 0.75 μm to 4 μm, 1 μm to 3.5 μm, and including 1.5 μm to 3.5 μm.

[0124] Figure 6 A flow cytometry particle sorter 600 according to an exemplary embodiment of the present invention is shown. The particle sorter 600 includes a flow cytometer 610, a controller / processor 690, and a memory 695. The flow cytometer 610 includes one or more excitation lasers 615a-615c, a focusing lens 620, a flow chamber (i.e., a flow cell) 625, a forward scattering detector 630, a side scattering detector 635, a fluorescence collecting lens 640, one or more beam splitters 645a-645g, one or more bandpass filters 650a-650e, one or more long-pass (“LP”) filters 655a-655b, and one or more fluorescence detectors 660a-660f.

[0125] The 615a-c laser is excited to emit light in the form of a laser beam. Figure 6 In the example particle sorter, the laser beams emitted from excitation lasers 615a-615c have wavelengths of 488 nm, 633 nm, and 325 nm, respectively. The laser beams are first guided through one or more beam splitters 645a and 645b. Beam splitter 645a transmits 488 nm light and reflects 633 nm light. Beam splitter 645b transmits UV light (light with a wavelength range of 10 to 400 nm) and reflects both 488 nm and 633 nm light.

[0126] The laser beam is then directed to a focusing lens 620, which focuses the beam onto a portion of the flow stream containing the sample particles within a flow chamber 625. The flow chamber is part of a fluid system that typically directs one particle at a time to the focused laser beam for interrogation. The flow chamber may include a flow cell in a benchtop cytometer or a nozzle tip in an airflow cytometer.

[0127] Light from the laser beam interacts with particles in the sample through diffraction, refraction, reflection, scattering, and absorption at different wavelengths, depending on the characteristics of the particles (e.g., their size, internal structure, and the presence of one or more fluorescent molecules attached to or naturally present on or in the particles). Fluorescence emission, along with diffracted, refracted, reflected, and scattered light, can be routed through one or more of beam splitters 645a-645g, bandpass filters 650a-650e, longpass filters 655a-655b, and fluorescence collecting lenses 640 to one or more of forward scattering detectors 630, side scattering detectors 635, and fluorescence detectors 660a-660f.

[0128] A fluorescence collecting lens 640 collects light emitted from particle-laser beam interactions and directs that light to one or more beamsplitters and filters. Bandpass optical filters, such as bandpass filters 650a-650e, allow a narrow range of wavelengths to pass through the filter. For example, bandpass filter 650a is a 510 / 20 filter. The first number represents the center of the spectral band. The second number provides the range of the spectral band. Thus, a 510 / 20 filter extends 10 nm on each side of the center of the spectral band, or from 500 nm to 520 nm. Short-pass filters transmit light with wavelengths equal to or shorter than a specific wavelength. Long-pass optical filters, such as long-pass filters 655a-655b, transmit light with wavelengths equal to or longer than a specific wavelength. For example, long-pass filter 655a, as a 670 nm long-pass filter, transmits light with wavelengths equal to or longer than 670 nm. Filters are typically selected to optimize the detector's specificity for a particular fluorescent dye. Filters can be configured such that the spectral band of the light transmitted to the detector is close to the emission peak of the fluorescent dye.

[0129] Beam splitters direct light of different wavelengths in different directions. A beam splitter is characterized by filter properties, such as short-pass and long-pass characteristics. For example, beam splitter 645g is a 620SP beam splitter, meaning that beam splitter 645g transmits light with wavelengths of 620 nm or shorter and reflects light with wavelengths longer than 620 nm in different directions. In one embodiment, beam splitters 645a-645g may include optical mirrors, such as dichroic mirrors.

[0130] A forward scattering detector 630 is positioned slightly off-axis from the direct beam passing through the flow cell and is configured to detect diffracted light, the excitation light which propagates primarily in the forward direction through or around the particle. The intensity of the light detected by the forward scattering detector depends on the overall size of the particle. The forward scattering detector may include a photodiode. A side scattering detector 635 is configured to detect refracted and reflected light from the surface and internal structures of the particle and tends to increase with increasing particle structural complexity. Fluorescence emission from fluorescent molecules associated with the particle can be detected by one or more fluorescence detectors 660a-660f. The side scattering detector 635 and the fluorescence detector may include photomultiplier tubes. The signal detected at the forward scattering detector 630, the side scattering detector 635, and the fluorescence detector can be converted into an electrical signal (voltage) by the detectors. This data can provide information about the sample.

[0131] Those skilled in the art will recognize that the flow cytometer described in the embodiments of the present invention is not limited to... Figure 6 The flow cytometer described herein 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 at various wavelengths and with various different configurations.

[0132] In operation, the cytometer is controlled by a controller / processor 690, and measurement data from the detector can be stored in memory 695 and processed by the controller / processor 690. Although not explicitly shown, the controller / processor 690 is coupled to the detector to receive output signals from the detector and can also be coupled to the electrical and electromechanical components of the flow cytometer 600 to control the laser, fluid flow parameters, etc. Input / output (I / O) functionality 697 may also be provided in the system. Memory 695, controller / processor 690, and I / O 697 may be provided entirely as an integrated part of the flow cytometer 610. In this embodiment, a display may also form part of the I / O functionality 697 for presenting experimental data to the user of the cytometer 600. Alternatively, some or all of memory 695, controller / processor 690, and I / O functionality may be part of one or more external devices (e.g., a general-purpose computer). In some embodiments, some or all of memory 695 and controller / processor 690 may communicate wirelessly or wired with the cytometer 610. The controller / processor 690, together with the memory 695 and I / O 697, can be configured to perform various functions related to the preparation and analysis of flow cytometry experiments.

[0133] Figure 6The system shown includes six different detectors that detect fluorescence in six different wavelength bands (which may be referred to herein as “filter windows” for a given detector), as defined by the construction of filters and / or separators in the beam path from flow cell 625 to each detector. Different fluorescent molecules used in flow cytometry experiments will emit light in their own characteristic wavelength bands. Specific fluorescent labels used in the experiment and their associated fluorescence emission bands can be selected to generally coincide with the filter windows of the detectors. However, as more detectors are provided and more labels are used, a perfect correspondence between filter windows and fluorescence emission spectra is impossible. Generally, while the peak of the emission spectrum of a particular fluorescent molecule may lie within the filter window of a particular detector, some of the emission spectrum of that label will also overlap with the filter windows of one or more other detectors. This can be referred to as overflow. I / O 697 can be configured to receive data on a flow cytometry experiment having a set of fluorescent labels and multiple cell populations with multiple labels, each cell population having a subset of the multiple labels. I / O 697 can also be configured to receive biological data that assigns one or more identifiers to one or more cell populations, marker density data, emission spectral data, data that assigns tags to one or more markers, and cytometer configuration data. Flow cytometry experimental data, such as marker spectral properties and flow cytometry configuration data, can also be stored in memory 695. Controller / processor 690 can be configured to evaluate one or more assignments of tags to identifiers.

[0134] In some embodiments, the target particle sorter is a flow cytometry system using the droplet deflector described above, the droplet deflector being configured to apply a known deflection force. In some embodiments, the target particle sorter is a flow cytometry system. Suitable flow cytometry systems may include, but are not limited to, those in Ormerod (ed.), Flow Cytometry: A Practical Approach, Oxford Univ. Press (1997); Jaroszeski et al. (eds.), Flow Cytometry Protocols, Methods in Molecular Biology No. 91, Humana Press (1997); Practical Flow Cytometry, 3rd ed., Wiley-Liss (1995); Virgo et al. (2012) Ann Clin Biochem. Jan; 49(pt 1): 17-28; Linden et al., Semin Throm Hemost. 2004 Oct; 30(5): 502-11; Alison et al. J Pathol, 2010 Dec; 222(4): 335-344; and Herbig et al. (2007) Crit Rev Ther Drug The contents of Carrier Syst. 24(3): 203-255, the disclosure of which is incorporated herein by reference. In some cases, the flow cytometry systems of interest include the BD Biosciences FACSCanto™ II flow cytometer, BDDAccuri™ flow cytometer, BD Biosciences FACSCelesta™ flow cytometer, BD Biosciences FACSLyri™ flow cytometer, BD Biosciences FACSVers™ flow cytometer, BD Biosciences FACSymphon™ flow cytometer, BD Biosciences LSRFortess™ flow cytometer, BD Biosciences LSRFortes™ X-20 flow cytometer, and BD Biosciences FACSCalibur™ cell sorter, BD Biosciences FACSCount™ cell sorter, BD Biosciences FACSLyric™ cell sorter, and BD Biosciences Via™ cell sorter, BD Biosciences Influx. TMCell sorting instrument, BDBiosciences Jazz TM Cell sorter, BD Biosciences Aria TM Cell sorter and BD Biosciences FACSMelody TM Cell sorting instruments, etc. In some instances, the cell sorting instrument is the BD FACSymphony. TM S6 Cell Sorter; BD FACSMelody TM Cell sorting instrument; BD FACSAria TM III Cell Sorter; BD FACSAria TM Fusion Cell Sorter; BD FACSJazz TM Or BD Influx TM Cell sorting instrument.

[0135] In some instances, the target particle sorter is a flow cytometry system configured to image particles in a flowing stream using fluorescence imaging with radio frequency labeled emission (FIRE), as described, for example, in Diebold, et al. Nature Photonics Vol. 7 (10); 806-810 (2013) and U.S. Patent Nos. 9,423,353; 9,784,661; 9,983,132; 10,006,852; 10,078,045; 10,036,699; 10,222,316; 10,288,546; 10,324,019; 10,4 The disclosures of U.S. Patent Publications 08,758; 10,451,538; 10,620,111; and 2017 / 0133857; 2017 / 0328826; 2017 / 0350803; 2018 / 0275042; 2019 / 0376895 and 2019 / 0376894 are incorporated herein by reference.

[0136] In some embodiments, the target system is a flow cytometry system, such as those described in U.S. Patent Nos. 10,663,476; 10,620,111; 10,613,017; 10,605,713; 10,585,031; 10,578,542; 10,578,469; 10,481,074; 10,302,545; 10,145,793; 10,113,967; 10,006,852; 9,952,076; 9,933,341; 9,726,527; 9,453,789; 9,200,334; and 9,097,640. The contents of the references 0; 9,095,494; 9,092,034; 8,975,595; 8,753,573; 8,233,146; 8,140,300; 7,544,326; 7,201,875; 7,129,505; 6,821,740; 6,813,017; 6,809,804; 6,372,506; 5,700,692; 5,643,796; 5,627,040; 5,620,842; 5,602,039; 4,987,086; 4,498,766 are incorporated herein by reference.

[0137] The method of deflecting droplets in a droplet stream by applying a known deflection force

[0138] Aspects of this disclosure include methods for deflecting droplets in a droplet stream using a droplet deflector configured to apply a known offset deflection force to the droplet stream. As described in more detail above, applying a “offset deflection force” means, for example, a deflection force that deflects droplets in the droplet stream, which may include a “side-to-side” and a “forward” direction orthogonal to the horizontal plane of the droplet stream, and in some cases, an offset deflection force is applied after rotating the angle by which the deflection force is applied to the droplet stream about the longitudinal axis of the droplet stream. As mentioned above, applying a “known offset deflection force” means applying a deflection force to the droplets in the droplet stream, the deflection force being offset by design or a predetermined amount.

[0139] The method according to some embodiments includes illuminating an interrogation point of a flow cell with a light source, detecting the light from the interrogation point with a detector, generating a droplet stream from fluid flowing out of the flow cell using a droplet generator, and deflecting the droplets of the droplet stream with a droplet deflector configured to apply a known deflection force to the droplet stream. Deflecting the droplets according to the target method, including sorting particles (e.g., cells), results in improved sorting efficiency, such that fewer sample particles are wasted during sample sorting (i.e., intentionally deflecting droplets containing particles such as cells to unintended droplet deposition sites, causing them to remain unsorted). In some cases, sorting efficiency can be improved, allowing for the collection and sorting of more cell phenotypic variations when using the target method. When used as part of flow cytometry sample sorting, the target method can improve the yield of particle sorting.

[0140] In some embodiments, the droplet stream contains a sample as a biological sample, and the method includes sorting and collecting two or more different types of cells such that, when practicing the target method, the sample containing particles is illuminated with a light source and the light from the sample is detected by a light detection system having one or more photodetectors. In some embodiments, the sample is a biological sample. The term “biological sample” is used in its conventional sense to refer to a subset of the whole organism, plant, fungal, or animal tissue, cell, or component that may be found in blood, mucus, lymph, synovial fluid, cerebrospinal fluid, saliva, bronchoalveolar lavage fluid, amniotic fluid, sheep cord blood, urine, vaginal fluid, and semen in certain instances. Thus, “biological sample” refers both to a subset of a natural organism or its tissues and to homogenates, lysates, or extracts prepared from a subset of an organism or its tissues, including, but not limited to, plasma, serum, cerebrospinal fluid, lymph, skin sections, respiratory tract, gastrointestinal tract, cardiovascular and genitourinary tract, tears, saliva, milk, blood cells, tumors, and organs. Biological samples can be any type of organic tissue, including healthy tissue and diseased tissue (e.g., cancerous tissue, malignant tissue, necrotic tissue, etc.). In some embodiments, biological samples are liquid samples, such as blood or blood derivatives, such as plasma, tears, urine, semen, etc. In some instances, the sample is a blood sample, including whole blood, such as blood obtained from venipuncture or finger prick (the blood may or may not be mixed with any reagents, such as preservatives, anticoagulants, etc., before testing).

[0141] In some embodiments, the source of the sample is "mammal" or "mammal," terms widely used to describe organisms belonging to the class Mammalia, including Carnivora (e.g., dogs and cats), Rodentia (e.g., mice, guinea pigs, and rats), and primates (e.g., humans, chimpanzees, and monkeys). In some instances, the subject is a human. The method can be applied to samples obtained from human subjects of both sexes and at any developmental stage (i.e., newborns, infants, adolescents, teenagers, and adults), and in some embodiments, the human subject is an adolescent, teenager, or adult. While the invention can be applied to samples from human subjects, it should be understood that these methods can also be performed on samples from other animal subjects (i.e., in "non-human subjects"), such as, but not limited to, birds, mice, rats, dogs, cats, livestock, and horses.

[0142] In an embodiment, the query point of the flow cell is illuminated by light from a light source. When practicing the target method, for example, a sample (e.g., in the flow stream of a flow cytometer) may be illuminated in the flow cell with light from a light source. In some embodiments, the light source is a broadband light source that emits light with a wide wavelength range, such as across 50 nm or greater, for example 100 nm or greater, for example 150 nm or greater, for example 200 nm or greater, for example 250 nm or greater, for example 300 nm or greater, for example 350 nm or greater, for example 400 nm or greater, and including through 500 nm or greater. For example, a suitable broadband light source emits light with wavelengths from 200 nm to 1500 nm. Another example of a suitable broadband light source includes a light source that emits light with wavelengths from 400 nm to 1000 nm. When the method involves illumination with a broadband light source, the broadband light source protocol of interest may include, but is not limited to, halogen lamps, deuterium arc lamps, xenon arc lamps, stable fiber-coupled broadband light sources, broadband LEDs with continuous spectra, superluminescent diodes, semiconductor light-emitting diodes, broadband LED white light sources, multi-LED integrated white light sources, and other broadband light sources or any combination thereof.

[0143] In other embodiments, the method includes illumination with a narrowband light source emitting a specific wavelength or a narrow wavelength range, such as a light source emitting light in a narrow wavelength range, such as 50 nm or less, for example 40 nm or less, for example 30 nm or less, for example 25 nm or less, for example 20 nm or less, for example 15 nm or less, for example 10 nm or less, for example 5 nm or less, for example 2 nm or less, and including a light source emitting light of a specific wavelength (i.e., monochromatic light). When the method includes illumination with a narrowband light source, the narrowband light source protocol of interest may include, but is not limited to, narrow-wavelength LEDs, laser diodes, or broadband light sources coupled to one or more optical bandpass filters, diffraction gratings, monochromators, or any combination thereof.

[0144] In some embodiments, the method includes irradiating the interrogation point with one or more lasers. As described above, the type and number of lasers will vary depending on the sample and the light to be collected, and may be gas lasers, such as helium-neon lasers, argon lasers, krypton lasers, xenon lasers, nitrogen lasers, CO2 lasers, CO lasers, argon-fluorine (ArF) excimer lasers, krypton-fluorine (KrF) excimer lasers, xenon chloride (XeCl) excimer lasers, or xenon-fluorine (XeF) excimer lasers, or combinations thereof. In other instances, the method includes irradiating the flow stream with a dye laser, such as a stilbene, coumarin, or rhodamine laser. In still other instances, the method includes irradiating the flow stream with 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, or combinations thereof. In other instances, the method involves irradiating the flow with a solid-state laser, such as a ruby ​​laser, an Nd:YAG laser, an NdCrYAG laser, an Er:YAG laser, an Nd:YLF laser, an Nd:YVO4 laser, an Nd:YCa4O(BO3)3 laser, an Nd:YCOB laser, a titanite sapphire laser, a thulium YAG laser, a ytterbium YAG laser, a ytterbium oxide laser, or a cerium-doped laser, or combinations thereof.

[0145] The sample can be illuminated with one or more of the aforementioned light sources, such as two or more light sources, three or more light sources, four or more light sources, five or more light sources, and including ten or more light sources. The light sources can include any combination of light source types. For example, in some embodiments, the method includes illuminating the query point of the flow cell with a laser array, such as an array having one or more gas lasers, one or more dye lasers, and one or more solid-state lasers.

[0146] The sample can be illuminated with wavelengths ranging from 200 nm to 1500 nm, such as 250 nm to 1250 nm, 300 nm to 1000 nm, 350 nm to 900 nm, and including 400 nm to 800 nm. For example, when the light source is a broadband light source, the query point of the flow cell can be illuminated with wavelengths from 200 nm to 900 nm. In other instances, when the light source comprises multiple narrowband light sources, the sample can be illuminated with specific wavelengths in the range of 200 nm to 900 nm. For example, the light source can be multiple narrowband LEDs (1 nm to 25 nm), each LED independently emitting light in the wavelength range of 200 nm to 900 nm. In other embodiments, the narrowband light source comprises one or more lasers (e.g., a laser array) and illuminates the sample with specific wavelengths ranging from 200 nm to 700 nm, such as lasers as described above that have gas lasers, excimer lasers, dye lasers, metal vapor lasers, and solid-state lasers.

[0147] When using more than one light source, the query point of the flow cell can be illuminated simultaneously or sequentially by the light source or a combination thereof. For example, each of the light sources can simultaneously illuminate the query point of the flow cell. In other embodiments, each of the light sources can sequentially illuminate the query point of the flow cell. When using more than one light source to sequentially illuminate the query point of the flow cell, the duration for which each light source illuminates the query point can be independently 0.001 microseconds or more, for example, 0.01 microseconds or more, for example, 0.1 microseconds or more, for example, 1 microsecond or more, for example, 5 microseconds or more, for example, 10 microseconds or more, for example, 30 microseconds or more, and including 60 microseconds or more. For example, the method may include illuminating the sample with a light source (e.g., a laser) for a duration ranging from 0.001 microseconds to 100 microseconds, for example, 0.01 microseconds to 75 microseconds, for example, 0.1 microseconds to 50 microseconds, for example, 1 microsecond to 25 microseconds, and including 5 microseconds to 10 microseconds. In embodiments where two or more light sources sequentially illuminate the query point, the duration for which the sample is illuminated by each light source can be the same or different.

[0148] The time interval between each light source illumination can also vary as needed, independently separated by delays of 0.001 microseconds or more, such as 0.01 microseconds or more, 0.1 microseconds or more, 1 microsecond or more, 5 microseconds or more, 10 microseconds or more, 15 microseconds or more, 30 microseconds or more, and including 60 microseconds or more. For example, the time interval between each light source illumination can range from 0.001 microseconds to 60 microseconds, such as 0.01 microseconds to 50 microseconds, 0.1 microseconds to 35 microseconds, 1 microsecond to 25 microseconds, and including 5 microseconds to 10 microseconds. In some embodiments, the time interval between each light source illumination is 10 microseconds. In embodiments where the query point of the flow cell is sequentially illuminated by more than two (i.e., three or more) light sources, the delays between each light source illumination can be the same or different.

[0149] The query point of the flow cell can be illuminated continuously or at discrete intervals. In some instances, the method includes continuously illuminating the query point of the flow cell with a light source. In other instances, the query point of the flow cell is illuminated with a light source at discrete intervals, such as every 0.001 milliseconds, every 0.01 milliseconds, every 0.1 milliseconds, every 1 millisecond, every 10 milliseconds, every 100 milliseconds, and including every 1000 milliseconds, or other intervals.

[0150] Depending on the light source, the inquiry point of the flow cell can be illuminated at a distance, said distance varying, for example, 0.01 mm or more, 0.05 mm or more, 0.1 mm or more, 0.5 mm or more, 1 mm or more, 2.5 mm or more, 5 mm or more, 10 mm or more, 15 mm or more, 25 mm or more, and including 50 mm or more. Furthermore, the illumination angle can also vary, ranging from 10° to 90°, for example, 15° to 85°, 20° to 80°, 25° to 75°, and including 30° to 60°, for example, at a 90° angle.

[0151] In some embodiments, the method includes illuminating the interrogation point of the flow cell with two or more frequency-shifted beams of light. A beam generator component having a laser and an acousto-optic device for frequency-shifting the laser can be used. In these embodiments, the method includes illuminating the acousto-optic device with a laser. Depending on the desired wavelength of the light generated in the output laser beam (e.g., for illuminating a sample in the flow stream), the laser can have a specific wavelength varying from 200 nm to 1500 nm, such as 250 nm to 1250 nm, 300 nm to 1000 nm, 350 nm to 900 nm, and including 400 nm to 800 nm. The acousto-optic device can be illuminating with one or more lasers, such as two or more lasers, three or more lasers, four or more lasers, five or more lasers, and including ten or more lasers. The lasers can include any combination of laser types. For example, in some embodiments, the method includes illuminating the acousto-optic device with an array of lasers, such as an array having one or more gas lasers, one or more dye lasers, and one or more solid-state lasers.

[0152] When using more than one laser, the acousto-optic device can be illuminated simultaneously or sequentially by the lasers or combinations thereof. For example, the acousto-optic device can be illuminated simultaneously by each of the lasers. In other embodiments, the acousto-optic device is illuminated sequentially by each laser. When the acousto-optic device is illuminated sequentially by more than one laser, the duration for which each laser illuminates the acousto-optic device can be independently 0.001 microseconds or more, for example, 0.01 microseconds or more, for example, 0.1 microseconds or more, for example, 1 microsecond or more, for example, 5 microseconds or more, for example, 10 microseconds or more, for example, 30 microseconds or more, and including 60 microseconds or more. For example, the method may include illuminating the acousto-optic device with lasers for a duration ranging from 0.001 microseconds to 100 microseconds, for example, 0.01 microseconds to 75 microseconds, for example, 0.1 microseconds to 50 microseconds, for example, 1 microsecond to 25 microseconds, and including 5 microseconds to 10 microseconds. In embodiments where the acousto-optic device is illuminated sequentially by two or more lasers, the duration for which the acousto-optic device is illuminated by each laser can be the same or different.

[0153] The time interval between each laser irradiation can also vary as needed, and is independently separated by delays of 0.001 microseconds or more, such as 0.01 microseconds or more, 0.1 microseconds or more, 1 microsecond or more, 5 microseconds or more, 10 microseconds or more, 15 microseconds or more, 30 microseconds or more, and including 60 microseconds or more. For example, the time interval between each light source irradiation can range from 0.001 microseconds to 60 microseconds, such as 0.01 microseconds to 50 microseconds, 0.1 microseconds to 35 microseconds, 1 microsecond to 25 microseconds, and including 5 microseconds to 10 microseconds. In some embodiments, the time interval between each laser irradiation is 10 microseconds. In embodiments where the acousto-optic device is irradiated sequentially by more than two (i.e., three or more) lasers, the delay between each laser irradiation can be the same or different.

[0154] The acousto-optic device can be illuminated continuously or at discrete intervals. In some instances, the method involves illuminating the acousto-optic device continuously with a laser. In other instances, the acousto-optic device is illuminated with a laser at discrete intervals, such as every 0.001 milliseconds, every 0.01 milliseconds, every 0.1 milliseconds, every 1 millisecond, every 10 milliseconds, every 100 milliseconds, and including every 1000 milliseconds, or some other interval.

[0155] According to the laser, the acousto-optic device can be illuminated at a distance that varies, for example, 0.01 mm or more, 0.05 mm or more, 0.1 mm or more, 0.5 mm or more, 1 mm or more, 2.5 mm or more, 5 mm or more, 10 mm or more, 15 mm or more, 25 mm or more, and including 50 mm or more. Furthermore, the illumination angle can also vary, ranging from 10° to 90°, for example, 15° to 85°, 20° to 80°, 25° to 75°, and including 30° to 60°, for example, at a 90° angle.

[0156] In an embodiment, the method includes applying radio frequency (RF) drive signals to an acousto-optic device to generate an angle-deflected laser beam. Two or more RF drive signals may be applied to the acousto-optic device to generate an output laser beam having a desired number of angle-deflected laser beams, such as three or more RF drive signals, four or more RF drive signals, five or more RF drive signals, six or more RF drive signals, seven or more RF drive signals, eight or more RF drive signals, nine or more RF drive signals, ten or more RF drive signals, fifteen or more RF drive signals, twenty-five or more RF drive signals, fifty or more RF drive signals, and including one hundred or more RF drive signals.

[0157] The angle-deflecting laser beams generated by the radio frequency (RF) drive signals each have an intensity based on the amplitude of the applied RF drive signal. In some embodiments, the method includes applying an RF drive signal with an amplitude sufficient to produce an angle-deflecting laser beam with a desired intensity. In some instances, each applied RF drive signal independently has an amplitude of about 0.001V to about 500V, for example, about 0.005V to about 400V, for example, about 0.01V to about 300V, for example, about 0.05V to about 200V, for example, about 0.1V to about 100V, for example, about 0.5V to about 75V, for example, about 1V to 50V, for example, about 2V to 40V, for example, 3V to about 30V, and including about 5V to about 25V. In some embodiments, each applied radio frequency drive signal has a frequency 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, and includes about 5 MHz to about 50 MHz.

[0158] In these embodiments, the angle-deflecting laser beams in the output laser beam are spatially separated. Depending on the applied radio frequency drive signal and the desired illumination distribution of the output laser beam, the angle-deflecting laser beams can be separated by 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, for example, 500 μm or more, for example, 1000 μm or more, and including 5000 μm or more. In some embodiments, the angle-deflecting laser beams overlap, for example, with adjacent angle-deflecting laser beams along the horizontal axis of the output laser beam. The overlap between adjacent angle-deflected laser beams (e.g., overlap of beam points) can be 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, and includes 100 μm or more.

[0159] In some instances, the flow stream is illuminated with an angularly deflected beam of multiple frequency-shifted light, and cells in the flow stream are imaged using radio frequency labeled emission (FIRE) fluorescence imaging to generate frequency-coded images, as described, for example, in Diebold, et al. Nature Photonics Vol. 7(10); 806-810 (2013) and U.S. Patent Nos. 9,423,353; 9,784,661; 9,983,132; 10,006,852; 10,078,045; 10,036,699; 10,222,316; 10,288,546; 10,324,019; 10,4 The disclosures of U.S. Patent Publications 08,758; 10,451,538; 10,620,111; and 2017 / 0133857; 2017 / 0328826; 2017 / 0350803; 2018 / 0275042; 2019 / 0376895 and 2019 / 0376894 are incorporated herein by reference.

[0160] As described above, in some embodiments, light from the interrogation point of the flow cell is fed to a detector as described in more detail below, and in some embodiments, it may be measured by multiple photodetectors. In some embodiments, the method includes measuring light collected within a wavelength range (e.g., 200 nm–1000 nm). For example, the method may include the spectrum of light collected within one or more wavelength ranges of 200 nm–1000 nm. In other embodiments, the method includes measuring light collected at one or more specific wavelengths. For example, the collected light may be measured at 450 nm, 518 nm, 519 nm, 561 nm, 578 nm, 605 nm, 607 nm, 625 nm, 650 nm, 660 nm, 667 nm, 670 nm, 668 nm, 695 nm, 710 nm, 723 nm, 780 nm, 785 nm, 647 nm, 617 nm, and any combination thereof. In some embodiments, the method includes measuring the wavelength of light corresponding to the fluorescence peak wavelength of the fluorophore. In some embodiments, the method includes measuring the light collected across the entire fluorescence spectrum of each fluorophore in a sample flowing in a flow stream.

[0161] The collected light can be measured continuously or at discrete intervals. In some instances, the method involves continuously measuring the light. In other instances, the light can be measured at discrete intervals, such as once every 0.001 milliseconds, every 0.01 milliseconds, every 0.1 milliseconds, every 1 millisecond, every 10 milliseconds, every 100 milliseconds, and including every 1000 milliseconds, or some other interval.

[0162] The collected light measurements may be performed once or more during the target method, for example, two or more, three or more, five or more, and including ten or more. In some embodiments, light propagation is measured two or more times, and in some instances the data are averaged.

[0163] The light from the point of inquiry can be measured at one or more wavelengths, such as five or more different wavelengths, ten or more different wavelengths, 25 or more different wavelengths, 50 or more different wavelengths, 100 or more different wavelengths, 200 or more different wavelengths, 300 or more different wavelengths, and includes light collected measured at 400 or more different wavelengths.

[0164] The method according to this disclosure further includes generating a droplet flow from the fluid exiting the flow cell using a droplet generator. As mentioned above, the droplet generator can be any convenient device for generating a droplet flow from the fluid exiting the flow cell. In some instances, the droplet generator is an oscillating transducer, such as a piezoelectric oscillator.

[0165] The method according to this disclosure further includes deflecting droplets in a droplet stream using a droplet deflector configured to apply a known deflection force to the droplet stream. As described above, in order to sort target droplets (e.g., target droplets containing particles of interest), the analyzed droplet stream is affected by a known deflection force of the droplet deflector (as described above).

[0166] As described in more detail above, in some embodiments, the droplet deflector includes a first plate and a second plate configured to be offset from each other. In this embodiment, the first plate and the second plate can be configured to be adjustablely offset from each other. In an example, the first plate and the second plate can be configured to be adjustablely offset from each other relative to a horizontal plane. In this example, the horizontal plane is perpendicular to the axis of the droplet flow (i.e., the longitudinal axis).

[0167] In one embodiment, the first plate includes an elongated portion configured to allow the first plate to be adjustably offset relative to a horizontal plane relative to a second plate. In this embodiment, the elongated portion of the first plate includes a positioning screw configured to allow the first plate to be adjustably offset relative to a horizontal plane relative to a second plate.

[0168] As described above, in this example, the first plate and the second plate are configured to be adjustablely offset from each other by more than 0 mm to 5 mm. In this example, the first plate and the second plate are configured to be adjustablely offset from each other in increments determined based on the thread pitch of the positioning screws, which are configured to adjustably offset the first plate from the second plate.

[0169] In one example, when measured at a distance of 140 mm below the lowest point of the first deflector plate, the known offset deflection force is sufficient to shift the droplet deposition position by 2 mm or more. Therefore, as described above, the known offset deflection force includes components on the "side-to-side" and "front-to-back" axes in a horizontal plane orthogonal to the longitudinal axis of the flow. In other examples, the known offset deflection force is sufficient to shift the droplet deposition position by one droplet diameter or less.

[0170] In some cases, the droplet deflector according to this disclosure is configured to deflect droplets into multiple zones. In this case, the zone may include a collection container. In other cases, the collection container may be a multi-well plate. In still other cases, the multi-well plate contains 1536 or fewer wells. In some instances, the zone includes collection tubes. In such instances, each collection tube has a diameter of 1.8 mm or less.

[0171] As described above, the droplet deflector of the target method may include a first plate and a second plate configured to be parallel to each other. That is, even when offset from each other, the first plate and the second plate are placed facing each other in a parallel manner. In other cases, the first plate and the second plate are configured to be adjustablely rotated to face each other. That is, as the offset between the first plate and the second plate is adjusted, the first plate and the second plate can be rotated accordingly so that the plates face each other.

[0172] In some cases, the second plate includes an elongated portion configured to allow the second plate to be adjustably offset relative to the horizontal plane relative to the first plate. In this case, the elongated portion of the second plate may include a locating screw configured to allow the second plate to be adjustably offset relative to the horizontal plane relative to the first plate.

[0173] As described in more detail above, in some embodiments, the droplet deflector further includes an actuator (e.g., a motor) configured to adjust the offset between the first and second plates. As described above, in some embodiments, the actuator (e.g., a motor) is operatively connected to a feedback subsystem. In such embodiments, the feedback subsystem may include a controller operatively connected to the actuator (e.g., the motor) and connected to a detector configured to detect the distance of droplet offset in the droplet flow. In other embodiments, the feedback subsystem is configured to iteratively adjust the offset between the first and second plates.

[0174] In this embodiment, the first and second plates are metallic. A voltage applied to each of the metal plates causes a droplet flowing through it to be accelerated and deflected at multiple different angles based on the droplet's charge and the polarity of the charge. The voltage applied to the first and second metal plates to transfer the charged particles can be 10 mV or greater, such as 25 mV or greater, such as 50 mV or greater, such as 100 mV or greater, such as 250 mV or greater, such as 500 mV or greater, such as 750 mV or greater, such as 1000 mV or greater, such as 2500 mV or greater, such as 5000 mV or greater, such as 10000 V or greater, such as 15000 V or greater, such as 25000 V or greater, such as 50000 V or greater, and includes 100000 V or greater. In some embodiments, the voltage applied to the first and second metal plates is from 0.5 kV to 15 kV, for example, from 1 kV to 15 kV, from 1.5 kV to 12.5 kV, and including 2 kV to 10 kV. Therefore, the electric field strength between the metal plates ranges from 0.1 V / m to 1 × 10⁻⁶. 7 V / m, for example 0.5V / m to 5×10 6 For example, 1V / m to 1×10 6 V / m, for example, 5V / m to 5×10 5 V / m, for example, 10V / m to 1×10 5 V / m and includes 50V / m up to 5×10 4 V / m, for example 1×10 5 V / m to 2×10 6 V / m.

[0175] In this example, the metal plates are spaced 1 mm or more apart. In other cases, the metal plates are spaced 3 mm or more apart. In this example, the first and second plates are rectangular.

[0176] In some embodiments, methods for sorting sample components include using a particle sorting module to sort particles (e.g., cells in a biological sample), as described in, for example, U.S. Patent Publication No. 2017 / 0299493, filed March 28, 2017, the disclosure of which is incorporated herein by reference. In some embodiments, a sorting decision module having multiple sorting decision units is used to sort cells in the sample, as described in, for example, U.S. Provisional Patent Application No. 62 / 803,264, filed February 8, 2019, the disclosure of which is incorporated herein by reference.

[0177] Particle sorting module

[0178] Various aspects of this disclosure include a particle sorting module. In embodiments, the particle sorting module includes a droplet deflector as described herein, configured to apply a known offset deflection force to a droplet stream and, for example, to transfer droplets containing the particles to be analyzed to a receiving location. As described in more detail above, applying an “offset deflection force” means, for example, a deflection force that transfers droplets in a droplet stream, can include a “side-to-side” and a “front-to-back” direction orthogonal to the horizontal plane of the droplet stream, and in some cases, the deflection force is offset by applying the deflection force after rotating the angle at which the deflection force is applied to the droplet stream about the longitudinal axis of the droplet stream. As described above, applying a “known offset deflection force” means applying a deflection force to the droplets in the droplet stream, the deflection force being designed or predetermined to be offset.

[0179] The transfer of the droplet of interest to the containment location can be achieved by a droplet deflector via, for example, by applying an electrostatic field to electrostatically charge the droplet and deflect the charged droplet from the flow. In this example, the voltage applied to the first and second plates of the droplet deflector in the target particle sorting module can be 10mV or greater, for example 25mV or greater, for example 50mV or greater, for example 75mV or greater, for example 100mV or greater, for example 250mV or greater, for example 500mV or greater, for example 750mV or greater, for example 1V or greater, for example 2.5V or greater, for example 5V or greater, for example 10V or greater, for example 25V or greater, for example 50V or greater and including 100V or greater, for example 500V or greater, for example 1000V or greater, for example 5000V or greater, for example 10000V or greater, for example 15000V or greater, for example 25000V or greater, for example 50000V or greater and including 100000V or greater. In some embodiments, the voltage applied to each set of parallel metal plates is 0.5kV to 15kV, for example 1kV to 15kV, for example 1.5kV to 12.5kV, and includes 2kV to 10kV.

[0180] In embodiments, the particle sorting module according to this disclosure can be used to sort components of a sample, such as cells in a biological sample. The term "sorting," used herein in its conventional sense, refers to separating components of a sample (e.g., cells, non-cellular particles, such as biological macromolecules) and, in some instances, transferring the separated components to a receiving location having one or more containers, as described below. For example, the target particle sorting module can be configured to sort samples having two or more components, such as three or more components, such as four or more components, such as five or more components, such as ten or more components, such as fifteen or more components, and includes sorting samples having 25 or more components. One or more sample components may be separated from the sample and transferred to containers, such as two or more sample components, such as three or more sample components, such as four or more sample components, such as five or more sample components, such as ten or more sample components, and includes 15 or more sample components that may be separated from the sample and transferred to containers at the receiving location.

[0181] The particle sorting module can be configured to generate a flow of analyzed droplets and deflect each analyzed droplet from the flow of analyzed droplets to a deflected droplet containment location (i.e., droplet deposition location). As used herein, the term "deflected droplet containment location" (i.e., droplet deposition location or location) refers to a location downstream of the droplet deflector (relative to the droplet flow) where sorted droplets containing the cells of interest can be collected after being deflected by the droplet deflector. As described above, droplets in the flow can be measured radially across a plane orthogonal to the longitudinal axis of the flow, and can be transferred by a distance of 0.001 mm or more from their normal trajectory along the longitudinal axis of the droplet flow, for example, 0.005 mm or more, for example, 0.01 mm or more, for example, 0.05 mm or more, for example, 0.1 mm or more, for example, 0.5 mm or more, for example, 1 mm or more, for example, 2 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, for example, 30 mm or more, for example, 35 mm or more, and including 50 mm or more. For example, droplets in a droplet stream can travel distances from 0.001 mm to 100 mm, such as 0.005 mm to 95 mm, 0.001 mm to 90 mm, 0.05 mm to 85 mm, 0.01 mm to 80 mm, 0.05 mm to 75 mm, 0.1 mm to 70 mm, 0.5 mm to 65 mm, 1 mm to 60 mm, 5 mm to 55 mm, and including 10 mm to 50 mm. Therefore, the droplet receiving location can be 0.001 mm or more from the longitudinal axis of the flow, for example, 0.005 mm or more, for example, 0.01 mm or more, for example, 0.05 mm or more, for example, 0.1 mm or more, for example, 0.5 mm or more, for example, 1 mm or more, for example, 2 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, for example, 30 mm or more, for example, 35 mm or more, and includes 50 mm or more from the longitudinal axis of the flow. The droplet receiving position of the target particle sorting module can be offset from the front-to-back axis of a horizontal plane perpendicular to the longitudinal axis of the flow, for example, an offset of one droplet diameter or less to 5 mm on the front-to-back axis, for example, one droplet diameter or less to 4 mm, for example, two droplet diameters to 3 mm, including two and a half droplet diameters to 2.5 mm.

[0182] In one embodiment, the target particle sorting module includes a droplet deflector comprising a first plate and a second plate configured to be offset from each other. In some embodiments, the first and second plates are configured to be adjustablely offset from each other. In one example, the first and second plates are configured to be adjustablely offset from each other relative to a horizontal plane. In other examples, the horizontal plane is perpendicular to the axis of the droplet flow.

[0183] In some embodiments of the particle sorter module, the first plate includes an elongated portion configured to allow the first plate to be adjustably offset relative to a horizontal plane relative to a second plate. In this embodiment, the elongated portion of the first plate may include a positioning screw configured to allow the first plate to be adjustably offset relative to a horizontal plane relative to a second plate.

[0184] In this example, the first and second plates of the particle sorting module are configured to be adjustablely offset from each other by more than 0 mm to 5 mm. In this example, the first and second plates are configured to be adjustablely offset from each other by an increment determined based on the thread pitch of the positioning screws configured to adjustably offset the first plate from the second plate.

[0185] In the example of the particle sorting module, the known deflection force is sufficient to offset the droplet deposition position by 2 mm or more, for example, when measured at a distance of 140 mm below the lowest point of the first deflection plate. Therefore, as described above, the known deflection force includes components on the "side-to-side" and "front-to-back" axes in a horizontal plane orthogonal to the longitudinal axis of the flow. In other examples, the known deflection force is sufficient to offset the droplet deposition position by one droplet diameter or less.

[0186] In some cases, the droplet deflector of the particle sorting module according to this disclosure is configured to deflect droplets into multiple partitions. In this case, the partitions may include collection containers. In other cases, the collection container may be a multi-well plate. In still other cases, the multi-well plate contains 1536 or fewer wells. In some instances, the partitions include collection tubes. In such instances, each collection tube has a diameter of 1.8 mm or less.

[0187] As described above, the droplet deflector of the target particle sorting module may include a first plate and a second plate configured to be parallel to each other. That is, even when offset from each other, the first plate and the second plate remain oriented in a parallel configuration. In other cases, the first plate and the second plate are configured to be adjustablely rotated to face each other. That is, as the offset between the first plate and the second plate is adjusted, the first plate and the second plate can be rotated accordingly so that the plates face each other.

[0188] In some cases, the second plate of the droplet deflector includes an elongated portion configured to allow the second plate to be adjustably offset relative to the horizontal plane relative to the first plate. In this case, the elongated portion of the second plate may include a positioning screw configured to allow the second plate to be adjustably offset relative to the horizontal plane relative to the first plate.

[0189] As described in more detail above, in some embodiments, the droplet deflector of the particle sorting module further includes an actuator (e.g., a motor) configured to adjust the offset between the first and second plates. As described above, in some embodiments, the actuator (e.g., a motor) is operatively connected to a feedback subsystem. In such embodiments, the feedback subsystem may include a controller operatively connected to the actuator (e.g., the motor) and to a detector configured to detect the offset distance of the droplet flow. In still other embodiments, the feedback subsystem is configured to iteratively adjust the offset between the first and second plates.

[0190] In one embodiment, the particle sorting module includes a droplet deflector, wherein the first plate and the second plate are metallic. A voltage can be applied to each metal plate of the droplet deflector, causing droplets flowing through them to be accelerated and deflected at multiple different angles based on the droplet's charge and the polarity of the charge. The voltage applied to the first and second metal plates to transfer the charged droplets can be 10 mV or greater, such as 25 mV or greater, such as 50 mV or greater, such as 100 mV or greater, such as 250 mV or greater, such as 50 mV or greater, such as 500 mV or greater, such as 750 mV or greater, such as 1000 mV or greater, such as 2500 mV or greater, such as 50000 V or greater, such as 10000 V or greater, such as 15000 V or greater, such as 25000 V or greater, such as 50000 V or greater, and includes 100000 V or greater. In some embodiments, the voltage applied to the first and second metal plates is from 0.5 kV to 15 kV, for example, from 1 kV to 15 kV, from 1.5 kV to 12.5 kV, and includes 2 kV to 10 kV. Therefore, the electric field strength between the metal plates ranges from 0.1 V / m to 1 × 10⁻⁶. 7 V / m, for example 0.5V / m to 5×10 6 For example, 1V / m to 1×10 6 V / m, for example, 5V / m to 5×10 5 V / m, for example, 10V / m to 1×10 5 V / m and includes 50V / m up to 5×10 4 V / m, for example 1×10 5 V / m to 2×10 6 V / m.

[0191] In this example, the metal plates of the droplet deflector are spaced 1 mm or more apart. In other examples, the metal plates are spaced 3 mm or more apart. In this example, the first and second plates are rectangular.

[0192] practicality

[0193] Particle sorters, methods, and particle sorting modules can be used in a variety of applications where it is necessary to sort particle components (e.g., cells) from samples (e.g., biological samples) in a fluid medium. In some embodiments, the particle sorters, methods, and particle sorting modules described herein can be used for flow cytometry characterization of biological samples labeled with fluorescent tags. In other embodiments, particle sorters, methods, and particle sorting modules can be used for the spectral analysis of emitted light. Furthermore, targeted particle sorters, methods, and particle sorting modules can be used to improve the efficiency of sorting samples (e.g., in a flowing stream). Improving the efficiency of sorting samples means that fewer sample particles (e.g., cells) may be wasted when sorting samples using targeted particle sorter methods and particle sorting modules (i.e., depositing particles, such as cells, so that they are not used). When droplets containing target particles (e.g., target cells) are deflected but not contained in a designated partition (e.g., a collection container), a lack of alignment between the deflected droplets and the partition occurs because the applied deflection force is not a known offset deflection force. Specifically, target particle sorters and methods can improve sorting efficiency and, in particular, reduce the number of wasted cells. In some instances, sorting efficiency can be improved to allow for the collection and sorting of more variations of particles when using the target particle sorter, method, and particle sorter module. Particle variations refer to, for example, cell phenotypes, such that a large number of different cell phenotypes can be sorted when using the embodiments described in this disclosure. The embodiments described in this disclosure can be used in flow cytometers that wish to provide improved cell sorting efficiency, enhanced particle collection, particle charging efficiency, more precise particle charging, and enhanced particle deflection during cell sorting.

[0194] Embodiments of this disclosure also reveal applications for cells that may need to be prepared from biological samples for research, laboratory testing, or therapeutic purposes. In some embodiments, the targeted methods and apparatus may facilitate the acquisition of single cells prepared from a target fluid or tissue biological sample. For example, the targeted methods and systems facilitate the acquisition of cells from fluid or tissue samples for use as research or diagnostic samples for diseases such as cancer. Similarly, the targeted methods and systems facilitate the acquisition of cells from fluid or tissue samples for therapeutic purposes. Compared to conventional flow cytometry systems, the methods and apparatus of this disclosure allow for the separation and collection of cells from biological samples (e.g., organs, tissues, tissue fragments, fluids) with increased efficiency and low cost.

[0195] Notwithstanding the appended claims, this disclosure is also defined by the following terms:

[0196] 1. A particle sorter, comprising:

[0197] Flow pool;

[0198] A light source used to illuminate the inquiry point of the flow cell;

[0199] A detector used to detect light from the point of inquiry;

[0200] A droplet generator, used to produce a droplet flow from fluid exiting a flow cell; and

[0201] A droplet deflector, configured to apply a known deflection force to the droplet stream.

[0202] 2. The particle sorter according to Clause 1, wherein the droplet deflector includes a first plate and a second plate configured to be offset from each other.

[0203] 3. The particle sorter according to Clause 2, wherein the first plate and the second plate are configured to be adjustablely offset from each other.

[0204] 4. The particle sorter according to Clause 3, wherein the first plate and the second plate are configured to be adjustablely offset from each other relative to a horizontal plane.

[0205] 5. The particle sorter according to Clause 4, wherein the horizontal plane is perpendicular to the axis of the droplet stream.

[0206] 6. The particle sorter according to Clause 4, wherein the first plate includes an elongated portion configured to allow the first plate to be adjustably offset relative to the horizontal plane relative to the second plate.

[0207] 7. The particle sorter according to Clause 6, wherein the extended portion of the first plate includes a positioning screw configured to allow the first plate to be adjustably offset relative to the horizontal plane relative to the second plate.

[0208] 8. The particle sorter according to any one of clauses 3 to 7, wherein the first plate and the second plate are configured to be offset from each other by more than 0 mm to 5 mm.

[0209] 9. The particle sorter according to Clause 7, wherein the first plate and the second plate are configured to be adjustablely offset from each other in increments determined by the thread pitch of the positioning screws.

[0210] 10. A particle sorter according to any one of clauses 1 to 9, wherein the known deflection force is sufficient to offset the droplet deposition position by 2 mm or more.

[0211] 11. A particle sorter according to any one of clauses 1 to 10, wherein a known deflection force is sufficient to offset the droplet deposition position by one droplet diameter or less.

[0212] 12. The particle sorter according to any one of clauses 1 to 11 further includes a plurality of partitions configured to contain droplets deflected by droplet deflectors.

[0213] 13. The particle sorter according to Clause 12, wherein the partition includes a collection container.

[0214] 14. The particle sorter according to Clause 13, wherein the collection container is a multi-well plate.

[0215] 15. The particle sorter according to Clause 14, wherein the multi-well plate comprises 1536 or fewer wells.

[0216] 16. The particle sorter according to Clause 12, wherein the partition includes a collection tube.

[0217] 17. The particle sorter as described in Clause 12, wherein the diameter of each partition is 1.8 mm or less.

[0218] 18. The particle sorter according to any one of clauses 2 to 9, wherein the first plate and the second plate are configured to be parallel to each other.

[0219] 19. The particle sorter according to any one of clauses 3 to 9, wherein the first plate and the second plate are configured to be adjustablely rotated to face each other.

[0220] 20. The particle sorter according to Clause 6, wherein the second plate includes an elongated portion configured to allow the second plate to be adjustably offset relative to the first plate relative to a horizontal plane.

[0221] 21. The particle sorter according to Clause 20, wherein the extended portion of the second plate includes a positioning screw configured to allow the second plate to be adjustably offset relative to the first plate relative to a horizontal plane.

[0222] 22. The particle sorter according to any one of clauses 3 to 9, wherein the droplet deflector further includes an actuator configured to adjust the offset between the first plate and the second plate.

[0223] 23. The particle sorter according to Clause 22, wherein the actuator is operatively connected to the feedback subsystem.

[0224] 24. The particle sorter according to Clause 23, wherein the feedback subsystem includes a controller operatively connected to the actuator and to a detector configured to detect the offset distance of droplets in the droplet stream.

[0225] 25. The particle sorter according to any one of clauses 23 to 24, wherein the feedback subsystem is configured to iteratively adjust the offset between the first plate and the second plate.

[0226] 26. The particle sorter according to any one of clauses 2 to 9, wherein the first plate and the second plate are metallic.

[0227] 27. The particle sorter according to Clause 26, wherein the metal plates are spaced apart by 1 mm or more.

[0228] 28. The particle sorter according to Clause 26, wherein the metal plates are spaced apart by 3 mm or more.

[0229] 29. The particle sorter according to any one of clauses 2 to 9, wherein the first plate and the second plate are rectangular.

[0230] 30. The particle sorter according to any one of clauses 1 to 29, wherein the light source is a laser.

[0231] 31. A method comprising:

[0232] Illuminate the inquiry point in the flow cell with a light source;

[0233] The light from the point of inquiry is detected using a detector;

[0234] A droplet generator is used to produce droplet flow from the fluid exiting the flow cell; and

[0235] The droplets of a droplet stream are deflected using a droplet deflector configured to apply a known deflection force to the droplet stream.

[0236] 32. The method according to Clause 31, wherein the droplet deflector includes a first plate and a second plate configured to be offset from each other.

[0237] 33. The method according to Clause 32, wherein the first plate and the second plate are configured to be adjustablely offset from each other.

[0238] 34. The method according to Clause 33, wherein the first plate and the second plate are configured to be adjustablely offset from each other relative to the horizontal plane.

[0239] 35. The method according to Clause 34, wherein the horizontal plane is perpendicular to the axis of the droplet flow.

[0240] 36. The method according to Clause 34, wherein the first plate includes an elongated portion configured to allow the first plate to be adjustably offset relative to the horizontal plane relative to the second plate.

[0241] 37. The method according to Clause 36, wherein the extended portion of the first plate includes a positioning screw configured to allow the first plate to be adjustably offset relative to the horizontal plane relative to the second plate.

[0242] 38. The method according to any one of clauses 33 to 37, wherein the first plate and the second plate are configured to be offset from each other by more than 0 mm to 5 mm.

[0243] 39. The method according to Clause 37, wherein the first plate and the second plate are configured to be adjustablely offset from each other in increments determined by the thread pitch of the positioning screws.

[0244] 40. The method according to any one of clauses 31 to 39, wherein the known deflection force is sufficient to deflect the droplet deposition position by 2 mm or more.

[0245] 41. The method according to any one of clauses 31 to 40, wherein the known deflection force is sufficient to offset the droplet deposition position by one droplet diameter or less.

[0246] 42. The method according to any one of clauses 31 to 41, wherein the droplet deflector is further configured to deflect the droplet into a plurality of partitions.

[0247] 43. The method according to Clause 42, wherein the partition includes a collection container.

[0248] 44. The method according to Clause 43, wherein the collection container is a multi-well plate.

[0249] 45. The method according to Clause 44, wherein the multi-well plate comprises 1536 or fewer wells.

[0250] 46. ​​The method according to Clause 42, wherein the partition includes a collection tube.

[0251] 47. The method described in Clause 42, wherein the diameter of each partition is 1.8 mm or less.

[0252] 48. The method according to any one of clauses 32 to 39, wherein the first plate and the second plate are configured to be parallel to each other.

[0253] 49. The method according to any one of clauses 32 to 39, wherein the first plate and the second plate are configured to rotate adjustablely to face each other.

[0254] 50. The method according to Clause 36, wherein the second plate includes an elongated portion configured to allow the second plate to be adjustably offset relative to the first plate relative to a horizontal plane.

[0255] 51. The method according to Clause 50, wherein the elongated portion of the second plate includes a positioning screw configured to allow the second plate to be adjustably offset relative to the first plate relative to a horizontal plane.

[0256] 52. The method according to any one of clauses 33 to 39, wherein the droplet deflector further includes an actuator configured to adjust the offset between the first plate and the second plate.

[0257] 53. The method according to Clause 52, wherein the actuator is operatively connected to the feedback subsystem.

[0258] 54. The method according to Clause 53, wherein the feedback subsystem includes a controller operatively connected to the actuator and to a detector configured to detect the offset distance of droplets in the droplet flow.

[0259] 55. The method according to any one of clauses 52 to 54, wherein the feedback subsystem is configured to iteratively adjust the offset between the first plate and the second plate.

[0260] 56. The method according to any one of clauses 32 to 39, wherein the first plate and the second plate are metallic.

[0261] 57. The method according to Clause 56, wherein the metal plates are spaced apart by 1 mm or more.

[0262] 58. The method according to Clause 56, wherein the metal plates are spaced apart by 3 mm or more.

[0263] 59. The method according to any one of clauses 32 to 39, wherein the first plate and the second plate are rectangular.

[0264] 60. The method according to any one of clauses 31 to 59, wherein the flow is irradiated by a laser.

[0265] 61. A particle sorting module including a droplet deflector configured to apply a known offset deflection force to a droplet stream.

[0266] 62. The particle sorting module according to Clause 61, wherein the droplet deflector includes a first plate and a second plate configured to be offset from each other.

[0267] 63. The particle sorting module according to Clause 62, wherein the first plate and the second plate are configured to be adjustablely offset from each other.

[0268] 64. The particle sorting module according to Clause 63, wherein the first plate and the second plate are configured to be adjustablely offset from each other relative to a horizontal plane.

[0269] 65. The particle sorting module according to Clause 64, wherein the horizontal plane is perpendicular to the axis of the droplet stream.

[0270] 66. The particle sorting module according to Clause 64, wherein the first plate includes an elongated portion configured to allow the first plate to be adjustably offset relative to the horizontal plane relative to the second plate.

[0271] 67. The particle sorting module according to Clause 66, wherein the extended portion of the first plate includes a positioning screw configured to allow the first plate to be adjustably offset relative to the horizontal plane relative to the second plate.

[0272] 68. The particle sorting module according to any one of clauses 63 to 67, wherein the first plate and the second plate are configured to be offset from each other by more than 0 mm to 5 mm.

[0273] 69. The particle sorting module according to Clause 67, wherein the first plate and the second plate are configured to be adjustablely offset from each other in increments determined by the thread pitch of the positioning screws.

[0274] 70. A particle sorting module according to any one of clauses 61 to 69, wherein the known deflection force is sufficient to offset the droplet deposition position by 2 mm or more.

[0275] 71. A particle sorting module according to any one of clauses 61 to 70, wherein the known deflection force is sufficient to offset the droplet deposition position by one droplet diameter or less.

[0276] 72. The particle sorting module according to any one of clauses 61 to 71, wherein the droplet deflector is further configured to deflect droplets into multiple partitions.

[0277] 73. The particle sorting module according to Clause 72, wherein the partition includes a collection container.

[0278] 74. The particle sorting module according to Clause 73, wherein the collection container is a multi-well plate.

[0279] 75. The particle sorting module according to Clause 74, wherein the multi-well plate comprises 1536 or fewer wells.

[0280] 76. The particle sorting module according to Clause 75, wherein the partition includes a collection tube.

[0281] 77. The particle sorting module according to Clause 72, wherein the diameter of each partition is 1.8 mm or less.

[0282] 78. The particle sorting module according to any one of clauses 62 to 69, wherein the first plate and the second plate are configured to be parallel to each other.

[0283] 79. The particle sorting module according to any one of clauses 63 to 69, wherein the first plate and the second plate are configured to rotate adjustablely to face each other.

[0284] 80. The particle sorting module according to Clause 66, wherein the second plate includes an elongated portion configured to allow the second plate to be adjustably offset relative to the first plate relative to a horizontal plane.

[0285] 81. The particle sorting module according to Clause 80, wherein the extended portion of the second plate includes a positioning screw configured to allow the second plate to be adjustably offset relative to the first plate relative to a horizontal plane.

[0286] 82. The particle sorting module according to any one of clauses 63 to 69, wherein the droplet deflector further includes an actuator configured to adjust the offset between the first plate and the second plate.

[0287] 83. The particle sorting module according to Clause 82, wherein the actuator is operatively connected to the feedback subsystem.

[0288] 84. The particle sorting module according to Clause 83, wherein the feedback subsystem includes a controller operatively connected to the actuator and to a detector configured to detect the offset distance of droplets in the droplet stream.

[0289] 85. A particle sorting module according to any one of clauses 83 to 84, wherein the feedback subsystem is configured to iteratively adjust the offset between the first plate and the second plate.

[0290] 86. The particle sorting module according to any one of clauses 62 to 69, wherein the first plate and the second plate are metallic.

[0291] 87. The particle sorting module according to Clause 86, wherein the metal plates are spaced apart by 1 mm or more.

[0292] 88. The particle sorting module according to Clause 86, wherein the metal plates are spaced apart by 3 mm or more.

[0293] 89. The particle sorting module according to any one of clauses 62 to 69, wherein the first plate and the second plate are rectangular.

[0294] Although the foregoing invention has been described in some detail by way of illustration and example for the purpose of clarity, it will be apparent to those skilled in the art, based on the teachings of the invention, that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

[0295] Therefore, the foregoing merely illustrates the principles of the invention. It should be understood that, although not explicitly described or shown herein, those skilled in the art will be able to design various devices embodying the principles of the invention and included within its spirit and scope. Furthermore, all examples and conditional language listed herein are primarily intended to assist the reader in understanding the principles of the invention and the inventor's concepts for contributing to the field, and should be interpreted as not being limited to these specifically listed examples and conditions. Moreover, all statements herein referencing the principles, aspects, and embodiments of the invention and their specific examples are intended to cover their structural and functional equivalents. Furthermore, these equivalents are intended to include both currently known equivalents and future-developed equivalents, i.e., any element developed that performs the same function regardless of its structure. Furthermore, nothing disclosed herein is intended to be offered to the public, whether or not such disclosure is expressly recited in the claims.

[0296] Therefore, the scope of the invention is not intended to be limited to the exemplary embodiments shown and described herein. Rather, the scope and spirit of the invention are embodied in the appended claims. In the claims, 35U.SC §112(f) or 35U.SC §112(6) is explicitly defined as referring to the limitation in the claim only when the exact phrase “means as” or the exact phrase “step as” is used at the beginning of such limitation in the claim; if such an exact phrase is not used in the limitation of the claim, then 35U.SC §112(f) or 35U.SC §112(6) is not referred to.

Claims

1. A particle sorter, comprising: Flow pool; A light source used to illuminate the inquiry point of the flow cell; A detector used to detect light from the point of inquiry; A droplet generator, used to produce droplet streams from fluid flowing out of a flow cell; and A droplet deflector configured to apply a known deflection force to a droplet stream, wherein the droplet deflector includes a first plate and a second plate configured to be adjustablely offset from each other relative to a horizontal plane perpendicular to the axis of the droplet stream; The first plate includes an elongated portion configured to allow the first plate to be adjustably offset relative to the horizontal plane relative to the second plate; The extended portion of the first plate includes a positioning screw, which is configured to allow the first plate to be adjustably offset relative to the horizontal plane relative to the second plate; Among them, the known deflection forces are sufficient to shift the droplet deposition position by one droplet diameter or less.

2. The particle sorter of claim 1 further includes a plurality of partitions configured to accommodate droplets deflected by droplet deflectors.

3. The particle sorter according to claim 2, wherein, The partition includes a collection container.

4. The particle sorter according to claim 1, wherein, The droplet deflector also includes an actuator configured to adjust the offset between the first plate and the second plate.

5. The particle sorter according to claim 4, wherein, The actuator is operatively connected to the feedback subsystem.

6. The particle sorter according to claim 5, wherein, The feedback subsystem includes a controller operatively connected to the actuator and to a detector configured to detect the distance of droplet deflection in the droplet flow.

7. The particle sorter according to claim 3, wherein, The collection container includes a multi-well plate or a collection pipe.

8. A particle sorting method, comprising: Illuminate the inquiry point in the flow cell with a light source; The light from the point of inquiry is detected using a detector; A droplet generator is used to produce droplet flow based on the fluid flowing out of the flow cell; and A droplet deflector is used to deflect the droplets of a droplet stream, the droplet deflector being configured to apply a known deflection force to the droplet stream, wherein the droplet deflector includes a first plate and a second plate configured to be adjustablely offset from each other relative to a horizontal plane perpendicular to the axis of the droplet stream. The first plate includes an elongated portion configured to allow the first plate to be adjustably offset relative to the horizontal plane relative to the second plate; The extended portion of the first plate includes a positioning screw, which is configured to allow the first plate to be adjustably offset relative to the horizontal plane relative to the second plate; Among them, the known deflection forces are sufficient to shift the droplet deposition position by one droplet diameter or less.

9. A particle sorting module comprising a droplet deflector configured to apply a known offset deflection force to a stream of droplets, wherein the droplet deflector includes a first plate and a second plate configured to be adjustablely offset from each other relative to a horizontal plane perpendicular to the axis of the droplet stream; in, The first plate includes an elongated portion configured to allow the first plate to be adjustably offset relative to the horizontal plane relative to the second plate; The extended portion of the first plate includes a positioning screw, which is configured to allow the first plate to be adjustably offset relative to the horizontal plane relative to the second plate; Among them, the known deflection forces are sufficient to shift the droplet deposition position by one droplet diameter or less.

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