Particle sorting module, system, and method of use having an alignment window.

Abstract

To provide a particle sorting module with an alignment window, systems, and methods of use thereof.SOLUTION: The present disclosure includes a particle sorting module having an opening that is configured for visualizing droplets of a deflected flow stream. Particle sorting modules according to certain embodiments include a housing having a proximal end, a distal end and a wall therebetween having an opening positioned in the wall that is configured for aligning the flow stream with one or more sample containers at the distal end. Systems and methods for aligning a flow stream with one or more sample containers and sorting particles of a sample (e.g., a biological sample) are also provided. Kits having one or more of the particle sorting modules suitable for coupling with a particle sorting system and for practicing the subject methods are also described.SELECTED DRAWING: Figure 1

Description

[[BACKGROUND ART]]

[0001] Flow-based particle sorting systems such as sorting flow cytometers are used to sort particles in a fluid sample based on at least one measured characteristic of the particles.

[0002] In a flow-based particle sorting system, particles such as molecules, analyte-bound beads, or individual cells in a fluid suspension pass through a stream via a detection region where a sensor detects particles of the type to be sorted contained in the stream. Upon detecting a particle of the type to be sorted, the sensor triggers a sorting mechanism that selectively isolates the particle of interest.

[0003] Particle detection is generally performed by exposing particles to irradiation light from one or more lasers and passing a fluid stream through a detection region where the light scattering characteristics and fluorescence characteristics of the particles are measured. Particles or components thereof can be labeled with fluorescent dyes to facilitate detection, and a number of different particles or components can be detected simultaneously by labeling different particles or components with spectrally distinct fluorescent dyes. Detection is performed using one or more photosensors to facilitate independent measurement of the fluorescence from each different fluorescent dye.

[0004] To sort particles in a sample, a droplet charging mechanism charges a droplet of the flow stream that contains a particle of the type to be sorted with an electric charge at the break-off point of the flow stream. Droplets are passed through an electrostatic field and are deflected into one or more collection containers based on the polarity and magnitude of the charge of the droplet. Uncharged droplets are not deflected by the electrostatic field. [[SUMMARY OF THE INVENTION]]

[0005] Aspects of the present disclosure include a particle sorting module having an opening configured to visualize droplets in a deflected flowstream. A particle sorting module according to a particular embodiment comprises a housing having a proximal end, a distal end, and a wall between them, the wall having an opening located within the wall, configured to align the flowstream with one or more sample containers at the distal end. In some embodiments, the opening is located in the wall downstream from a droplet deflector that deflects droplets in the flowstream into one or more sample containers. In some examples, the opening is located 0.5 cm to 5 cm from the droplet deflector. In other examples, the opening is located 0.5 cm to 5 cm from the sample container at the distal end of the particle sorting module. In some embodiments, the opening includes an optical adjustment component, such as a focusing lens, for propagating light from an illumination source onto the deflected flowstream. In other embodiments, the optical adjustment component is a transparent optical window. In certain examples, a transparent optical window may include one or more reference identifiers, such as one or more markings associated with the boundaries of one or more sample containers at the distal end of the housing. In some examples, the reference identifiers include a marking indicating the center of the distal end of the housing. In certain examples, the particle sorting module comprises a waste liquid container coupled to the distal end and two sample containers. In some embodiments, the transparent optical window includes markings associated with the boundaries of the waste liquid container and the two sample containers. In certain embodiments, the particle sorting module of interest comprises a flow cell nozzle located at the proximal end of the housing and a sample inspection area that is in fluid communication with this flow cell nozzle. In some examples, the sample inspection area has a cuvette (e.g., a glass or plastic cuvette). In some particle sorting modules, the cuvette is co-molded. In other particle sorting modules, the cuvette is fixed with adhesive. In yet another particle sorting module, the cuvette is integral with the particle sorting module. The particle sorting module may also have one or more of the following at its proximal end: a sample inlet and a sheath fluid inlet.

[0006] Aspects of the present disclosure also include systems for aligning a flowstream with one or more sample containers and separating particles from a sample (e.g., a biological sample). A system according to a particular embodiment comprises, for example, a particle sorting module having an opening for visualizing droplets of a deflected flowstream, as described above, and an imaging sensor configured to capture one or more images of the deflected flowstream through the opening. In some embodiments, the system comprises a laser for illuminating the flowstream through the opening. The opening may include one or more optical adjustment components, such as a focusing lens or a transparent optical window. In a particular embodiment, the system comprises a processor having a memory operably coupled to the processor, which includes stored instructions, which, when executed by the processor, cause the processor to capture an image of the opening of the particle sorting module to obtain an imaged opening, and to calculate the pixel position of the boundary within the imaged opening for each of the ports at the distal end of the housing along the horizontal axis. In these embodiments, calculating the pixel position may include calculating the total number of pixels across the entire imaged aperture along the horizontal axis and multiplying the total number of pixels by a predetermined ratio. In a particular example, the predetermined ratio is calculated by dividing the number of pixels at the boundary of each port within the imaged aperture by the total number of pixels across the entire imaged aperture along the horizontal axis. In some embodiments, the system includes a memory with stored instructions, which, when executed by the processor, cause the processor to align the flow stream with one of the ports at the distal end of the housing. In other embodiments, the system includes a memory with stored instructions, which, when executed by the processor, cause the processor to align the flow stream with the center of one of the ports at the distal end of the housing. In yet another embodiment, the system includes a memory with stored instructions, which, when executed by the processor, cause the processor to align the flow stream with the center of the distal end of the housing.The system of interest is configured to separate particles of a sample (e.g., a biological sample) in a flowstream. In some embodiments, the system further comprises a sample fluid delivery subsystem and a sheath fluid delivery subsystem that fluidly communicates with the inlet at the proximal end of the particle separation module. In certain examples, a waste liquid tank is coupled to the outlet from the particle separation module.

[0007] Aspects of the present disclosure also include methods for aligning a flowstream with one or more ports at the distal end of a particle sorting module. Methods according to certain embodiments include capturing an image through an opening in a particle sorting module configured to visualize droplets of the flowstream, calculating the pixel position of the boundary of each port at the distal end within the imaged opening along the horizontal axis, and aligning the flowstream with the ports based on the calculated pixel position of the boundary of each port within the imaged opening. In some embodiments, calculating the pixel position includes calculating the total number of pixels in the entire imaged opening along the horizontal axis, and multiplying the total number of pixels by a predetermined ratio. In certain examples, the method further includes calculating the predetermined ratio by dividing the number of pixels of the boundary of each port within the imaged opening by the total number of pixels in the entire imaged opening along the horizontal axis. In some embodiments, the method includes illuminating the flowstream through the opening with a light source such as a laser. Methods according to certain examples also include sorting particles in the flowstream. In some embodiments, the method includes irradiating a sample having particles in a flow stream within an inspection area of ​​a particle sorting module with a light source, detecting light from the sample, and applying a deflection force to the particles in the flow stream at the distal end of the particle sorting module to separate the particles into one or more sample containers. In certain embodiments, the sample is a biological sample, and the method includes sorting and collecting two or more different types of cells.

[0008] Kits having one or more of the particle sorting modules of the subject are also provided. A kit according to a particular embodiment comprises a particle sorting module having an opening configured to visualize droplets of a deflected flowstream and one or more sample collection containers for coupling with the particle sorting module. The kit of interest may also comprise a droplet deflector. In some embodiments, the particle sorting module has one or more of a sample inlet and a sheath fluid inlet, and the kit may comprise one or more connectors for coupling the particle sorting module to a sample fluid delivery system and a sheath fluid delivery system. [Brief explanation of the drawing]

[0009] The present invention can be best understood from the following detailed description when read in conjunction with the accompanying drawings. The drawings include the following figures.

[0010] [Figure 1] An exemplary calculation for determining the boundary of the sample collection port at the distal end of the particle sorting module, according to a specific embodiment, is shown. [Figure 2] This shows a particle sorting module having an opening configured to visualize droplets in a deflected flowstream, according to a specific embodiment. [Modes for carrying out the invention]

[0011] Aspects of the present disclosure include particle sorting modules having openings configured to visualize droplets of a deflected flowstream. A particle sorting module according to a particular embodiment comprises a housing having a proximal end, a distal end, and a wall between them, the wall having openings located within the wall, configured to align the flowstream with one or more sample containers at the distal end. Systems and methods for aligning a flowstream with one or more sample containers and sorting particles of a sample (e.g., a biological sample) are also provided. Kits comprising one or more particle sorting modules suitable for coupling with a particle sorting system and for carrying out the methods of the subject are also described.

[0012] Before the present invention is described in detail, it should be understood that the present invention is not limited to the specific embodiments described and is therefore naturally modifiable. Furthermore, since the scope of the present invention is limited only by the appended claims, it should also be understood that the terms used herein are merely for describing specific embodiments and are not intended to limit them.

[0013] Where a range of values ​​is provided, unless the context explicitly indicates otherwise, it is understood that each intermediate value between the upper and lower limits of that range, up to one-tenth of the lower limit unit, and any other described values ​​or intermediates within the range described herein are included in the invention. The upper and lower limits of these smaller ranges may independently be included in smaller ranges and are likewise included in the invention, according to any specifically excluded limits in the described range. Where a described range includes one or both limits, ranges excluding either or both of those included limits are also likewise included in the invention.

[0014] In this specification, the term “approximately” is used to indicate a specific range of numbers. The term “approximately” is used in this specification to provide literal support for the exact number that precedes it, as well as for numbers that are close to or approximate it. When determining whether a number is close to or approximates a specifically stated number, the close or approximate unstated number may be a number that, in the context in which it is presented, provides a substantial equivalent of the specifically stated number.

[0015] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art in which the present invention pertains. Any methods and materials similar or equivalent to those described herein may also be used in carrying out or testing the present invention, but representative exemplary methods and materials are described below.

[0016] All publications and patents cited herein 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 herein by reference to disclose and describe methods and / or materials, and publications are cited in relation to those methods and / or materials. Any citation of a publication relates to its disclosure prior to the filing date, and the present invention should not be construed as acknowledging that such publication has no prior rights on the grounds of prior invention. Furthermore, the publication date provided may differ from the actual publication date, and the actual publication date may need to be independently verified.

[0017] Where used herein and in the appended claims, the singular forms “a,” “an,” and “the” refer to multiple subjects unless explicitly indicated otherwise in the context. Furthermore, it should be noted that claims may be drafted to exclude any alternative elements. Therefore, this statement is intended to function as an antecedent for the use of exclusive terms such as “solely” and “only,” or for the use of “negative” limitation, in relation to the enumeration of claim elements.

[0018] As will become apparent to those skilled in the art by reading this disclosure, each of the individual embodiments described and illustrated herein has separate components and features that can be readily separated from or combined with any of the features of several other embodiments without departing from the scope or spirit of the invention. Any described method may be carried out in the order of the described events or in any other logically possible order.

[0019] Apparatus and methods have been, or will be, described with a functional description for grammatical fluidity, but unless explicitly stated under Section 112 of the U.S. Patent Act, the claims should not necessarily be interpreted as being limited by a “means” or “step” limitation, but should be granted the full scope of the meaning and equivalents of the definitions provided by the claims under the doctrine of legal equivalents, and if the claims are explicitly stated under Section 112 of the U.S. Patent Act, they should be granted the full legal equivalents under Section 112 of the U.S. Patent Act.

[0020] As summarized above, the Disclosure provides a particle sorting module for sorting particulate components of a sample, such as cells in a biological sample. In a further description of embodiments of the Disclosure, a particle sorting module having an opening configured to visualize droplets of a deflected flow stream (for example, to align a deflected flow stream with one or more sample collection containers) is first described in more detail. Next, a particle sorting system and method for separating particles in a sample is described. Kits comprising one or more particle sorting modules suitable for coupling with a particle sorting system and for carrying out the method of the subject are also provided.

[0021] Particle sorting module with openings for visualizing flow stream As summarized above, aspects of the present disclosure include a particle sorting module having an opening configured to visualize droplets in a deflected flowstream. In embodiments, the particle sorting module of the subject is configured to sort components of a sample, such as cells in a biological sample. The term “sorting” is used herein in a general sense to mean separating components of a sample (e.g., non-cellular particles such as cells and biomolecules) and, in some examples, delivering the separated components to one or more ports coupled to a sample collection container, as described below. For example, the particle sorting module of the subject may be configured to sort samples having two or more components, including sorting samples having, for example, three or more components, for example, four or more components, for example, five or more components, for example, ten or more components, for example, fifteen or more components, and twenty-five or more components. One or more sample components may be separated from the sample and delivered to a sample collection container, for example, 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 fifteen or more sample components may be separated from the sample and delivered to a sample collection container at the receiving site.

[0022] In embodiments, the particle sorting module includes an opening (e.g., as described in more detail below) for visualizing droplets deflected by deflection forces applied from one or more droplet deflectors. In some embodiments, the particle sorting module is configured to generate a stream of analyzed droplets and deflect each analyzed droplet from the stream of analyzed droplets to a deflected droplet receiving position. As used herein, the term “deflected droplet receiving position” refers to a location downstream from the droplet deflector where sorted droplets containing particles of interest (e.g., cells) can be collected after being deflected by the droplet deflector plate. In embodiments, droplets in a flow stream are deflected from their normal trajectory along the longitudinal axis of the flow stream. In some examples, droplets in a flow stream are deflected from their normal trajectory at a breakoff point in the flow stream. A “breakoff point” means a point in the flow stream where a continuous flow stream begins to form separate droplets. In other examples, droplets in a flowstream are deflected from their normal trajectory at locations downstream of the breakoff point, including locations 50 mm or more downstream of the breakoff point in the flowstream, where the droplets are deflected from their normal trajectory by a droplet deflector.

[0023] In the embodiment, the droplet is deflected from its normal trajectory by a distance of 0.001 mm or more when measured radially across a plane perpendicular to the longitudinal axis of the flowstream, including, 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 50 mm or more, when measured radially. For example, droplets in a flowstream can be spaced as far as 0.001mm to 100mm, including, for example, 0.005mm to 95mm, 0.001mm to 90mm, 0.05mm to 85mm, 0.01mm to 80mm, 0.05mm to 75mm, 0.1mm to 70mm, 0.5mm to 65mm, 1mm to 60mm, 5mm to 55mm, and 10mm to 50mm. Therefore, droplets within the flowstream are deflected from their trajectory along the longitudinal axis of the flowstream by an angle of 0.01° to 90°, including, for example, 0.05° to 85°, 0.1° to 80°, 0.5° to 75°, 10° to 70°, 15° to 65°, 20° to 60°, 25° to 55°, and 30° to 50°.

[0024] The particle sorting module of interest may include one or more sample collection ports at the droplet receiving location (e.g., at the distal end of the particle sorting module). Each sample collection port includes an opening (i.e., an open space through which the droplet passes) for receiving droplets from the flowstream. In embodiments, each opening of the sample collection port at the distal end of the particle sorting module described herein may independently be 0.001 mm or more from the longitudinal axis of the flowstream, including, 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 50 mm or more. For example, each opening of the sample collection port at the distal end of the particle sorting module may be in the range of 0.001 mm to 100 mm, including, for example, 0.01 mm to 90 mm, for example, 0.1 mm to 80 mm, for example, 0.5 mm to 70 mm, for example, 1 mm to 60 mm, and 5 mm to 50 mm. Each opening of the sample collection port at the distal end of the particle sorting module may be at a different angle with respect to the longitudinal axis of the flowstream, independently of each other, including, for example, 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 30° to 50°. In embodiments where the distal end of the particle sorting module includes two or more sample collection ports, the spacing between the openings of each sample collection port may be 0.001 mm or more from the longitudinal axis of the flowstream, independently including, 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 50 mm or more.For example, the spacing between the openings of each sample collection port can vary independently from 0.001 mm to 100 mm, including, for example, 0.01 mm to 90 mm, 0.1 mm to 80 mm, 0.5 mm to 70 mm, 1 mm to 60 mm, and 5 mm to 50 mm.

[0025] In certain examples, the opening of each sample collection port may include a cover, such as a valve, which can be reversibly closed as needed. For example, the opening of a sample collection port may include a valve configured to remain closed until a flow stream is detected. In other examples, the opening of a sample collection port may include a valve configured to remain closed until a sample collection container is coupled to the sample collection port. In yet another example, the opening of a sample collection port may include a valve configured to close after a predetermined period (for example, to collect only droplets from a specific portion of the sample). In yet another example, the opening of a sample collection port may include a valve configured to close after it is detected that a predetermined volume has been collected by the sample collection container coupled to the sample collection port. In certain examples, the opening of a sample collection port may be configured to close in response to a user input command.

[0026] The opening of the sample collection port at the distal end of the particle sorting module of interest may be any preferred shape, and the shape of the sample collection port opening of interest may include, for example, linear shapes such as squares, rectangles, trapezoids, triangles, and hexagons, curved shapes such as circles and ellipses, and irregular shapes such as a parabolic bottom joined to a planar top. In certain embodiments, the opening of the sample collection port has a circular shape. In other embodiments, the opening of the sample collection port has an elliptical shape. In yet another embodiment, the opening of the sample collection port has a square or rectangular shape.

[0027] The size of the opening of each sample collection port can vary independently within a range of 0.1 cm to 5 cm, including, for example, 0.25 cm to 4.5 cm, 0.5 cm to 4 cm, 0.75 cm to 3.5 cm, and 1 cm to 3 cm. If the opening of the sample collection port is circular, the opening may have a diameter in the range of 0.1 cm to 5 cm, including, for example, 0.25 cm to 4.5 cm, 0.5 cm to 4 cm, 0.75 cm to 3.5 cm, and 1 cm to 3 cm.

[0028] The particle sorting module of interest may include one or more sample collection ports at the distal end of the housing, for example, two or more, for example, three or more, for example, four or more, for example, five or more, for example, six or more, for example, seven or more, for example, eight or more, for example, nine or more, and ten or more sample collection ports. If the sample collection ports are coupled to sample collection containers (e.g., waste liquid collection containers), the particle sorting module may include one or more sample collection containers coupled to the particle sorting module housing, for example, two or more, for example, three or more, for example, four or more, for example, five or more, for example, six or more, for example, seven or more, for example, eight or more, for example, nine or more, and ten or more sample collection containers.

[0029] The sample collection container may be coupled to the sample collection port at the distal end of the particle sorting module by any convenient mounting protocol. In some embodiments, the sample collection container is mechanically coupled to the sample collection port by means of a Luer lock connection, sterile tube welding, or by screwing it to the sample collection port. In other embodiments, the sample collection container is fixed to the sample collection port by permanent or non-permanent adhesive. In yet another embodiment, the sample collection container is co-molded with the sample collection port. In yet another embodiment, the sample collection container is integrated with the particle sorting module housing such that the sample collection container and the particle sorting module housing form a single unit. In yet another example, the sample collection container may be fluidically coupled to the sample collection port, for example, via tubes configured to transport the sorted droplets, and such embodiments may provide sterile removal of the sorted droplets by cutting across the fluid transport structure, such as the tubes. Suitable sample collection containers for collecting droplets from a flowstream include, but are not limited to, test tubes, conical tubes, multi-compartment containers such as microtiter plates (e.g., 96-well plates), centrifuge tubes, culture tubes, microtubes, caps, cuvettes, bottles, linear polymer containers, and bags.

[0030] Diverting droplets of interest towards a sample collection container (e.g., through a sample collection port) can be achieved by a droplet deflector through the application of an electrostatic field, thereby electrostatically charging the droplets and deflecting the charged droplets from the flow stream. The voltage applied to the metal plate of the droplet deflector of the subject particle sorting module may be 10mV or more, including, for example, 25mV or more, for example, 50mV or more, for example, 75mV or more, for example, 100mV or more, for example, 250mV or more, for example, 500mV or more, for example, 750mV or more, for example, 1V or more, for example, 2.5V or more, for example, 5V or more, for example, 10V or more, for example, 25V or more, for example, 50V or more, and 100V or more, for example, 500V or more, for example, 1000V or more, for example, 5000V or more, for example, 10000V or more, for example, 15000V or more, for example, 25000V or more, for example, 50000V or more, and 100000V or more. In a particular embodiment, the voltage applied to each set of parallel metal plates is 0.5kV to 15kV, including, for example, 1kV to 15kV, for example, 1.5kV to 12.5kV, and 2kV to 10kV.

[0031] Particles in the flowstream include, but are not limited to, U.S. Patent Nos. 9,952,076, 9,933,341, 9,726,527, 9,453,789, 9,200,334, 9,097,640, 9,095,494, 9,092,034, 8,975,595, 8,753,573, 8,233,146, 8,140,300, 7,544,326, 7,201,875, and The cells may be deflected by any convenient deflector plate protocol, including the cell sorting deflector plates described in Nos. 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, and 5,602,039, and these disclosures are incorporated herein by reference in their entirety.In certain embodiments, the deflector plate is used with BD Biosciences FACSCanto® II flow cytometer, BD Accuri® flow cytometer, BD Biosciences FACSCelesta® flow cytometer, BD Biosciences FACSLyric® flow cytometer, BD Biosciences FACSVerse® flow cytometer, BD Biosciences FACSymphony® flow cytometer, BD Biosciences LSRFortessa® flow cytometer, BD Biosciences LSRFortess® 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® cell sorter, BD Biosciences Jazz® cell sorter, BD Biosciences Aria® cell sorter, and BD Includes charged plates for sorting cells in a flow stream, such as those used in flow cytometry systems, including the Biosciences FACSMelody® cell sorter.

[0032] The openings in the particle sorting module described herein are configured to visualize deflected droplets, and in certain examples, a sample collection container is provided at the droplet receiving position. As summarized above, in some embodiments, the particle sorting module comprises a housing having a proximal end, a distal end, and a wall between them, the wall having an opening located within the wall for visualizing deflected droplets. The opening may be of any preferred shape, and the shape of the visualization opening of interest may include, but is not limited to, linear shapes such as squares, rectangles, trapezoids, triangles, and hexagons, curved shapes such as circles and ellipses, and irregular shapes such as a parabolic bottom joined to a planar top. In certain embodiments, the opening in the wall of the particle sorting module housing has a circular shape. In other embodiments, the opening in the wall of the particle sorting module housing has an elliptical shape. In yet another embodiment, the opening in the wall of the particle sorting module housing has a square or rectangular shape.

[0033] The size of the opening in the wall of the particle sorting module housing may vary, and in some embodiments, has a width ranging from 0.5 cm to 10 cm, such as 5 cm, including for example 1 cm to 9.5 cm, such as 1.5 cm to 9 cm, such as 2 cm to 8.5 cm, such as 2.5 cm to 8 cm, such as 3 cm to 7.5 cm, such as 3.5 cm to 7 cm, such as 4 cm to 6.5 cm, and 4 cm to 6 cm, and has a length ranging from 1 cm to 20 cm, such as 10 cm, including for example 1.5 cm to 19.5 cm, such as 2 cm to 19 cm, such as 2.5 cm to 18.5 cm, such as 3 cm to 18 cm, such as 3.5 cm to 17.5 cm, such as 4 cm to 17 cm, such as 4.5 cm to 16.5 cm, and 5 cm to 15 cm. In certain embodiments, the opening in the wall of the particle sorting module housing is circular, and the diameter of the opening ranges from 0.5 cm to 10 cm, such as 5 cm, including for example 1 cm to 9.5 cm, such as 1.5 cm to 9 cm, such as 2 cm to 8.5 cm, such as 2.5 cm to 8 cm, such as 3 cm to 7.5 cm, such as 3.5 cm to 7 cm, such as 4 cm to 6.5 cm, and 4 cm to 6 cm. As will be described in more detail below, in certain examples, an image is captured through the opening in the particle sorting module, and the number of pixels across the horizontal axis of the opening is calculated. Depending on the size of the opening and each pixel size, the opening in the wall of the particle sorting module housing may vary, and in some examples, for example 5×10 1 pixels to 5×10 5 pixels, for example 1×10 2 pixels to 1×10 5 pixels, for example 5×10 2 pixels to 5×10 4 pixels, and 1×10 3 pixels to 1×10 4 pixels, and is configured to provide an image having a pixel range of 1×10 1 pixels to 1×10 6 pixels.

[0034] The opening in the wall of the particle sorting module housing is located between the droplet deflector and the distal end of the housing. Depending on the length of the particle sorting module, in some embodiments the upper boundary of the opening is located between 1 cm and 24.5 cm, between 1.5 cm and 24 cm, between 2 cm and 23.5 cm, between 2.5 cm and 23 cm, between 3 cm and 22.5 cm, between 3.5 cm and 22 cm, between 4 cm and 21.5 cm, between 4.5 cm and 21 cm, and between 0.5 cm and 25 cm from the bottom of the droplet deflector, including the case where the upper boundary of the opening in the wall of the particle sorting module housing is located between 5 cm and 20 cm from the bottom of the droplet deflector. In some embodiments, the lower boundary (i.e., bottom) of the opening in the wall of the particle sorting module housing is located 0.5 cm to 10 cm from the distal end of the housing, including, for example, 1 cm to 9.5 cm, 1.5 cm to 9 cm, 2 cm to 8.5 cm, 2.5 cm to 8 cm, 3 cm to 7.5 cm, 3.5 cm to 7 cm, 4 cm to 6.5 cm, and 4 cm to 6 cm, for example, 5 cm.

[0035] In some embodiments, an opening in the wall of the particle sorting module housing includes one or more optical adjustment components. "Optical adjustment" means that the light propagated through the opening, or the light collected from the flow stream irradiated through the opening, is modified as needed. The optical adjustment component can be any convenient device or structure that provides the desired modification of light, and may include, but is not limited to, optical windows (e.g., clear windows), lenses, mirrors, pinholes, slits, gratings, optical refractors, and any combination thereof. An opening in the wall of the particle sorting module housing may optionally include one or more optical adjustment components in the sample inspection area, for example, two or more, for example, three or more, for example, four or more, and five or more optical adjustment components.

[0036] In some examples, openings in the walls of the particle sorting module housing include transparent optical windows. “Optically transparent” means that the optical window does not restrict the propagation of at least one wavelength of light passing through it. In some embodiments, the optical window is optically transparent to one or more of ultraviolet, visible, and near-infrared light. In one embodiment, the optical window is transparent to ultraviolet light. In another embodiment, the optical window is transparent to visible light. In yet another embodiment, the optical window is transparent to near-infrared light. In yet another embodiment, the optical window is transparent to ultraviolet and visible light. In yet another embodiment, the optical window is transparent to visible and near-infrared light. In yet another embodiment, the optical window is transparent to ultraviolet, visible, and near-infrared light. Depending on the desired light propagation characteristics, transparent optical windows may be formed from any suitable material, such as quartz, glass, silica, titanium dioxide, zinc oxide, silicon nitride, carbon black, aluminum oxide, or other optically transparent polymers, but not limited to, polymers containing optically transparent polymers such as acrylic, acrylic / styrene, cycloolefin polymers, polycarbonate, polyester, and polystyrene. The optically transparent region may include, for example, over 10% or more, over 25% or more, over 50% or more, over 75% or more, over 90% or more, over 95% or more, and over 99% or more of the optical window, and may extend over 5% or more of the optical window. In certain embodiments, the entire optical window (i.e., 100%) is optically transparent. For example, the optically transparent region may extend over 5% to 100% of the optical window, including, for example, 10% to 90%, for example, 20% to 80%, for example, 30% to 70%, and 40% to 60%.

[0037] In certain embodiments, the optical window includes one or more reference identifiers. In some examples, the reference identifiers may include markings associated with the boundaries of the deflected flowstream. For example, the markings define the deflection path of the flowstream. These markings may be associated, for example, with the charge of the droplet deflection plate or the applied voltage to the droplet deflection plate. In other examples, the markings are associated with the boundaries of one or more ports at the distal end of the housing. For example, the optical window may include reference identifiers that define the boundaries of one or more ports coupled to the sample collection container. In some examples, the optical window includes reference identifiers for aligning the flowstream with one or more of the ports. For example, the optical window may include reference identifiers that indicate the location of the waste liquid collection port at the distal end of the particle sorting module housing. In certain embodiments, the optical window includes reference identifiers that indicate the center of the distal end of the housing. For example, the optical window may include reference identifiers for aligning the flowstream with the center of the distal end of the particle sorting module housing.

[0038] In some embodiments, the openings within the particle sorting module housing include optical adjustment components for adjusting the light irradiated onto the deflected flowstream by a light source (e.g., a laser). For example, optical adjustment may involve increasing the dimensions of the light, increasing the focal length of the light, or collimating the light. In some examples, optical adjustment is an expansion protocol for increasing the dimensions of the light (e.g., beam spot), such as increasing the dimensions by 5% or more, such as increasing the dimensions by 10% or more, such as 25% or more, such as 50% or more, and such as 75% or more. In other embodiments, optical adjustment includes focusing the collected light to reduce the dimensions of the light, such as reducing the dimensions of the beam spot by 5% or more, such as 10% or more, such as 25% or more, such as 50% or more, and such as 75% or more. In certain embodiments, optical adjustment includes collimating the light. The term “collimate” is used in a general sense to refer to optically adjusting the collinearity of light propagation, or reducing divergence from a common propagation axis by light. In some cases, collimation involves narrowing the spatial cross-section of a light beam (for example, reducing the beam profile of a laser).

[0039] In some embodiments, the opening within the particle sorting module housing includes a focusing lens having a magnification of 0.1 to 0.95, including, for example, a magnification of 0.2 to 0.9, a magnification of 0.3 to 0.85, a magnification of 0.35 to 0.8, a magnification of 0.5 to 0.75, and a magnification of 0.55 to 0.7, for example, a magnification of 0.6. For example, in certain examples, the focusing lens is a bichromatic reduction lens having a magnification of about 0.6. The focal length of the focusing lens can vary in the range of 5 mm to 20 mm, including, for example, focal lengths in the range of 6 mm to 19 mm, for example, 7 mm to 18 mm, for example, 8 mm to 17 mm, for example, 9 mm to 16 mm, and 10 mm to 15 mm. In certain embodiments, the focusing lens has a focal length of about 13 mm.

[0040] In other embodiments, the optical adjustment component is a collimator. The collimator can be any convenient collimating protocol, such as one or more mirrors or curved lenses, or a combination thereof. For example, in certain examples, the collimator is a single collimating lens. In other examples, the collimator is a collimating mirror. In yet another example, the collimator includes two lenses. In yet another example, the collimator includes a mirror and a lens. When the collimator includes one or more lenses, the focal lengths of the collimating lenses can vary in the range of 5mm to 40mm, including, for example, focal lengths in the range of 6mm to 37.5mm, 7mm to 35mm, 8mm to 32.5mm, 9mm to 30mm, 10mm to 27.5mm, 12.5mm to 25mm, and 15mm to 20mm.

[0041] In some embodiments, the particle sorting module also includes a flow cell nozzle having a nozzle orifice configured to allow a flow stream to flow through the flow cell nozzle, and one or more droplet deflectors of the subject described above, configured to apply a deflection force to the flow stream flowing between them. The particle sorting module of the subject includes a flow cell nozzle having an orifice for propagating a fluid sample to a sample inspection area, and in some embodiments, the flow cell nozzle includes a proximal cylindrical portion defining a longitudinal axis and a distal frustoconical portion terminating at a flat surface having a nozzle orifice, which is perpendicular to the longitudinal axis. The length of the proximal cylindrical portion (measured along the longitudinal axis) can vary in the range of 1 mm to 15 mm, including, for example, 1.5 mm to 12.5 mm, e.g., 2 mm to 10 mm, e.g., 3 mm to 9 mm, and 4 mm to 8 mm. The length of the distal frustoconical portion (measured along the longitudinal axis) can also vary in the range of 1 mm to 10 mm, including, for example, 2 mm to 9 mm, 3 mm to 8 mm, and 4 mm to 7 mm. The diameter of the flow cell nozzle chamber can vary in some embodiments in the range of 1 mm to 10 mm, including, for example, 2 mm to 9 mm, 3 mm to 8 mm, and 4 mm to 7 mm.

[0042] In certain examples, the nozzle chamber does not include a cylindrical portion, and the entire flow cell nozzle chamber is formed in a frustoconical shape. In these embodiments, the length of the frustoconical nozzle chamber (measured along the longitudinal axis lateral to the nozzle orifice) may range from 1 mm to 15 mm, including, for example, 1.5 mm to 12.5 mm, e.g., 2 mm to 10 mm, e.g., 3 mm to 9 mm, and 4 mm to 8 mm. The diameter of the proximal portion of the frustoconical nozzle chamber may range from 1 mm to 10 mm, including, for example, 2 mm to 9 mm, e.g., 3 mm to 8 mm, and 4 mm to 7 mm.

[0043] In the embodiment, the sample flowstream originates from an orifice at the distal end of the flow cell nozzle. Depending on the desired characteristics of the flowstream, the flow cell nozzle orifice can be any suitable shape, and the cross-sectional shape of interest can be, but is not limited to, linear cross-sectional shapes such as square, rectangular, trapezoidal, triangular, and hexagonal; curved cross-sectional shapes such as circular and elliptical; and irregular shapes such as a parabolic bottom joined to a planar top. In a particular embodiment, the flow cell nozzle of interest has a circular orifice. The nozzle orifice size can vary in some embodiments from 1 μm to 20000 μm, including, for example, 2 μm to 17500 μm, 5 μm to 15000 μm, 10 μm to 12500 μm, 15 μm to 10000 μm, 25 μm to 7500 μm, 50 μm to 5000 μm, 75 μm to 1000 μm, 100 μm to 750 μm, and 150 μm to 500 μm. In certain embodiments, the nozzle orifice is 100 μm.

[0044] In some embodiments, the flow cell nozzle includes a sample injection port configured to supply the sample to the flow cell nozzle. In embodiments, the sample injection system is configured to provide a suitable flow of the sample into the flow cell nozzle chamber. Depending on the desired characteristics of the flowstream, the rate at which the sample is delivered into the flow cell nozzle chamber by the sample injection port may be 1 μL / second or more, including, for example, 2 μL / second or more, 3 μL / second or more, 5 μL / second or more, 10 μL / second or more, 15 μL / second or more, 25 μL / second or more, 50 μL / second or more, 100 μL / second or more, 150 μL / second or more, 200 μL / second or more, 250 μL / second or more, 300 μL / second or more, 350 μL / second or more, 400 μL / second or more, 450 μL / second or more, and 500 μL / second or more. For example, the sample flow rate may be in the range of 1 μL / sec to approximately 500 μL / sec, including, for example, 2 μL / sec to approximately 450 μL / sec, for example, 3 μL / sec to approximately 400 μL / sec, for example, 4 μL / sec to approximately 350 μL / sec, for example, 5 μL / sec to approximately 300 μL / sec, for example, 6 μL / sec to approximately 250 μL / sec, for example, 7 μL / sec to approximately 200 μL / sec, for example, 8 μL / sec to approximately 150 μL / sec, for example, 9 μL / sec to approximately 125 μL / sec, and 10 μL to approximately 100 μL / sec.

[0045] The sample injection port may be an orifice located within the wall of the nozzle chamber, or a conduit located at the proximal end of the nozzle chamber. If the sample injection port is an orifice located within the wall of the nozzle chamber, the sample injection port orifice may be any preferred shape, and the cross-sectional shape of interest may include, but is not limited to, linear cross-sectional shapes such as square, rectangular, trapezoidal, triangular, and hexagonal; curved cross-sectional shapes such as circular and elliptical; and irregular shapes such as a parabolic bottom joined to a planar top. In certain 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 certain examples, it may have an opening in the range of 0.1 mm to 5.0 mm, including, for example, 0.2 mm 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 1.25 mm to 1.75 mm, for example, 1.5 mm.

[0046] In certain examples, the sample injection port is a conduit located at the proximal end of the flow cell nozzle chamber. For example, the sample injection port may be a conduit positioned to have an orifice aligned with the flow cell nozzle orifice. When the sample injection port is a conduit positioned in line with the flow cell nozzle orifice, the cross-sectional shape of the sample injection tube can be any preferred shape, and the cross-sectional shape of interest may include, but is not limited to, linear cross-sectional shapes such as squares, rectangles, trapezoids, triangles, and hexagons; curved cross-sectional shapes such as circles and ellipses; and irregular shapes such as a parabolic bottom joined to a planar top. The orifice of the conduit may vary depending on its shape, and in certain examples, it may have an opening in the range of 0.1 mm to 5.0 mm, including, for example, 0.2 mm 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 1.25 mm to 1.75 mm, for example, 1.5 mm. The shape of the tip of the sample injection port may be the same as or different from the cross-sectional shape of the sample injection tube. For example, the orifice of the sample injection port may include a slanted tip with a bevel angle in the range of 1° to 10°, including, for example, bevel angles of 2° to 9°, for example, 3° to 8°, for example, 4° to 7°, and 5°.

[0047] In some embodiments, the flow cell nozzle also 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 flow of sheath fluid to the flow cell nozzle chamber, for example, together with the sample, to create a layered flow stream of sheath fluid surrounding the sample flow stream. Depending on the desired characteristics of the flow stream, the velocity of the sheath fluid delivered to the flow cell nozzle chamber may be 25 μL / s or more, including, for example, 50 μL / s or more, 75 μL / s or more, 100 μL / s or more, 250 μL / s or more, 500 μL / s or more, 750 μL / s or more, 1000 μL / s or more, and 2500 μL / s or more. For example, the flow rate of the sheath fluid may be in the range of 1 μL / sec to approximately 500 μL / sec, including, for example, 2 μL / sec to approximately 450 μL / sec, for example, 3 μL / sec to approximately 400 μL / sec, for example, 4 μL / sec to approximately 350 μL / sec, for example, 5 μL / sec to approximately 300 μL / sec, for example, 6 μL / sec to approximately 250 μL / sec, for example, 7 μL / sec to approximately 200 μL / sec, for example, 8 μL / sec to approximately 150 μL / sec, for example, 9 μL / sec to approximately 125 μL / sec, and 10 μL / sec to approximately 100 μL / sec.

[0048] In some embodiments, the sheath fluid injection port is an orifice located within the wall of the nozzle chamber. The orifice of the sheath fluid injection port can be any preferred shape, and the cross-sectional shape of interest is not limited to, but includes, for example, linear cross-sectional shapes such as square, rectangular, trapezoidal, triangular, and hexagonal; curved cross-sectional shapes such as circular and elliptical; and irregular shapes such as a parabolic bottom joined to a planar top. The size of the sample injection port orifice can vary depending on the shape, and in certain examples, it may have an opening in the range of 0.1 mm to 5.0 mm, including, 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 1.25 mm to 1.75 mm, for example, 1.5 mm.

[0049] The particle sorting module also includes a sample inspection area that is in fluid communication with the flow cell nozzle orifice. As will be described in more detail below, the sample flowstream originates from the orifice at the distal end of the flow cell nozzle, and particles in the flowstream can be irradiated by a light source in the sample inspection area of ​​the particle sorting module. The size of the inspection area of ​​the particle sorting module may vary depending on the characteristics of the flow nozzle, such as the size of the nozzle orifice and the size of the sample injection port. In embodiments, the inspection area may have a width of 0.01 mm or more, including, 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, 3 mm or more, for example, 5 mm or more, and 10 mm or more. The length of the inspection area can also vary, and in some examples, it can vary along a range of 0.01 mm or more, including, for example, 0.1 mm or more, for example, 0.5 mm or more, for example, 1 mm or more, for example, 1.5 mm or more, for example, 2 mm or more, for example, 3 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, and 50 mm or more.

[0050] The inspection area on the particle sorting module may be configured to facilitate the irradiation of a planar cross-section of the generated flowstream, or to facilitate the irradiation of a diffusion field (e.g., by a diffusion laser or lamp) of a predetermined length. In some embodiments, the inspection area on the particle sorting module includes a transparent window to facilitate the irradiation of a predetermined length of the generated flowstream, such as 1 mm or more, 2 mm or more, 3 mm or more, 4 mm or more, 5 mm or more, and 10 mm or more. Depending on the light source used to irradiate the generated flowstream (as described later), the inspection area of ​​the particle sorting module may be configured to allow light in the range of 100 nm to 1500 nm to pass through, for example, 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 500 nm to 800 nm. Therefore, the particle sorting module of the inspection area may be formed from any transparent material that allows a desired range of wavelengths to pass through, and such materials include, but are not limited to, optical glass, borosilicate glass, Pyrex® glass, ultraviolet quartz, infrared quartz, sapphire, and, among other polymeric plastic materials, polycarbonate, polyvinyl chloride (PVC), polyurethane, polyether, polyamide, polyimide, or other polymeric plastic materials, including polyester, copolymers of these thermoplastics such as PETG (glycol-modified polyethylene terephthalate), and, but are not limited to, poly(ethylene terephthalate) (PET), bottle-grade PET (copolymer made based on monoethylene glycol, terephthalic acid, and other comonomers such as isophthalic acid, cyclohexanedimethanol), poly(butylene terephthalate) (PBT), and poly(hexamethylene terephthalate), poly(ethylene adipate), poly(1,Poly(alkylene adipates) such as 4-butylene adipate and poly(hexamethylene adipate), poly(alkylene severates) such as poly(ethylene severate), poly(alkylene sebacate) such as poly(ethylene sebacate), poly(ε-caprolactone) and poly(β-propiolactone), poly(alkylene isophthalates) such as poly(ethylene isophthalate), poly(alkylene 2,6-naphthalene dicarboxylate) such as poly(ethylene 2,6-naphthalene dicarboxylate), poly(alkylene sulfonyl-4,4'-dibenzoate) such as poly(ethylene sulfonyl-4,4'-dibenzoate), poly(p-phenylene ethylene dicarboxylate) such as poly(p-phenylene alkylenedicarboxylate), poly(trans-1,4-cyclo Lactic acid polymers and copolymers such as poly(trans-1,4-cyclohexanediylalkylenedicarboxylate) such as hexanediylethylenedicarboxylate, poly(1,4-cyclohexane-dimethyleneethylenedicarboxylate) such as poly(1,4-cyclohexane-dimethyleneethylenedicarboxylate), poly([2.2.2]-bicyclooctane-1,4-dimethyleneethylenedicarboxylate) such as poly([2.2.2]-bicyclooctane-1,4-dimethylenealkylenedicarboxylate), lactic acid polymers and copolymers such as (S)-polylactide, (R,S)-polylactide, poly(tetramethylglycolide), and poly(lactide-co-glycolide), and bisphenol A, 3,3'-dimethylbisphenol A, 3,3',5,5'-tetrachlorobisphenol A, 3,3',5,Examples include polycarbonates of 5'-tetramethylbisphenol A, polyamides such as poly(p-phenylene terephthalamide), polyesters such as polyethylene terephthalate, and polyesters such as Mylar® polyethylene terephthalate. In some embodiments, the particle sorting module of interest includes a cuvette positioned within the sample inspection area. In embodiments, the cuvette can transmit light in the range of 100 nm to 1500 nm, including, for example, 150 nm to 1400 nm, for example, 200 nm to 1300 nm, for example, 250 nm to 1200 nm, for example, 300 nm to 1100 nm, for example, 350 nm to 1000 nm, for example, 400 nm to 900 nm, and 500 nm to 800 nm.

[0051] In some embodiments, the particle sorting module has a sealed housing. The term “sealed” means that each component of the particle sorting module is completely contained within the housing, and the components are sealed or isolated from the surrounding environment. In other words, the components within the sealed housing are not exposed to or in contact with the external environment. In some embodiments, the components contained within the housing are isolated from the gaseous environment of the surrounding environment (i.e., not exposed to gases outside the housing). In other embodiments, the components contained within the housing are isolated from the fluid environment of the surrounding environment (i.e., not exposed to any fluids present outside the housing). In yet another embodiment, the components contained within the housing are sterilized and isolated from live bacteria or other microorganisms present in the surrounding environment (i.e., sterile).

[0052] The size of the particle sorting module housing can vary in length from 10cm to 100cm, including, for example, 15cm to 95cm, 20cm to 90cm, 25cm to 85cm, 30cm to 80cm, 35cm to 75cm, and 40cm to 60cm. The width of the particle sorting module housing can range from 1cm to 25cm, including, for example, 2cm to 20cm, 3cm to 15cm, and 5cm to 10cm.

[0053] The housing may be formed from any suitable material compatible with the fluid sample (e.g., a biological sample), such as metal, glass (e.g., Pyrex glass, borosilicate glass), ceramic, or plastic. In certain embodiments, the particle sorting module housing may be formed from plastic, such as rigid plastic, polymer, or thermoplastic material. Suitable plastics, for example, may include, but are not limited to, polycarbonate, polyvinyl chloride (PVC), polyurethane, polyether, polyamide, polyimide, or other polymeric plastic materials, as well as copolymers of these thermoplastics such as PETG (glycol-modified polyethylene terephthalate). In certain embodiments, the particle sorting module housing is formed from polyester, and the polyester of interest is not limited to, but includes, poly(ethylene terephthalate) (PET), bottle-grade PET (copolymers made based on monoethylene glycol, terephthalic acid, and other comonomers such as isophthalic acid, cyclohexanedimethanol), poly(butylene terephthalate) (PBT), and poly(alkylene terephthalate) such as poly(hexamethylene terephthalate), poly(ethylene adipate), poly(1,4-butylene adipate), and poly(hexamethylene adipate), poly(alkylene adipate) such as poly(ethylene severate), and poly(ethylene seba Poly(alkylene sebacate), poly(ε-caprolactone) and poly(β-propiolactone), poly(ethylene isophthalate), poly(ethylene 2,6-naphthalene dicarboxylate), poly(ethylene sulfonyl-4,4'-dibenzoate), poly(p-phenylene ethylene dicarboxylate), poly(trans-1,4-cyclohexanediyl ethylene dicarboxylate), poly(1,Examples include poly(1,4-cyclohexane-dimethylene alkylenedicarboxylate) such as 4-cyclohexane-dimethylene ethylenedicarboxylate, poly([2.2.2]-bicyclooctane-1,4-dimethylene ethylenedicarboxylate) such as poly([2.2.2]-bicyclooctane-1,4-dimethylene alkylenedicarboxylate), lactic acid polymers and copolymers such as (S)-polylactide, (R,S)-polylactide, poly(tetramethyl glycolide), and poly(lactide-co-glycolide), as well as polycarbonates of bisphenol A, 3,3'-dimethylbisphenol A, 3,3',5,5'-tetrachlorobisphenol A, 3,3',5,5'-tetramethylbisphenol A, polyamides such as poly(p-phenylene terephthalamide), polyesters such as polyethylene terephthalate, and polyesters such as Mylar® polyethylene terephthalate.

[0054] In some embodiments, one or more of the outer walls of the particle sorting module housing have aligners for connecting the housing to a particle sorting system (as described in more detail below). For example, the housing may have two or more walls having aligners for connecting the housing to a particle sorting system, including, for example, three or more walls and four or more walls having aligners. In a particular embodiment, the housing has one wall having aligners for connecting the housing to a particle sorting system. Each wall having aligners may include one or more aligners, including, for example, two or more aligners, for example, three or more aligners, for example, four or more aligners, for example, five or more aligners, for example, seven or more aligners, and ten or more aligners. In a particular embodiment, the particle sorting device includes an outer wall having three aligners.

[0055] Any suitable type of aligner may be employed, such as alignment projections, alignment rails, alignment notches, alignment grooves, alignment slots, alignment countersinks, alignment counterbores, alignment recesses, alignment holes, or combinations thereof. For example, in some examples, the outer wall of the housing includes one or more projections, such as pins, dowels, or bumps. In certain embodiments, the aligner is a pin, such as a ball-tip pin. In other examples, the outer wall of the housing includes one or more recesses, such as holes or notches. In certain examples, the outer wall of the housing includes one or more alignment projections and one or more alignment recesses.

[0056] The shape of the aligner can vary, and the cross-sectional shape of interest may include, but is not limited to, linear cross-sectional shapes such as squares, rectangles, trapezoids, triangles, and hexagons; curved cross-sectional shapes such as circles and ellipses; and irregular shapes such as a parabolic base coupled to a planar top. In some embodiments, the aligner is cylindrical. In other embodiments, the aligner is spherical. In yet another embodiment, the aligner is polygonal, such as a square or rectangle. In certain embodiments, the aligner is a cylindrical pin with a spherical tip (e.g., a ball-tip pin). Each aligner may have the same or different shapes (as described in more detail below) depending on the coupling register of the particle sorting system. In some embodiments, each aligner has the same shape. In other embodiments, each aligner has a different shape. In yet another embodiment, two or more aligners have the same shape, and one or more aligners have a different shape.

[0057] In some embodiments, the outer wall of the housing includes one or more fasteners for maintaining the particle sorting module in contact with the particle sorting system (as described in more detail below). Preferred fasteners may include, but are not limited to, magnets, hook-and-loop fasteners, Velcro®, non-permanent adhesives, or combinations thereof. In certain embodiments, the outer wall of the housing includes one or more magnets for coupling with one or more magnets on the register of the particle sorting system. In these embodiments, alignment between the particle sorting module and the register can be achieved by coupling the magnets on the register with the magnets on the outer wall of the particle sorting module housing. In some embodiments, the particle sorting module includes both an aligner and magnets. In other embodiments, the aligner includes one or more magnets, such as projections with magnets at their tips (e.g., ball-tipped pin projections). In certain embodiments, the aligner is a magnet such as a magnetic projection, a magnetic ball at the tip of a pin projection, or a magnet positioned within a hole or recess. The term “magnet” is used herein in a general sense to refer to a magnetic material having a permanent magnetic field such that the magnetic field from the magnet does not substantially decrease over time. For example, magnets include iron alloy materials containing aluminum, nickel, and cobalt (i.e., Alnico magnets), ceramic or ferrite magnets, samarium cobalt magnets (e.g., SmCo5), neodymium alloy (NdFeB) magnets (e.g., Nd2Fe 14 B) These may be rare earth magnets or combinations thereof. Depending on the size of the magnet, the magnetic field generated by the magnet of interest positioned at the proximal end of the connector will range from 0.01T to 10T, or 0.01T to 5T, or 0.01T to 2T, or 0.1T to 2T, or 0.1T to 1.5T, or 0.1T to 1T.

[0058] In certain embodiments, the particle sorting module is a sealed particle sorting module, such as the one described in U.S. Patent Publication No. 2017 / 0299493, filed on March 28, 2017, whose disclosure is incorporated herein by reference.

[0059] In certain embodiments, the particle sorting module of interest includes one or more sets of integrated droplet deflectors. The droplet deflector of interest may include two or more sets of parallel metal plates, for example, three or more sets, four or more sets, five or more sets, six or more sets, seven or more sets, eight or more sets, nine or more sets, and ten or more sets of parallel metal plates. The parallel metal plates of the droplet deflector of the subject may be formed from any suitable metal capable of generating an electric field, 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 thereof, as well as alloys, e.g., 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-chromium-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., (e.g., bronze), copper-nickel alloy, copper-tungsten alloy, copper-gold-silver alloy, copper-nickel-iron alloy, copper-manganese-tin alloy, copper-aluminum-zinc-tin alloy, copper-gold alloy, gold alloy, gold-silver alloy, indium alloy, indium-tin alloy, indium-tin oxide alloy, iron alloy, iron-chromium alloy (e.g., steel), iron-chromium-nickel alloy (e.g., stainless steel), iron-silicon alloy, iron-chromium-molybdenum alloy, iron-carbon alloy, iron-boron alloy, iron - Magnesium alloy, iron-manganese alloy, iron-molybdenum alloy, iron-nickel alloy, iron-phosphorus alloy, iron-titanium alloy, iron-vanadium alloy, lead alloy, lead-antimony alloy, lead-copper alloy, lead-tin alloy, lead-tin-antimony alloy, nickel alloy, nickel-manganese-aluminum-silicon alloy, nickel-chromium alloy, nickel-copper alloy, nickel-molybdenum-chromium-tungsten alloy, nickel-copper-iron-manganese alloy, nickel-carbon alloy,This may include nickel-chromium-iron alloys, nickel-silicon alloys, nickel-titanium alloys, silver alloys, silver-copper alloys (e.g., sterling silver), silver-copper-germanium alloys (e.g., argentium sterling silver), silver-gold alloys, silver-copper-gold alloys, silver-platinum alloys, tin alloys, tin-copper-antimony alloys, tin-lead-copper alloys, tin-lead-antimony alloys, titanium alloys, titanium-vanadium-chromium alloys, titanium-aluminum alloys, titanium-aluminum-vanadium alloys, zinc alloys, zinc-copper alloys, zinc-aluminum-magnesium-copper alloys, zirconium alloys, zirconium-tin alloys, or combinations thereof.

[0060] The parallel metal plates of the droplet deflector of the subject may be any preferred shape, such as circular, elliptical, semicircular, crescent, star, square, triangular, rhombus, pentagon, hexagon, heptagon, octagon, rectangle, or other preferred polygons. In certain embodiments, the parallel metal plates are rectangular. As will be described in more detail below, in certain examples, the parallel metal plates are twisted, such as a twisted rectangle having a twist angle of 5° or more, including twist angles of 10° or more, 15° or more, 20° or more, 25° or more, 30° or more, 35° or more, 40° or more, 45° or more, 50° or more, 55° or more, and 60° or more.

[0061] The dimensions may vary depending on the shape of the metal plate. In some embodiments, each metal plate has a width in the range of 0.5 mm to 10 mm, including widths in the ranges of 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 4.5 mm to 6 mm. The length also varies in the range of 10 mm to 500 mm, including widths in the ranges of 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 50 mm to 100 mm. In certain embodiments, the metal plate is an asymmetric polygon in which the first end has a width smaller than the width of the second end. The width at each end may range from 0.01 mm to 10 mm, including, for example, 0.05 mm to 9.5 mm, for example, 0.1 mm to 9 mm, for example, 0.5 mm to 8.5 mm, for example, 1 mm to 8 mm, for example, 2 mm to 8 mm, for example, 2.5 mm to 7.5 mm, and 3 mm to 6 mm. In certain embodiments, the cover bar is an asymmetric polygon having a first end with a width of 1 to 10 mm and a second end with a width of 2 to 5 mm. For example, the cover bar may be an asymmetric polygon having a first end with a width of 5 mm and a second end with a width of 10 mm. In embodiments, the surface area of ​​each metal plate may vary as desired, for example, 0.5 to 14 cm². 2 For example, 0.75~13cm 2 For example, 1-12 cm 2 For example, 1.5-11cm 2 , and 2-10cm 2 Including, 0.25-15cm 2 It could be within the range.

[0062] In certain embodiments, the particle sorting module includes one or more droplet deflectors, such as those described in U.S. Patent Publication No. 2018 / 0095022, filed on June 14, 2017, which is incorporated herein by reference.

[0063] In some embodiments, the outer wall of the housing includes one or more electrical connections configured for electrical connectivity between the particle sorting module and the particle sorting system. For example, the outer housing may include two or more electrical connections, such as three or more electrical connections, four or more electrical connections, five or more electrical connections, and ten or more electrical connections. In some embodiments, the electrical connections provide power to the droplet deflector plate. Any convenient electrical connections may be employed, such as conductive pins, pads, wires, or coils protruding from or recessed within the outer wall of the housing. In certain embodiments, the particle sorting module of interest includes five or more electrical pins.

[0064] Particle sorting system Aspects of the present disclosure also include systems for separating particulate components of a sample, such as cells in a biological sample. A system according to a particular embodiment includes a particle sorting module having an opening configured to visualize droplets in a deflected flowstream, and an imaging sensor configured to capture one or more images through the opening (e.g., the boundary of a sample collection port at the distal end of the particle sorting module).

[0065] In embodiments, the imaging sensor may be any suitable device capable of capturing optical images and converting them into electronic data signals, such as a charge-coupled device, a semiconductor charge-coupled device (CCD), an active pixel sensor (APS), a complementary metal-oxide-semiconductor (CMOS) image sensor, or an N-type metal-oxide-semiconductor (NMOS) image sensor, but is not limited to the following. In some embodiments, the imaging sensor is a CCD camera. For example, the camera may be an electron-multiplier CCD (EMCCD) camera or an enhanced CCD (ICCD) camera. In other embodiments, the imaging sensor is a CMOS camera.

[0066] The number of image sensors in the subject system may vary as needed. For example, the subject system may include one or more image sensors, including, for example, two or more image sensors, for example, three or more image sensors, for example, four or more image sensors, for example, five or more image sensors, and ten or more image sensors. In certain embodiments, the system includes one image sensor. In other embodiments, the system includes two image sensors. When the system includes two or more image sensors, each image sensor may be oriented (when referenced in the XY plane) relative to the other at angles in the range of 10° to 90°, including, for example, 15° to 85°, for example, 20° to 80°, for example, 25° to 75°, and 30° to 60°. In certain embodiments, each image sensor is oriented orthogonal to each other (when referenced in the XY plane). For example, when the subject system includes two image sensors, the first image sensor is oriented orthogonal to the second image sensor (when referenced in the XY plane).

[0067] If the subject system includes two or more image sensors, each image sensor may be the same or a combination of sensors. For example, if the subject system includes two image sensors, in some embodiments, the first image sensor is a CCD type device and the second image sensor is a CMOS type device. In other embodiments, both the first and second image sensors are CCD type devices. In yet another embodiment, both the first and second image sensors are CMOS type devices.

[0068] In some embodiments, the imaging sensor is fixed and maintains a single position with respect to an opening in the particle sorting module housing. In other embodiments, the imaging sensor may be configured to move, for example, along the path of a deflected flow stream. For example, the imaging sensor may be configured to move upstream and downstream along a deflected flow stream and capture images in multiple detection fields through an opening in the particle sorting module housing. When the imaging sensor is configured to move, it may move continuously. In other embodiments, the imaging sensor may move in discrete intervals, for example, including increments of 1 mm or more, e.g., increments of 2 mm or more, and increments of 5 mm or more.

[0069] An imaging sensor can be configured to capture images continuously or at discrete intervals. In some examples, the imaging sensor of interest is configured to capture images continuously. In other examples, the imaging sensor is configured to measure at discrete intervals, such as capturing images of a flow stream at intervals including every 0.001 milliseconds, every 0.01 milliseconds, every 0.1 milliseconds, every 1 millisecond, every 10 milliseconds, every 100 milliseconds, and every 1000 milliseconds, or some other interval.

[0070] As will be described in more detail below, the image sensor is configured to capture one or more images of a deflected flow stream. For example, the image sensor may be configured to capture two or more images of a deflected flow stream, including, for example, three or more images, for example, four or more images, for example, five or more images, for example, ten or more images, for example, fifteen or more images, and twenty-five or more images. If multiple images are captured, the processor may include, if desired, a digital imaging processing algorithm for combining the multiple images.

[0071] Depending on the desired image resolution, the image sensor may have an exposure time of 100ms or less, including exposure times of, for example, 75ms or less, 50ms or less, 25ms or less, 10ms or less, 5ms or less, 1ms or less, 0.1ms or less, 0.01ms or less, 0.001ms or less, 0.0001ms or less, 0.00001ms or less, and 0.000001ms or less when fully reading out the sensor. For example, the exposure time of the image sensor may be in the range of 0.0001ms to 10ms, including, for example, 0.001ms to 5ms, 0.01ms to 2ms, and 0.1ms to 1ms.

[0072] In certain embodiments, the imaging sensor in the subject system may have 1M or more active pixels, such as 1.5M or more, 2M or more, 2.5M or more, or 3M or more. In certain embodiments, the pixels correspond to actual physical dimensions of about 0.3 μm. Depending on the detection field, in some examples the imaging sensor is about 150 mm 2 ~approx. 175mm 2 , about 175mm 2 ~about 200mm 2 , about 200mm 2 ~approx. 225mm 2 , about 225mm 2 ~about 250mm 2 , about 250mm 2 ~about 300mm 2 , about 300mm 2 ~400mm 2 Approximately 400mm 2 ~approximately 500mm 2 Approximately 500mm 2 ~about 750mm 2 , about 750mm 2 ~approximately 1000mm 2 , or approximately 1000mm 2 The above, etc., 150mm 2 It has the above sensor area.

[0073] The imaging sensor can be positioned at any suitable distance from the opening in the particle sorting module housing, as long as the sensor can capture an image of the deflected flow stream through the opening. For example, the imaging sensor can be positioned at 0.01 mm or more from the opening in the particle sorting module housing, including, 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 2.5 mm or more, for example 5 mm or more, for example 10 mm or more, for example 15 mm or more, for example 25 mm or more, and 50 mm or more from the opening in the particle sorting module housing.

[0074] In some examples, the image sensor also includes an optical adjustment component. The optical adjustment component may be configured to modify the captured image as needed, such as increasing or decreasing the dimensions to be captured, or increasing the optical resolution of the image. In some examples, the optical adjustment is an expansion protocol configured to increase the size of the detection field captured by the image sensor, for example, by 5% or more, for example, 10% or more, for example, 25% or more, for example, 50% or more, and 75% or more of the detection field of the image sensor. In other examples, the optical adjustment is a reduction protocol configured to decrease the size of the detection field captured by the image sensor, for example, by 5% or more, for example, 10% or more, for example, 25% or more, for example, 50% or more, and 75% or more of the detection field captured by the image sensor. In certain embodiments, the optical adjustment is a resolution enhancement protocol configured to improve the resolution of the captured image, for example, by 5% or more, for example, 10% or more, for example, 25% or more, for example, 50% or more, and 75% or more of the resolution of the captured image. Image capture by the image sensor can be adjusted using any convenient optical adjustment protocol, including but not limited to lenses, mirrors, filters, and combinations thereof. In certain embodiments, the image sensor includes a focusing lens. The focusing lens may be, for example, a reduction lens. In other embodiments, the focusing lens is a magnifying lens.

[0075] The imaging sensors of this disclosure may also include one or more wavelength separators. The term “wavelength separator” is used herein in a general sense to refer to an optical protocol for separating polychromatic light into its component wavelengths for detection. Wavelength separation according to a particular embodiment may involve selectively passing or blocking specific wavelengths or wavelength ranges of polychromatic light. To separate the wavelengths of light, transmitted light may pass through any convenient wavelength separation protocol, including, but not limited to, tinted glass, bandpass filters, interference filters, dichroic mirrors, diffraction gratings, monochromators, and combinations thereof, among other wavelength separation protocols. The system may include one or more wavelength separators, for example, two or more, for example, three or more, for example, four or more, for example, five or more, and ten or more wavelength separators. In one embodiment, the imaging sensor includes one bandpass filter. In another embodiment, the imaging sensor includes two or more bandpass filters. In yet another embodiment, the imaging sensor includes two or more bandpass filters and a diffraction grating. In yet another embodiment, the image sensor includes a plurality of bandpass filters and a monochromator. In a particular embodiment, the image sensor includes a plurality of bandpass filters and a diffraction grating configured in a filter wheel setup. If the image sensor includes two or more wavelength separators, the wavelength separators may be used individually or in series to separate multicolor light into component wavelengths. In some embodiments, the wavelength separators are arranged in series. In other embodiments, the wavelength separators are arranged individually so that one or more measurements are performed using each of the wavelength separators.

[0076] In some embodiments, the system includes one or more optical filters, such as one or more bandpass filters. For example, in some examples, the optical filters of interest are bandpass filters having a minimum bandwidth in the range of 2 nm to 100 nm, and bandpass filters having a minimum bandwidth in the range of 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 20 nm to 50 nm. Other examples of optical filters include long-pass filters that attenuate wavelengths of light below 1550nm, below 1500nm, below 1450nm, below 1400nm, below 1350nm, below 1300nm, below 1000nm, below 950nm, below 900nm, below 850nm, below 800nm, below 750nm, below 700nm, below 650nm, below 600nm, below 550nm, below 500nm, and below 450nm, such as long-pass filters that attenuate wavelengths of light below 1600nm. Further examples include short-pass filters that attenuate wavelengths of light above 250 nm, above 300 nm, above 350 nm, above 400 nm, above 450 nm, above 500 nm, above 550 nm, and above 600 nm, such as short-pass filters that attenuate wavelengths of light above 200 nm.

[0077] In other embodiments, the wavelength separator is a diffraction grating. The diffraction grating may be a transmission, dispersive, or reflective grating, but is not limited to the following. The preferred spacing of the diffraction grating can vary in the range of 0.01 μm to 10 μm, including, for example, 0.025 μm to 7.5 μm, e.g., 0.5 μm to 5 μm, e.g., 0.75 μm to 4 μm, e.g., 1 μm to 3.5 μm, and 1.5 μm to 3.5 μm, depending on the configuration of the light source, detection field, and imaging sensor, as well as any other optical adjustment protocols present (e.g., focusing lenses).

[0078] In some embodiments, each imaging sensor is operably coupled to one or more light sources for illuminating the flowstream through an opening in the particle sorting module housing. In some embodiments, the light source is a laser, such as a continuous wave laser. For example, the laser may be a diode laser, such as an ultraviolet diode laser, a visible diode laser, and a near-infrared diode laser. In some examples, the diode laser outputs light in the range of 375 nm to 1000 nm, including, for example, 405 nm to 875 nm, e.g., 450 nm to 800 nm, e.g., 500 nm to 650 nm, and 525 nm to 625 nm. In certain embodiments, the laser of interest includes a 405 nm diode laser. In other embodiments, the laser may be a helium-neon (HeNe) laser. In some examples, the laser is a gas laser such as a helium-neon laser, argon laser, krypton laser, xenon laser, nitrogen laser, CO2 laser, CO laser, argon-fluorine (ArF) excimer laser, krypton-fluorine (KrF) excimer laser, xenon-chlorine (XeCl) excimer laser, or xenon-fluorine (XeF) excimer laser, or a combination thereof. In other examples, the system of interest includes dye lasers such as a stilbene laser, coumarin laser, or rhodamine laser. In yet another example, the laser of interest includes metallic vapor lasers such as a helium-cadmium (HeCd) laser, a helium-mercury (HeHg) laser, a helium-selenium (HeSe) laser, a helium-silver (HeAg) laser, a strontium laser, a neon-copper (NeCu) laser, a copper laser, or a gold laser, and combinations thereof. Further examples include solid-state lasers such as ruby ​​lasers, Nd:YAG lasers, NdCrYAG lasers, Er:YAG lasers, Nd:YLF lasers, Nd:YVO4 lasers, Nd:YCa4O(BO3)3 lasers, Nd:YCOB lasers, titanium-sapphire lasers, slim YAG lasers, ytterbium YAG lasers, Yb2O3 lasers, or cerium-doped lasers, and combinations thereof.In certain embodiments, the light source is a low-power laser (for example, a 650nm laser outputting 5mW).

[0079] In other embodiments, the light source is a non-laser light source such as a broadband light source that emits light having a wide range of wavelengths, for example, 50 nm or more, such as 100 nm or more, 150 nm or more, 200 nm or more, 250 nm or more, 300 nm or more, 350 nm or more, 400 nm or more, and 500 nm or more. For example, one preferred broadband light source emits light having wavelengths from 200 nm to 1500 nm. Another embodiment of a preferred broadband light source includes a light source that emits light having wavelengths from 400 nm to 1000 nm. Among other broadband light sources, any convenient broadband light source protocol can be employed, such as halogen lamps, deuterium arc lamps, xenon arc lamps, stabilized fiber-coupled broadband light sources, broadband LEDs with continuous spectra, ultra-high brightness light-emitting diodes, semiconductor light-emitting diodes, broad-spectrum LED white light sources, multi-LED integrated white light sources, or any combination thereof. In certain embodiments, the light source for illuminating the flow stream through an opening in the particle sorting module during image capture includes an array of infrared LEDs.

[0080] In other embodiments, the light source is a narrowband light source that emits a specific wavelength or a narrow range of wavelengths. In some examples, the narrowband light source includes a light source that emits light having a narrow range of wavelengths, such as 50 nm or less, 40 nm or less, 30 nm or less, 25 nm or less, 20 nm or less, 15 nm or less, 10 nm or less, 5 nm or less, 2 nm or less, and a light source that emits light of a specific wavelength (i.e., monochromatic light). Any convenient narrowband light source protocol may be employed, such as a narrow-wavelength LED, a laser diode, or a broadband light source coupled with one or more optical bandpass filters, diffraction gratings, monochromators, or any combination thereof.

[0081] In embodiments, the imaging sensor is configured to capture an image in the detection field through an opening in the particle sorting module housing. The “detection field” refers to the field of view through the opening in the particle sorting module housing that is captured by the imaging sensor. The imaged detection field can vary. In some embodiments, the detection field is a predetermined length of the deflected flow stream. In other embodiments, the detection field is a predetermined portion of the opening in the particle sorting module housing. In some embodiments, the imaging sensor is configured to capture an image of the deflected flow stream originating from a droplet deflector in the detection field. In embodiments, the image detection field may extend to 0.001 cm or more of the deflected flow stream, including, for example, 0.005 cm or more, for example, 0.01 cm or more, for example, 0.05 cm or more, for example, 0.1 cm or more, for example, 0.5 cm or more, for example, 1 cm or more, for example, 2 cm or more, for example, 5 cm or more, and 10 cm or more.

[0082] In some embodiments, the detection field includes the location of the sample collection port at the distal end of the particle sorting module housing. In certain examples, the sample collection port is coupled to a sample collection container, and the imaging sensor is configured to capture the location of the sample collection container at the distal end of the housing. In certain embodiments, the imaging sensor is configured to capture an image through an opening in the particle sorting module housing to obtain an imaged opening, and a processor operably coupled to the imaging sensor is configured to calculate the position of the boundary within the imaged opening for each of the sample collection ports at the distal end of the housing, along the horizontal axis. In some examples, calculating the boundary position includes determining the distance between each boundary of the sample collection ports. For example, the processor may be configured to calculate the boundary of a first sample collection port based on an imaged opening located 0.1 mm to 100 mm from the leftmost edge of the image, along the horizontal axis, including, for example, 0.5 mm to 95 mm, for example, 1 mm to 90 mm, for example, 1.5 mm to 85 mm, for example, 2 mm to 80 mm, for example, 2.5 mm to 75 mm, and 3 mm to 70 mm. In another example, the processor may be configured to calculate the boundary of a second sample collection port based on an imaged aperture located 0.1 mm to 100 mm from the left edge of the image along the horizontal axis, including, for example, 0.5 mm to 95 mm, 1 mm to 90 mm, 1.5 mm to 85 mm, 2 mm to 80 mm, 2.5 mm to 75 mm, and 3 mm to 70 mm. In yet another example, the processor may be configured to calculate the boundary of a third sample collection port based on an imaged aperture located 0.1 mm to 100 mm from the left edge of the image along the horizontal axis, including, for example, 0.5 mm to 95 mm, 1 mm to 90 mm, 1.5 mm to 85 mm, 2 mm to 80 mm, 2.5 mm to 75 mm, and 3 mm to 70 mm.

[0083] In other embodiments, the processor may be configured to calculate based on imaged apertures, where the sample collection ports are spaced 0.1 mm to 10 mm apart from each other along the horizontal axis, including, for example, 0.5 mm to 9.5 mm, 1 mm to 9 mm, 1.5 mm to 8.5 mm, 2 mm to 8 mm, 2.5 mm to 7.5 mm, and 3 mm to 7 mm.

[0084] In yet another embodiment, the processor may be configured to calculate the boundary of the first sample collection port based on an imaged aperture located 0.1 mm to 100 mm from the right edge of the image along the horizontal axis, including, for example, 0.5 mm to 95 mm, 1 mm to 90 mm, 1.5 mm to 85 mm, 2 mm to 80 mm, 2.5 mm to 75 mm, and 3 mm to 70 mm. In yet another example, the processor may be configured to calculate the boundary of the second sample collection port based on an imaged aperture located 0.1 mm to 100 mm from the right edge of the image along the horizontal axis, including, for example, 0.5 mm to 95 mm, 1 mm to 90 mm, 1.5 mm to 85 mm, 2 mm to 80 mm, 2.5 mm to 75 mm, and 3 mm to 70 mm. In yet another example, the processor may be configured to calculate the boundary of the third sample collection port based on an imaged aperture located 0.1 mm to 100 mm from the right edge of the image along the horizontal axis, including, for example, 0.5 mm to 95 mm, 1 mm to 90 mm, 1.5 mm to 85 mm, 2 mm to 80 mm, 2.5 mm to 75 mm, and 3 mm to 70 mm.

[0085] In some embodiments, the system is configured to capture an image of the aperture of a particle sorting module to obtain an imaged aperture and convert the image of the aperture into a pixelated image. The term “pixelated” is used herein in a general sense to mean that the captured image consists of a large number of regularly spaced (e.g., grid-like) pixels, each pixel forming part of the captured image. In these embodiments, a processor operably coupled to the imaging sensor may be configured to calculate the pixel position within the imaged aperture boundary for each of the ports at the distal end of the housing along the horizontal axis.

[0086] In some embodiments, the processor may be configured to calculate the pixel position of the boundary of each sample collection port from the leftmost edge of the image, along the horizontal axis, based on the imaged aperture. For example, the position of the boundary of each sample collection port may be output as the number of pixels from the leftmost edge of the image, along the horizontal axis. In some examples, the position of the boundary of each sample collection port may range from 1 to 1000 pixels from the leftmost edge of the image of the aperture in the particle sorting module housing, along the horizontal axis, including, for example, 5 to 750 pixels, 10 to 500 pixels, and 25 to 250 pixels. In other examples, the position of the boundary of each sample collection port may be output as the ratio of the number of pixels along the horizontal axis to the total number of pixels in the entire imaged aperture. For example, the total number of pixels in the entire imaged aperture may be 100 pixels, and the position of each boundary of the sample collection port may be in a ratio in the range of 1 / 100 to 99 / 100, including ratios in the ranges of 5 / 100 to 95 / 100, 10 / 100 to 90 / 100, 15 / 100 to 85 / 100, 20 / 100 to 80 / 100, and 25 / 100 to 75 / 100.

[0087] In certain embodiments, the system of interest includes a processor having memory operably coupled to the processor, the memory containing instructions stored thereon, which, when executed by the processor, cause the processor to calculate the total number of pixels in the entire imaged aperture along the horizontal axis and multiply this total number of pixels by a predetermined ratio. In these embodiments, the boundaries of each sample collection port (e.g., left and right boundaries along the horizontal axis) are determined based on the imaged aperture, assigned a number of pixels, and multiplied by a predetermined ratio. In certain examples, the predetermined ratio is calculated by dividing the number of pixels in the imaged aperture at the boundary of each sample collection port by the total number of pixels in the entire imaged aperture along the horizontal axis.

[0088] Figure 1 shows an exemplary calculation for determining the boundary of the sample collection port at the distal end of a particle sorting module according to a particular embodiment. Figure 1 shows a particle sorting module 100 having a droplet deflector 101 that deflects the flow stream according to trajectories 101a, 101b, and 101c. The particle sorting module 100 includes an opening 102 for visualizing the flow stream having trajectories 101a, 101b, and 101c. The flow stream is irradiated with a laser 103. At the distal end of the particle sorting module 100 are sample collection ports 100L, 100C, and 100R.

[0089] The boundary of the first sample collection port (e.g., the first deflection port) at 100L is L1 and L r Defined by, the second sample collection port (e.g., wastewater port) 100C is C1 and C r Defined by, the third sample collection port (e.g., the second deflection port) 100R is R1 and R r It is defined by. In this embodiment, the total number of pixels (P) in the entire aperture is 100. The boundary of the sample collection port (L1 and L r , C1 and C r , as well as R1 and R r ) is calculated as P*(pixel position / total number of pixels).

[0090] If the system is configured to determine the positioning of the sample collection ports based on the imaged aperture, the processor may further include memory containing instructions for aligning the flow stream with one or more sample collection ports. For example, the memory may contain instructions for aligning the flow stream with the center of one of the sample collection ports based on the determined boundary. In one example, the system may be configured to align the flow stream with the sample collection ports. In another example, the system may be configured to align the flow stream with the center of the distal end of the housing along the horizontal axis.

[0091] Figure 2 shows a particle sorting module having an opening configured to visualize droplets in a deflected flowstream, according to a particular embodiment. As shown in Figure 2, the pixel positions of the sample collection port boundary, as well as the trajectories of the deflected droplets in the flowstream, are determined from the image captured through the opening.

[0092] A particle sorting system according to a particular embodiment also includes a sample input subsystem fluidly coupled to the inlet at the proximal end of the particle sorting module. In this embodiment, the sample input subsystem is configured to provide a suitable flow of the sample to the flow cell nozzle chamber within the particle sorting module. Depending on the desired characteristics of the flow stream generated from the flow nozzle, the rate at which the sample is transported to the particle sorting module by the sample input subsystem can be 1 μL / min or more, including, for example, 2 μL / min or more, 3 μL / min or more, 5 μL / min or more, 10 μL / min or more, 15 μL / min or more, 25 μL / min or more, 50 μL / min or more, and 100 μL / min or more. In some examples, the flow rate is 1 μL / min or more, including, for example, 2 μL / min or more, 3 μL / min or more, 5 μL / min or more, 10 μL / min or more, 15 μL / min or more, 25 μL / min or more, 50 μL / min or more, and 100 μL / min or more.

[0093] In some embodiments, the sample fluid subsystem includes a container, a cap, and one or more ports into the internal cavity of the container. The container has a distal end and a proximal end, with a wall between the distal and proximal ends, which together form an internal cavity within the container. In some embodiments, the outer wall of the container and the internal cavity have the same cross-sectional shape, and the cross-sectional shape of interest is not limited to, but includes, for example, curved cross-sectional shapes such as circular and elliptical shapes, straight cross-sectional shapes such as square, rectangular, trapezoidal, triangular, and hexagonal shapes, and irregular shapes such as, for example, a parabolic bottom joined to a planar top. The container may be formed from any suitable material, not limited to, but as described above, such as glass, metal, or plastics such as flexible or rigid plastics, polymers, or thermoplastic materials.

[0094] The size of the internal cavity of the container may vary depending on the size of the sample and the size of the particle sorting module. In some examples, the length of the internal cavity may range from 1 cm to 25 cm, including, for example, 2.5 cm to 22.5 cm, for example, 5 cm to 20 cm, for example, 7.5 cm to 17.5 cm, and 10 cm to 15 cm. The width of the internal cavity may range from 1 cm to 20 cm, including, for example, 2 cm to 17.5 cm, for example, 3 cm to 15 cm, for example, 4 cm to 12.5 cm, and 5 cm to 10 cm. If the internal cavity of the container has a cylindrical cross-section, the diameter may vary in some embodiments from 1 cm to 10 cm, including, for example, 2 cm to 9 cm, for example, 3 cm to 8 cm, and 4 cm to 7 cm. Therefore, the volume of the container may range from, for example, 5 to 250 cm³. 3 For example, 10-200cm 3 For example, 15-150cm 3 For example, 20-125cm 3 , and 25-100cm 3 Including, 1-500cm 3It can vary within this range. In some embodiments, the container for the sample input module is a tube having a volume in the range of 1 mL to 500 mL, including, for example, 2 mL to 400 mL, for example, 3 mL to 300 mL, for example, 4 mL to 200 mL, for example, 5 mL to 150 mL, and 10 mL to 100 mL.

[0095] In embodiments, the container of the sample input module also includes a cap configured to close the proximal end of the container. For example, the cap may be a screw cap, a snap cap, or a cap that connects to the container by permanent, semi-permanent, or non-permanent adhesive. In certain examples, the cap forms a fluid seal with the wall of the container. The cap may be part of an integrated container, including cases where the cap is molded, soldered, welded, or fixed to the container using permanent adhesive. In other embodiments, the cap is releasably attached to the container. “Releasably” means that the cap can be freely removed from the proximal end of the container and reattached to the proximal end of the container. If the cap is releasably attached to the container, the cap may be non-permanently fixed to the container by any convenient attachment protocol, including, but not limited to, hook-and-loop fasteners, latches, notches, grooves, pins, tethers, hinges, Velcro®, non-permanent adhesives, screws, or combinations thereof. In certain examples, the container includes a threaded outer wall that is screwed to the inner wall of the cap.

[0096] The cap may include one or more ports into the internal cavity of the container, for example, two or more ports, for example, three or more ports, for example, four or more ports, and five or more ports. In certain embodiments, the cap includes two ports. The ports may be any convenient ports configured to communicate with the internal cavity of the container in fluid or gas. In some embodiments, the cap includes a port configured to transport gas into the container to create positive pressure inside the container and to transport a sample fluid from inside the container to a particle sorting module through a second port. In some examples, the container includes a third opening in the cap to allow air to escape.

[0097] Depending on the desired function of the port, any suitable port configuration may be employed. Examples of ports include, among other types of ports, channels, orifices, channels with check valves, Luer taper fittings, and ports with breakable seals (e.g., single-use ports). In certain embodiments, the port is comprised of a Luer taper fitting such as a Luer lock or Luer slip. The port within the cap of the sample input module may be of any suitable shape. The cross-sectional shape of the port of interest is not limited to, but includes, for example, linear cross-sectional shapes such as squares, rectangles, trapezoids, triangles, and hexagons; curved cross-sectional shapes such as circles and ellipses; and irregular shapes such as a parabolic bottom joined to a planar top. The dimensions of the port may vary in some embodiments in the range of 1 mm to 100 mm, including, for example, 2 mm to 95 mm, 3 mm to 90 mm, 4 mm to 80 mm, 5 mm to 70 mm, 6 mm to 60 mm, and 10 mm to 50 mm. In some embodiments, the port is a circular orifice, and the diameter of the port is in the range of 1 mm to 100 mm, including, for example, 2 mm to 90 mm, 4 mm to 80 mm, 5 mm to 70 mm, 6 mm to 60 mm, and 10 mm to 50 mm. Therefore, depending on the shape of the port, the port inside the cap is, for example, 0.05 mm 2 ~200mm 2 For example, 0.1 mm2 ~150mm 2 For example, 0.5mm 2 ~100mm 2 For example, 1 mm 2 ~75mm 2 For example, 2mm 2 ~50mm 2 , and 5mm 2 ~25mm 2 Including 0.01 mm 2 ~250mm 2 It may have an opening within the range of [this range].

[0098] In some embodiments, the sample input subsystem includes one or more conduits that fluidly communicate with the internal cavity of the container through one or more ports in the cap. For example, the sample input module may include two or more conduits, for example, three or more conduits and five or more conduits. Each conduit includes a proximal end that contacts the internal cavity of the container and a distal end having an opening for inputting or outputting gas or fluid. In some examples, the sample input subsystem includes an inlet conduit for transporting gas into the container and an outlet conduit for transporting the sample fluid from the container to the particle sorting module. In other examples, the sample input subsystem includes two inlet conduits for transporting gas into the container and one outlet conduit for transporting the sample fluid from the container to the particle sorting module.

[0099] Each conduit may have an independently varying length, and each conduit may be 5 cm or more, including, for example, 7 cm or more, 10 cm or more, 25 cm or more, 30 cm or more, 50 cm or more, 75 cm or more, 100 cm or more, 250 cm or more, and 500 cm or more. The inner diameter of each conduit may also vary, including, for example, 0.75 mm or more, 1 mm or more, 1.5 mm or more, 2 mm or more, 5 mm or more, 10 mm or more, 25 mm or more, and 50 mm or more, and 0.5 mm or more. For example, depending on the desired flow rate for transporting fluid from the container to the particle sorting module, the inner diameter may range from 0.5 mm to 50 cm, including, for example, 1 mm to 25 mm and 5 mm to 15 mm.

[0100] Each conduit may be configured with one or more valves that can be opened or closed as needed. For example, each conduit may be configured with two or more valves, including, for example, three or more valves, four or more valves, and five or more valves. Any convenient valve protocol may be employed, but is not limited to, pinch valves, ball valves, butterfly valves, disc valves, clapper valves, check valves, ball check valves, diaphragm valves, lift check valves, inclined disc check valves, needle valves, piston valves, plug valves, poppet valves, and spool valves. In certain embodiments, an inlet conduit for transporting gas into a container may be configured with two pinch valves. To maintain the sterility of the sample, the inlet conduit from the gas source to the container may include a filter such as a high-efficiency particulate filter (HEPA) or a filter having pores of 50 μm or less, including, for example, 25 μm or less, 15 μm or less, 10 μm or less, 5 μm or less, 1 μm or less, 0.5 μm or less, 0.1 μm or less, 0.05 μm or less, 0.01 μm or less, and 0.001 μm or less.

[0101] In certain embodiments, the system further includes one or more gas sources in gas communication with the inlet conduit of the sample input subsystem vessel. In some examples, the gas source is a pressurized gas, such as a pressurized gas cylinder or a compressor, but is not limited to the following. In certain examples, the pressurized gas has a pressure of 15 psi or more, including 5 psi or more, including 20 psi or more, including 2 psi or more, and in some examples the pressure is 75 psi or more, including 25 psi or more, including 50 psi or more, or 100 psi or more, or 125 psi or more, including 150 psi or more. The pressurized gas can be any convenient type of gas suitable for creating positive pressure within the vessel of the sample input subsystem. For example, pressurized gases may include air, nitrogen, argon, etc.

[0102] In some embodiments, the sample input subsystem includes a sample agitator. Any convenient agitation protocol may be employed, including, but is not limited to, other agitation protocols such as a sonicator, mechanical or electric shaker. In certain embodiments, the sample agitator is an eccentric motion device having a stepping motor and bearings (to eliminate movement) along with an off-center drive. The sample agitator may be configured to agitate the sample input module for any desired duration, including, for example, 1 minute or more, 2 minutes or more, 5 minutes or more, 10 minutes or more, 15 minutes or more, 30 minutes or more, 60 minutes or more, 120 minutes or more, 240 minutes or more, and 480 minutes or more.

[0103] The sample input subsystem may also include a temperature controller, and the temperature of the sample in the subject's sample input subsystem may be maintained or changed (e.g., increased or decreased) as needed. For example, the temperature controller may be configured to maintain the temperature of the sample input subsystem in the range of -80°C to 100°C, including, for example, -75°C to 75°C, for example, -50°C to 50°C, for example, -25°C to 25°C, for example, -10°C to 10°C, and 0°C to 25°C. In certain embodiments, the subject's system includes a temperature sensor for measuring the temperature in the sample input subsystem container and a feedback monitor configured to allow the sample input module to operate in a closed-loop manner. For example, in some embodiments, the system may evaluate the temperature in the sample input subsystem, and the feedback monitor may adjust the temperature (e.g., increase or decrease the temperature in the sample input subsystem container in substantially real time as needed to automatically obtain more effective results).

[0104] A particle sorting system according to a particular embodiment also includes a sheath fluid delivery subsystem for delivering sheath fluid to a flow cell nozzle of a particle sorting module. The term “sheath fluid” is used herein in a general sense to refer to a fluid delivered through a conduit (e.g., within a flow cytometer) used to form a coaxial annular flow of the sample-containing fluid, causing a hydrodynamically focused flow of the particle-containing sample fluid at the center of the sheath fluid stream. The sheath fluid of interest may be any convenient buffering composition for use within a flow cytometer, and may include, but is not limited to, one or more salts such as potassium phosphate, potassium chloride, sodium phosphate, sodium chloride, preservatives, and chelating agents such as disodium ethylenediaminetetraacetate (EDTA). In an embodiment, the sheath fluid distribution system includes a fluid reservoir containing the sheath fluid and a conduit having a proximal end that fluid-communicates with the sheath fluid reservoir and a distal end that fluid-communicates with the sheath fluid that is introduced into the particle sorting module.

[0105] In some embodiments, the sheath fluid delivery subsystem includes a pressurized housing having a flexible container with a reservoir for sheath fluid, located within the housing. In other embodiments, the sheath fluid delivery subsystem includes a housing and a first flexible container and a second flexible container located within the housing. The first flexible container includes a fluid reservoir and a conduit having a proximal end and a distal end, the proximal end being fluidically coupled to the fluid reservoir and the distal end being configured to couple the conduit to a particle sorting module, and the second flexible container includes a gas reservoir and a port for gas communication with the gas reservoir. In these embodiments, the second flexible container is located within the housing together with the first flexible container and is configured to apply pressure to the fluid reservoir of the first flexible container in order to deliver the sheath fluid from the distal end of the conduit into the particle sorting module.

[0106] In certain embodiments, the particle sorting system of interest includes a sheath fluid delivery subsystem for transporting a sheath fluid to a particle sorting module, such as those described in concurrently pending PCT patent application PCT / US2016 / 048433, filed on 24 October 2016 and published as WO2017 / 040151, and U.S. patent application 14 / 365,602, filed on 28 March 2017 and currently issued as U.S. Patent No. 9,551,643 and U.S. Patent Publication No. 2017 / 0299493, and these disclosures are incorporated herein by reference in their entirety.

[0107] As summarized above, the subject system is configured to separate particulate components of a sample, such as a biological sample. In some embodiments, the system further includes a photodetection system configured to irradiate and identify particulate components of the sample in a flowstream, such as within the sample inspection area (e.g., within a cuvette) of the particle sorting module. In these embodiments, the system includes one or more light sources for irradiating the sample in the sample inspection area of ​​the flowstream. The light sources may be broadband light sources such as halogen lamps, deuterium arc lamps, xenon arc lamps, stabilized fiber-coupled broadband light sources, broadband LEDs with continuous spectra, ultra-high brightness light-emitting diodes, semiconductor light-emitting diodes, broad-spectrum LED white light sources, multi-LED integrated white light sources, or any combination thereof, among other broadband light sources.

[0108] In other embodiments, the light source is a narrowband light source that emits a specific wavelength or a narrow range of wavelengths, such as a narrow-wavelength LED, a laser diode, or a broadband light source coupled with one or more optical bandpass filters, diffraction gratings, monochromators, or any combination thereof.

[0109] In certain embodiments, the light source is a laser. In some examples, the system of subject matter 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-chlorine (XeCl) excimer lasers, or xenon-fluorine (XeF) excimer lasers, or combinations thereof. In other examples, the system of subject matter includes dye lasers such as stilbene lasers, coumarin lasers, or rhodamine lasers. In yet another example, the laser of interest includes metallic 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, and combinations thereof. Further examples include solid-state lasers such as ruby ​​lasers, Nd:YAG lasers, NdCrYAG lasers, Er:YAG lasers, Nd:YLF lasers, Nd:YVO4 lasers, Nd:YCa4O(BO3)3 lasers, Nd:YCOB lasers, titanium-sapphire lasers, slim YAG lasers, ytterbium YAG lasers, Yb2O3 lasers, or cerium-doped lasers, and combinations thereof.

[0110] The light source can be positioned at an angle to the flow stream in the range of 10° to 90°, including, for example, 15° to 85°, 20° to 80°, 25° to 75°, and 30° to 60°. In certain embodiments, the light source is positioned at a 90° angle to the sample.

[0111] In these embodiments, the system of interest also includes one or more detectors for detecting and measuring light from a flow stream. The detectors of interest may include, but are not limited to, optical sensors or photodetectors such as active pixel sensors (APS), avalanche photodiodes, image sensors, charge-coupled devices (CCDs), enhanced charge-coupled devices (ICCDs), light-emitting diodes, photon counters, bolometers, pyroelectric detectors, photoresistors, solar cells, photodiodes, photomultiplier tubes, phototransistors, quantum dot photoconductors, or photodiodes, as well as combinations thereof. In certain embodiments, transmitted light is measured by a charge-coupled device (CCD), semiconductor charge-coupled device (CCD), active pixel sensor (APS), complementary metal-oxide-semiconductor (CMOS) image sensor, or N-type metal-oxide-semiconductor (NMOS) image sensor. In some embodiments, the image sensor is a CCD camera. For example, the camera may be an electron-multiplier CCD (EMCCD) camera or an enhanced CCD (ICCD) camera. In other embodiments, the image sensor is a CMOS camera. When fluorescence or scattered light is measured by a CCD, the activity detection surface area of ​​the CCD is, for example, 0.05 cm². 2 ~9cm 2 For example, 0.1 cm 2 ~8cm 2 For example, 0.5 cm 2 ~7cm 2 , and 1cm 2 ~5cm 2 Including, for example, 0.01 cm 2 ~10cm 2 For example, the number of photodetectors in the subject system may vary as needed, for example, to include two or more, three or more, five or more, and ten or more photodetectors, for example, one or more. If the subject system includes two or more photodetectors, each photodetector may be the same, or the collection of two or more photodetectors may be a combination of different photodetectors.

[0112] The detector may be positioned at a certain distance from the flowstream, depending on the type of light source and the characteristics of the sample (e.g., particle size in the sample). For example, the detector may be positioned at a distance of 0.01 mm or more from the sample, including, for example, 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 50 mm or more from the sample. The detector may also be positioned at a varying angle with respect to the sample. For example, the detector may be positioned at an angle with respect to the flowstream in the range of 10° to 90°, including, for example, 15° to 85°, 20° to 80°, 25° to 75°, and 30° to 60°. In certain embodiments, the detector is positioned at a 90° angle with respect to the flowstream. In some embodiments, the system includes a detector positioned to detect forward scattered light from the flowstream. In other embodiments, the system includes a detector positioned to detect lateral scattered light from the flow stream. In yet another embodiment, the system includes a detector positioned to detect fluorescence from the flow stream.

[0113] Particle sorting systems according to some embodiments may include a display and an operator input device. The operator input device may be, for example, a keyboard, a mouse, etc. The processing module includes a processor that accesses memory having stored instructions for performing steps of the method of the subject. The processing module may include an operating system, a graphical user interface (GUI) controller, system memory, memory storage devices, and input / output controllers, cache memory, a data backup unit, and many other devices. The processor may be a commercially available processor, or one of other processors that are available or will become available. The processor runs an operating system, which interfaces with firmware and hardware in a well-known manner and facilitates the processor to coordinate and execute the functions of various computer programs that can be written in various programming languages ​​such as Java, Perl, C++, other high-level or low-level languages, and combinations thereof, as is known in the art. The operating system usually works in conjunction with the processor to coordinate and execute the functions of other components of the computer. The operating system also provides scheduling, input / output control, file and data management, memory management, and communication control and related services, according to all known technologies. The processor may be any preferred analog or digital system. In some embodiments, the processor includes analog electronic equipment that provides feedback control, such as negative feedback control.

[0114] System memory can be any of a variety of known or future memory storage devices. Examples include any commonly available random access memory (RAM), magnetic media such as resident hard disks or tapes, optical media such as read-write compact disks, flash memory devices, or other memory storage devices. Memory storage devices can be any of a variety of known or future devices, including compact disk drives, tape drives, removable hard disk drives, or disk drives. Such types of memory storage devices typically read from and / or write to program storage media (not shown), such as compact disks, magnetic tapes, removable hard disks, or magnetic disks, respectively. Any of these program storage media, or others currently in use or to be developed in the future, can be considered computer program products. As is understood, these program storage media typically store computer software programs and / or data. Computer software programs, also called computer control logic, are typically stored in system memory and / or program storage devices used in conjunction with memory storage devices.

[0115] In some embodiments, the computer program product is described in terms of a computer-usable medium having control logic (a computer software program including program code) stored therein. When the control logic is executed by the computer's processor, it causes the processor to perform the functions described herein. In other embodiments, some functions are implemented primarily in hardware, for example, using a hardware state machine. Implementations of hardware state machines for performing the functions described herein will be apparent to those skilled in the art.

[0116] Memory can be any suitable device on which the processor can store and retrieve data, such as magnetic, optical, or solid-state storage devices (including magnetic or optical disks, or tapes, or RAM, or any other suitable device, whether fixed or portable). The processor may include a general-purpose digital microprocessor preferably programmed from a computer-readable medium carrying the required program code. The programming can be provided to the processor remotely via a communication channel, or it can be pre-stored in memory or any other portable or fixed computer-readable storage medium, using one of those devices together with the memory. For example, a magnetic disk or optical disk can carry programming that can be read by a disk writer / reader. The system of the present invention also includes, for example, programming in the form of a computer program product, and algorithms for use in carrying out the above methods. The programming according to the present invention can be recorded on a computer-readable medium, for example, any medium that can be directly read and accessed by a computer. Such media include, but are not limited to, magnetic storage media such as magnetic disks, hard disk storage media, and magnetic tapes; optical storage media such as CD-ROMs; electrical storage media such as RAM and ROMs; portable flash drives; and hybrids of these categories such as magnetic / optical storage media.

[0117] The processor may also have access to a communication channel for communicating with the user at a remote location. A remote location means that the user does not have direct contact with the system, but rather relays input information to the input manager from an external device, such as a computer connected to any other suitable communication channel, including a wide area network ("WAN"), telephone network, satellite network, or mobile phone (i.e., smartphone).

[0118] In some embodiments, the systems according to this disclosure may be configured to include a communication interface. In some embodiments, the communication interface includes a receiver and / or transmitter for communicating with a network and / or another device. The communication interface may be configured for wired or wireless communication, including, but is not limited to, radio frequency (RF) communication (e.g., radio frequency identification (RFID)), Zigbee communication protocol, WiFi, infrared, wireless universal serial bus (USB), ultra-wideband (UWB), Bluetooth® communication protocol, and cellular communication such as code division multiple access (CDMA) or Global System for Mobile Communications (GSM).

[0119] In one embodiment, the communication interface is configured to include one or more communication ports, such as a physical port or interface, such as a USB port, an RS-232 port, or any other suitable electrical connection port, to enable data communication between the subject system and other external devices, such as computer terminals (e.g., in a clinic or hospital environment), configured for similar complementary data communication.

[0120] In one embodiment, the communication interface is configured for infrared communication, Bluetooth® communication, or any other suitable wireless communication protocol, enabling the system of the subject to communicate with other devices, such as computer terminals and / or networks, communicative mobile phones, personal digital assistants, or any other communication devices that the user may use in conjunction with them.

[0121] In one embodiment, the communication interface is configured to provide a connection for data transfer using the Internet Protocol (IP) via a mobile phone network, Short Message Service (SMS), a wireless connection to a personal computer (PC) on a local area network (LAN) connected to the Internet, or a Wi-Fi connection to the Internet via a Wi-Fi hotspot.

[0122] In one embodiment, the subject system is configured to communicate wirelessly with a server device via a communication interface using a common standard such as 802.11 or Bluetooth® RF protocol, or IrDA infrared protocol. The server device may be another portable device such as a smartphone, personal digital assistant (PDA), or notebook computer, or a larger device such as a desktop computer or appliance. In some embodiments, the server device has a display such as a liquid crystal display (LCD), as well as input devices such as buttons, a keyboard, a mouse, or a touchscreen.

[0123] In some embodiments, the communication interface is configured to communicate automatically or semi-automatically with a network or server device and data stored within the subject system, for example, in any data storage unit, using one or more of the communication protocols and / or mechanisms described above.

[0124] An output controller may include a controller for any of the various known display devices for presenting information to a user, whether human or machine, local or remote. If one of the display devices provides visual information, this information may typically be logically and / or physically organized as an array of pixels. A graphical user interface (GUI) controller may include any of the various known or future software programs for providing a graphical input and output interface between the system and the user, and for processing user input. Functional elements of a computer may communicate with each other via a system bus. Some of these communications may be achieved in alternative embodiments using a network or other type of remote communication. An output manager may also provide information generated by a processing module to a user at a remote location, for example, via the internet, telephone, or satellite network, according to known techniques. The presentation of data by the output manager may be carried out according to various known techniques. As some examples, the data may include SQL, HTML, or XML documents, email or other files, or other forms of data. The data may include Internet URL addresses so that the user can retrieve additional SQL, HTML, XML, or other documents or data from a remote source. One or more platforms present within the subject system are typically of a class of computers commonly referred to as servers, but may be any type of known or future computer platform. They may also be mainframe computers, workstations, or other computer types. They may be connected via other communication systems, including any known or future type of cabling or wireless systems, whether networked or not. They may be located in the same location or physically separated.Depending on the type and / or configuration of the selected computer platform, various operating systems may be adopted on any of the computer platforms. Suitable operating systems include Windows 10, Windows NT®, Windows XP, Windows 7, Windows 8, iOS, Sun Solaris, Linux®, OS / 400, Compaq Tru64 Unix, SGI IRIX, Siemens Reliant Unix, Ubuntu, and Zorin OS.

[0125] Aspects of the present disclosure further include a flow cytometry system having one or more particle sorting modules having openings for visualizing a deflected flow stream, as described above. Suitable flow cytometry systems and methods for sorting particles in a sample include, but are not limited to, 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 Examples include those listed in Carrier Syst. 24(3):203-255, which are incorporated herein by reference.In specific examples, flow cytometry systems of interest include: BD Biosciences FACSCanto® II flow cytometer, BD Accuri® flow cytometer, BD Biosciences FACSCelesta® flow cytometer, BD Biosciences FACSLyric® flow cytometer, BD Biosciences FACSVerse® flow cytometer, BD Biosciences FACSymphony® flow cytometer, BD Biosciences LSRFortessa® flow cytometer, BD Biosciences LSRFortess® X-20 flow cytometer, and BD Biosciences FACSCalibur® flow cytometer, BD Biosciences FACSCount® cell sorter, BD Biosciences FACSLyric® cell sorter, and BD Biosciences Via® cell sorter, BD Biosciences Influx® cell sorter, BD Biosciences Jazz® cell sorter, and BD Biosciences Examples include the Aria® cell sorting machine and the BD Biosciences FACSMelody® cell sorting machine.

[0126] In some embodiments, the particle sorting system of the subject is specified in U.S. Patent Nos. 9,952,076, 9,933,341, 9,726,527, 9,453,789, 9,200,334, 9,097,640, 9,095,494, 9,092,034, 8,975,595, 8,753,573, 8,233,146, 8,140,300, 7,544,326, and Flow cytometry systems such as those described in Patent Nos. 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, and 5,602,039, the disclosures of which are incorporated herein by reference in their entirety.

[0127] In some embodiments, the particle sorting system of interest is configured to sort particles using a sealed particle sorting module, such as that described in U.S. Patent Publication No. 2017 / 0299493, filed on 28 March 2017, the disclosure of which is incorporated herein by reference. For example, the particle sorting system of interest may include a register configured to be coupled to an aligner on the housing of the particle sorting module. The register may include one or more aligners complementary to the aligner on the housing of the particle sorting module. Furthermore, the particle sorting system may include one or more fasteners for maintaining physical contact between the particle sorting module and the particle sorting system by coupling one or more magnets on the particle sorting module housing to one or more magnets on the particle sorting system.

[0128] In certain embodiments, the subject particle sorting system is a flow cytometry system having an excitation module that generates multiple frequency-shifted beams of light using radio frequency multiplexing. In these embodiments, the laser light generator may include multiple lasers and one or more acousto-optic components (e.g., acousto-optic polarizers, acousto-optic frequency shifters) for generating multiple frequency-shifted comb beams. In certain examples, the subject system is a flow cytometry system having a laser excitation module as described in U.S. Patent No. 9,423,353, No. 9,784,661, and U.S. Patent Publication No. 2017 / 0133857 and No. 2017 / 0350803, the disclosures of which are incorporated herein by reference.

[0129] Method for aligning a deflected flowstream with a sample collection port Aspects of the present disclosure also include methods for aligning a flowstream with one or more sample collection ports at the distal end of a particle sorting module (as described above). Methods according to certain embodiments include capturing images through openings in a particle sorting module configured to visualize droplets of the flowstream, calculating the pixel positions of the boundaries within the imaged openings for each port at the distal end along the horizontal axis, and aligning the flowstream with the ports based on the calculated pixel positions of the boundaries for each port within the imaged openings. In embodiments, the method of the subject can be fully automated, for example, to reduce or completely eliminate the need for user input or manual determination of the boundaries of the sample collection ports, and for aligning a deflected flowstream with the sample collection ports. In other words, determining the boundaries of each sample collection port, and aligning a deflected flowstream with the sample collection ports, can be done with little or no human intervention or manual input by the user.

[0130] When carrying out the method according to a particular embodiment, one or more images are captured in the detection field through an opening in the particle sorting module housing. The “detection field” means the field of view through the opening in the particle sorting module housing that is captured by the imaging sensor. The imaged detection field can vary. In some embodiments, the detection field is a predetermined length of the deflected flow stream. In other embodiments, the detection field is a predetermined portion of the opening in the particle sorting module housing. In some embodiments, the imaging sensor is configured to capture an image of the deflected flow stream originating from a droplet deflector in the detection field. In embodiments, the image detection field may extend to 0.001 cm or more of the deflected flow stream, including, for example, 0.005 cm or more, for example, 0.01 cm or more, for example, 0.05 cm or more, for example, 0.1 cm or more, for example, 0.5 cm or more, for example, 1 cm or more, for example, 2 cm or more, for example, 5 cm or more, and 10 cm or more.

[0131] When capturing one or more images through an opening in the particle sorting module housing, the detection field is illuminated by a light source. In some embodiments, the detection field is illuminated by a broadband light source or a narrowband light source. Suitable broadband light source protocols include, but are not limited to, halogen lamps, deuterium arc lamps, xenon arc lamps, stabilized fiber-coupled broadband light sources, broadband LEDs with continuous spectra, ultra-high-brightness light-emitting diodes, semiconductor light-emitting diodes, broad-spectrum LED white light sources, multi-LED integrated white light sources, or any combination thereof. Suitable narrowband light sources include, but are not limited to, lasers, narrow-wavelength LEDs, laser diodes, or narrowband light sources coupled to one or more optical bandpass filters, diffraction gratings, monochromators, or any combination thereof.

[0132] Capturing one or more images through an opening in the particle sorting module housing may include, for example, illumination with two or more light sources, for example, a combination of light sources including three or more light sources, for example, four or more light sources, and five or more light sources. When an image is captured by illumination with two light sources, the method of the subject may include illumination with both light sources simultaneously. In other embodiments, capturing an image may include sequential illumination with two light sources. When two light sources are illuminated sequentially, the illumination time from each light source may be 0.001 seconds or more, independently including, for example, 0.01 seconds or more, for example, 0.1 seconds or more, for example, 1 second or more, for example, 5 seconds or more, for example, 10 seconds or more, for example, 30 seconds or more, and 60 seconds or more. In embodiments in which an image is captured by sequential illumination with two or more light sources, the duration of illumination from each light source may be the same or different.

[0133] Images through the openings of the particle sorting module housing can be captured continuously or at discrete intervals. In some examples, the method involves capturing images continuously. In other examples, the method involves capturing images at discrete intervals, such as capturing images of the flow stream every 0.001 milliseconds, every 0.01 milliseconds, every 0.1 milliseconds, every 1 millisecond, every 10 milliseconds, every 100 milliseconds, and every 1000 milliseconds, or at some other interval. For example, one or more images, including two or more images, three or more images, four or more images, five or more images, ten or more images, fifteen or more images, and twenty-five or more images, can be captured within the detection field.

[0134] Images through the aperture of the particle sorting module can be captured at any preferred distance (e.g., from the deflected flow stream) as long as usable images are captured. For example, images can be captured through the aperture in the particle sorting module housing at a distance of 0.01 mm or more from the deflected flow stream, including, for example, 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 50 mm or more. Images can be captured through the aperture at any angle. For example, images can be captured at an angle with respect to the axis of the deflected flow stream in the range of 10° to 90°, including, for example, 15° to 85°, for example, 20° to 80°, for example, 25° to 75°, and 30° to 60°. In certain embodiments, the image is captured at an angle of 90° with respect to the axis of the deflected flowstream.

[0135] In some embodiments, the method includes calculating the boundary position for each of the sample collection ports at the distal end of the housing along the horizontal axis, based on the image. In some examples, calculating the boundary position includes determining the distance between each boundary of the sample collection ports. For example, the method may include calculating the boundary of a first sample collection port based on an imaged aperture located 0.1 mm to 100 mm from the leftmost edge of the image along the horizontal axis, including, for example, 0.5 mm to 95 mm, for example, 1 mm to 90 mm, for example, 1.5 mm to 85 mm, for example, 2 mm to 80 mm, for example, 2.5 mm to 75 mm, and 3 mm to 70 mm. In other examples, the method may include calculating based on an imaged aperture located 0.1 mm to 100 mm from the left edge of the image along the horizontal axis, including, for example, 0.5 mm to 95 mm, for example, 1 mm to 90 mm, for example, 1.5 mm to 85 mm, for example, 2 mm to 80 mm, for example, 2.5 mm to 75 mm, and 3 mm to 70 mm. In yet another example, the method may include calculating based on an imaged aperture located 0.1 mm to 100 mm from the left edge of the image along the horizontal axis, including, for example, 0.5 mm to 95 mm, for example, 1 mm to 90 mm, for example, 1.5 mm to 85 mm, for example, 2 mm to 80 mm, for example, 2.5 mm to 75 mm, and 3 mm to 70 mm.

[0136] In other embodiments, the method includes calculating the spacing between each of the sample collection ports (i.e., the distance between the boundaries of each sample collection port). For example, the method may include calculating based on imaged apertures, where each sample collection port is spaced 0.1 mm to 10 mm apart along the horizontal axis, including, for example, 0.5 mm to 9.5 mm, 1 mm to 9 mm, 1.5 mm to 8.5 mm, 2 mm to 8 mm, 2.5 mm to 7.5 mm, and 3 mm to 7 mm.

[0137] In some examples, the method involves calculating based on an imaged aperture located 0.1 mm to 100 mm from the right edge of the image along the horizontal axis, with the boundary of the first sample collection port including, for example, 0.5 mm to 95 mm, 1 mm to 90 mm, 1.5 mm to 85 mm, 2 mm to 80 mm, 2.5 mm to 75 mm, and 3 mm to 70 mm. In other examples, the method involves calculating based on an imaged aperture located 0.1 mm to 100 mm from the right edge of the image along the horizontal axis, with the boundary of the second sample collection port including, for example, 0.5 mm to 95 mm, 1 mm to 90 mm, 1.5 mm to 85 mm, 2 mm to 80 mm, 2.5 mm to 75 mm, and 3 mm to 70 mm. In yet another example, the method involves calculating based on an imaged aperture where the boundary of the third sample collection port is located 0.1 mm to 100 mm from the right edge of the image along the horizontal axis, including, for example, 0.5 mm to 95 mm, 1 mm to 90 mm, 1.5 mm to 85 mm, 2 mm to 80 mm, 2.5 mm to 75 mm, and 3 mm to 70 mm.

[0138] In certain embodiments, the method includes generating a pixelated image. Any preferred image generation protocol may be employed, such that the pixelated image can be output in a digital format (e.g., editable) in certain examples. In these embodiments, the method may include calculating the pixel position of the boundary within the imaged opening for each port at the distal end, along the horizontal axis. In some examples, the method includes calculating the pixel position of the boundary for each sample collection port from the leftmost edge of the image along the horizontal axis, based on the imaged opening. In other examples, the method includes calculating the pixel position of the boundary for each sample collection port from the rightmost edge of the image along the horizontal axis, based on the imaged opening. In certain examples, the method includes outputting the number of pixels from either the leftmost or rightmost edge of the image, or both, along the horizontal axis. For example, the boundary position for each sample collection port may range from 1 to 1000 pixels from the leftmost or rightmost edge of the image of the opening in the particle sorting module housing, along the horizontal axis, including, for example, 5 to 750 pixels, 10 to 500 pixels, and 25 to 250 pixels. In other examples, the position of each sample collection port boundary is output as a ratio of the number of pixels along the horizontal axis to the total number of pixels in the entire imaged aperture. For example, the total number of pixels in the entire imaged aperture may be 100 pixels, and the position of each boundary for the sample collection port may be a ratio in the range of 1 / 100 to 99 / 100, including ratios in the ranges of 5 / 100 to 95 / 100, 10 / 100 to 90 / 100, 15 / 100 to 85 / 100, 20 / 100 to 80 / 100, and 25 / 100 to 75 / 100.

[0139] In some embodiments, calculating the pixel position of each boundary of the sample collection port involves calculating the total number of pixels across the entire imaged aperture along the horizontal axis, and multiplying the total number of pixels by a predetermined ratio. In these embodiments, the boundaries of each sample collection port (e.g., left and right boundaries along the horizontal axis) are determined based on the imaged aperture, assigned a number of pixels, and multiplied by a predetermined ratio.

[0140] In certain embodiments, the method includes aligning a flowstream with a sample collection port based on the calculated pixel position of the boundary for each sample collection port at the distal end of the particle sorting module housing. In some examples, one or more deflected flowstreams are aligned with the sample collection port. In other examples, an undefended flowstream (i.e., the longitudinal axis of the flowstream) is aligned with the center of the distal end of the particle sorting module. In one example, an undefended flowstream may be aligned with a waste liquid collection port at the distal end of the particle sorting module.

[0141] As described above, the flowstream can be deflected by applying a deflection force to the flowstream with a droplet deflector. The voltage applied to the metal plate of the droplet deflector can be adjusted as needed to align the deflected flowstream with the sample collection port having the position determined above. The voltage applied to the deflector plate to deflect charged particles can be 10mV or more, including, for example, 25mV or more, 50mV or more, 100mV or more, 250mV or more, 500mV or more, 750mV or more, 1000mV or more, 2500mV or more, 5000mV or more, 10000V or more, 15000V or more, 25000V or more, 50000V or more, and 100000V or more. In certain embodiments, the voltage applied to the deflector plate is 0.5kV to 15kV, including, for example, 1kV to 15kV, 1.5kV to 12.5kV, and 2kV to 10kV. Therefore, the electric field strength between the deflector plates is, for example, 0.5V / m to 5 × 10⁻¹⁰. 6 V / m, for example, 1 V / m ~ 1 × 10⁻¹⁰ 6 V / m, for example, 5V / m ~ 5 × 10 5 V / m, for example, 10V / m ~ 1 × 10 5 V / m, and 50V / m ~ 5 × 10 4 Includes V / m, for example, 1 × 10 5 V / m ~ 2 × 10 6V / m, etc., 0.1V / m ~ 1 × 10 7 It is in the range of V / m.

[0142] In some embodiments, the method includes sorting components of a sample using a subject particle sorting module. In certain examples, the sample is a biological sample, and the method includes sorting and collecting two or more different types of cells. In some embodiments, the sample is a biological sample. The term “biological sample” is used in its conventional sense to refer to a whole organism, plant, fungus, or, in certain examples, a subset of the tissues, cells, or components of an animal that may be found in blood, mucus, lymph, synovial fluid, cerebrospinal fluid, saliva, bronchoalveolar lavage, amniotic fluid, amniotic umbilical cord blood, urine, vaginal fluid, and semen. Thus, “biological sample” refers to both a natural organism or a subset of its tissues, as well as 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 urinary tract, tears, saliva, milk, blood cells, tumors, and organs. The biological sample can be any type of biological tissue, including both healthy tissue and diseased tissue (e.g., cancerous, malignant, necrotic, etc.). In certain embodiments, the biological sample is a liquid sample such as blood or its derivatives, e.g., plasma, tears, urine, semen, and in some examples, the sample is a blood sample including whole blood, such as blood obtained from a venipuncture or fingerstick (the blood may or may not be combined with any reagents such as preservatives and anticoagulants before the assay).

[0143] In certain embodiments, the source of the sample is “mammal” or “mammalian,” terms used broadly to refer to organisms within 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 examples, the subject is human. The method may be applied to samples obtained from human subjects of both sexes at any stage of development (i.e., neonates, infants, adolescents, and adults), and in certain embodiments, the human subject may be adolescent, adolescent, or adult. While the present invention may be applied to samples from human subjects, it should be understood that it may also be applied to samples from other animal subjects (i.e., “non-human subjects”), such as birds, mice, rats, dogs, cats, livestock, and horses, but is not limited to the present invention.

[0144] Cells of interest can be targeted for separation from the flow stream according to various parameters, such as phenotypic characteristics identified by attaching specific fluorescent labels to the cells of interest. In some embodiments, the system is configured to deflect analyzed droplets determined to contain target cells. Various cells can be targeted for sorting using the method of interest. Target cells of interest include, but are not limited to, stem cells, T cells, dendritic cells, B cells, granulocytes, leukemia cells, lymphoma cells, viral cells (e.g., HIV cells), NK cells, macrophages, monocytes, fibroblasts, epithelial cells, endothelial cells, and erythrocytes. Target cells of interest include cells having favorable cell surface markers or cell surface antigens that can be captured or labeled by favorable affinity agents or their complexes. For example, target cells may contain cell surface antigens such as CD11b, CD123, CD14, CD15, CD16, CD19, CD193, CD2, CD25, CD27, CD3, CD335, CD36, CD4, CD43, CD45RO, CD56, CD61, CD7, CD8, CD34, CD1c, CD23, CD304, CD235a, T cell receptor α / β, T cell receptor γ / Δ, CD253, CD95, CD20, CD105, CD117, CD120b, Notch4, Lgr5 (N-terminus), SSEA-3, TRA-1-60 antigen, disialoganglioside GD2, and CD71. In some embodiments, target cells are selected from HIV-containing cells, Treg cells, antigen-specific T cell populations, tumor cells, or hematopoietic progenitor cells (CD34+) from whole blood, bone marrow, or umbilical cord blood.

[0145] Methods for sorting components of a sample, according to several embodiments, include introducing a fluid sample having target particles into a flow nozzle of a particle sorting module. Exiting the flow nozzle, the particles pass substantially one at a time through a sample inspection area, during which each particle is irradiated by a light source, and, if necessary, measurements of light scattering parameters and fluorescence emission (e.g., two or more light scattering parameters and one or more fluorescence emission measurements) are recorded separately for each particle. The particles pass through the flowstream substantially one at a time in the flow path, through the sample inspection area in the particle sorting module where each particle is irradiated by a light source. Depending on the characteristics of the flowstream being inspected, more than 0.001 mm of the flowstream may be irradiated, for example, more than 0.005 mm, more than 0.01 mm, more than 0.05 mm, more than 0.1 mm, more than 0.5 mm, and more than 1 mm of the flowstream, etc. In certain embodiments, the method includes irradiating a planar cross section of the flowstream within the sample inspection area with, for example, a laser (as described above). In other embodiments, the method includes irradiating a sample inspection area with a flow stream of a predetermined length, corresponding to, for example, the irradiation profile of a diffuse laser beam or lamp.

[0146] A detector, such as a photomultiplier tube (or "PMT"), is used in series with the sensing region to record the light passing through each particle (sometimes called forward scattering), the light reflected perpendicular to the direction of the particle flow through the sensing region (sometimes called orthogonal or side scattering), and the fluorescence emitted from the particle, if labeled with a fluorescent marker, as the particle passes through the sensing region and is irradiated by an energy source. Each of forward scattering (or FSC), orthogonal scattering (SSC), and fluorescence emission (FL1, FL2, etc.) contains separate parameters for each particle (or each "event"). Thus, for example, two, three, or four parameters may be collected (and recorded) from a particle labeled with two different fluorescent markers.

[0147] As mentioned above, suitable light detection protocols include, but are not limited to, optical sensors or photodetectors such as active pixel sensors (APS), avalanche photodiodes, image sensors, charge-coupled devices (CCD), intensified charge-coupled devices (ICCD), light emitting diodes, photon counters, bolometers, pyroelectric detectors, photoresistors, solar cells, photodiodes, photomultiplier tubes, phototransistors, quantum dot photoconductors, or photodiodes, among other photodetectors, and combinations of the foregoing. In certain embodiments, light from a stream irradiated in a sample examination region of a particle sorting module is measured by a charge-coupled device (CCD), a semiconductor charge-coupled device (CCD), an active pixel sensor (APS), a complementary metal oxide semiconductor (CMOS) image sensor, or an N-type metal oxide semiconductor (NMOS) image sensor. In certain embodiments, the light is measured by a charge-coupled device (CCD). When light from the irradiated stream in the sample examination region of the particle sorting module is measured by a CCD, the active detection surface area of the CCD is, for example, 0.05 cm 2 to 9 cm 2 , for example, 0.1 cm 2 to 8 cm 2 , for example, 0.5 cm 2 to 7 cm 2 , and 1 cm 2 to 5 cm 2 , including, for example, 0.01 cm 2 to 10 cm 2 and the like, and may vary.

[0148] The method in certain embodiments also includes data acquisition, analysis, and recording using a computer, etc., such that multiple data channels record data from each detector for light scattering and fluorescence emitted by each particle as each particle passes through the sample inspection area of ​​the particle sorting module. In these embodiments, the analysis includes sorting and counting the particles so that each particle exists as a set of digitized parameter values. The system of the subject may be configured to trigger on selected parameters to distinguish particles of interest from background and noise. “Trigger” refers to a preset threshold for detecting a parameter and may be used as a means for detecting the passage of particles through a light source. Detection of events exceeding the threshold of the selected parameter triggers the acquisition of light scattering and fluorescence data for the particle. For particles or other components in the medium being analyzed that cause a response below the threshold, no data is acquired. The trigger parameter may be the detection of forward scattered light caused by a particle passing through a light beam. A flow cytometer then detects and collects the light scattering and fluorescence data for the particle.

[0149] Next, a specific subpopulation of interest is further analyzed by "gating" based on data collected for the entire population. The data is plotted to obtain the best possible separation of the subpopulation in order to select an appropriate gate. This procedure can be performed by plotting forward light scattering (FSC) versus side (i.e., orthogonal) light scattering (SSC) on a two-dimensional dot plot. Then, a subpopulation of particles (i.e., those cells within the gate) is selected, and particles not within the gate are excluded. If necessary, the gate can be selected by drawing a line around the desired subpopulation using a cursor on a computer screen. Then, only those particles within the gate are further analyzed by plotting other parameters of these particles, such as fluorescence. If necessary, the above analysis can be configured to yield a count of the particles of interest in the sample.

[0150] In certain examples, the particle sorting module is a sealed particle sorting module having one or more aligners for coupling to a particle sorting system, as described above. In these embodiments, in order to couple the particle sorting module to the particle sorting system, the aligners on the outer wall of the particle sorting module housing are positioned to contact the aligners on the registers of the particle sorting system. If present, one or more fasteners can be engaged when the aligners on the outer wall of the particle sorting module housing contact the aligners on the registers of the particle sorting system in order to secure the particle sorting module to the particle sorting system. After coupling the particle sorting module to the particle sorting system, a certain amount of fluid sample is injected into the particle sorting module for the analysis and cell sorting described above.

[0151] In certain embodiments, a method for sorting components of a sample includes sorting particles (e.g., cells in a biological sample) using a sealed particle sorting module, such as the one filed on 28 March 2017, whose disclosure is incorporated herein by reference in U.S. Patent Publication No. 2017 / 0299493.

[0152] kit Aspects of the present invention further include a kit, which comprises one or more particle sorting modules described herein. In some embodiments, the kit includes a particle sorting module having an opening configured to visualize droplets of a deflected flowstream and one or more sample collection containers for coupling with the particle sorting module. In some embodiments, the kit includes a droplet deflector, for example, a set of parallel metal plates, along with instructions for installing the droplet deflector within the particle sorting module. In some examples, the kit includes one or more connectors for coupling the particle sorting module to a sample fluid delivery system and a sheath fluid delivery system.

[0153] The various components of the kit may reside in separate containers, or some or all of them may be pre-assembled. For example, in some cases, one or more components of the kit, such as each particle sorting module, sample collection container, and droplet deflector, may reside in a sealed pouch, such as a sterile foil pouch or envelope.

[0154] In addition to the components described above, the subject kit may further include instructions for carrying out the subject method (in a particular embodiment). These instructions may be present in the subject kit in various forms, and one or more of these forms may be present in the kit. One form in which these instructions may be present is as printed information in the kit packaging, such as on a suitable medium or substrate, such as one or more sheets of paper on which the information is printed, in accompanying documents, etc. Yet another form of these instructions may be a computer-readable medium on which the information is recorded, such as a diskette, compact disc (CD), portable flash drive, etc. Yet another form in which these instructions may be present is a website address that can be used via the internet to access the information at a remote site.

[0155] Utility The subject matter, particle sorting modules, particle sorting systems, methods, and computer systems, are used in a variety of applications where it is desirable to analyze and sort particulate components in a sample in a fluid medium, such as a biological sample. Embodiments of this disclosure are used when it is desirable to provide a flow cytometer having improved cell sorting accuracy, enhanced particle collection, particle charging efficiency, more accurate particle charging, and improved particle deflection during cell sorting.

[0156] Embodiments of this disclosure also involve the use of cells prepared from biological samples in applications where such use may be desirable in research, laboratory testing, or therapeutics. In some embodiments, the methods and devices of this subject may facilitate the acquisition of individual cells prepared from target fluid or tissue biological samples. For example, the methods and systems of this subject facilitate the acquisition of cells from fluid or tissue samples used as research or diagnostic specimens for diseases such as cancer. Similarly, the methods and systems of this subject may facilitate the acquisition of cells from fluid or tissue samples used in therapeutics. The methods and devices of this disclosure enable the separation and collection of cells from biological samples (e.g., organs, tissues, tissue fragments, body fluids) with improved efficiency and lower cost compared to conventional flow cytometry systems. In certain embodiments, the particle sorting module of this subject is sealed, thereby providing improved sterility to the particle sorting system, enhancing the collection of higher purity samples in research and high-throughput laboratory testing, etc., while reducing the incidence of secondary contamination between analyzed samples.

[0157] Notwithstanding the attached claims, this disclosure is also clarified by the following note:

[0158] 1. A particle sorting module having an opening configured to visualize droplets in a deflected flowstream. 2. The particle sorting module is A housing comprising a proximal end, a distal end, and a wall between them, with an opening located within this wall, Droplet deflector and A particle sorting module as described in Appendix 1, comprising the features described above. 3. A particle sorting module as described in Appendix 2, wherein the opening is located downstream from the droplet deflector. 4. A particle sorting module as described in any one of the appendices 1 to 3, wherein one or more ports at the distal end of the housing are configured to connect to one or more sample collection containers. 5. The particle sorting module described in Appendix 4, wherein the opening is configured to align a flowstream with one or more ports.

[0159] 6. The particle sorting module described in Appendix 4, wherein the opening is located between the droplet deflector and the distal end of the housing. 7. A particle sorting module as described in Appendix 6, wherein the opening is located 0.5 cm to 5 cm from the distal end of the housing. 8. A particle sorting module as described in Appendix 6, wherein the opening is located 0.5 cm to 5 cm from the droplet deflector. 9. The opening is a particle sorting module as described in any one of the appendices 1 to 8, comprising optical adjustment components. 10. The particle sorting module as described in Appendix 9, comprising an optical adjustment component, which includes a focusing lens configured to focus light irradiation onto droplets from a deflected flowstream.

[0160] 11. The optical adjustment component is a particle sorting module described in any one of the appendices 1 to 10, which has a transparent optical window. 12. The particle sorting module described in Appendix 11, wherein the optical window comprises one or more reference identifiers. 13. The particle sorting module as described in Appendix 12, wherein the reference identifier is a marking, each marking associated with the boundary of one or more ports at the distal end of the housing. 14. A particle sorting module according to any one of appendices 4 to 13, further comprising a waste liquid collection container coupled to the distal end of the housing and two sample collection containers. 15. The particle sorting module as described in Appendix 14, wherein the opening is an optical window having a reference identifier associated with the boundary of each of the waste liquid collection container and the two sample collection containers.

[0161] 16. The reference identifier is a particle sorting module as described in any one of the appendices 13 to 15, which has a marking indicating the center of the distal end of the housing. 17. A flow cell nozzle, located at the proximal end of the housing and equipped with an orifice, The sample inspection area that communicates with the orifice of the flow cell nozzle and the fluid A particle sorting module further comprising one of the features described in Appendix 1 to 16. 18. A particle sorting module as described in Appendix 17, further comprising cuvettes positioned within the sample inspection area. 19. A particle sorting module as described in Appendix 18, wherein the cuvette is co-molded with the housing. 20. The cuvette is a particle sorting module made of glass, as described in Appendix 18 or 19.

[0162] 21. The cuvette is a particle sorting module made of plastic, as described in Appendix 18 or 19. 22. A particle sorting module as described in any one of the appendices 1 to 21, further comprising a sample inlet at the proximal end of the housing. 23. A particle sorting module as described in any one of the appendices 1 to 22, further comprising a sheath fluid inlet at the proximal end of the housing.

[0163] 24. A system, A particle sorting module having an opening configured to visualize droplets in a deflected flowstream, An imaging sensor configured to capture one or more images through an aperture A system equipped with these features. 25. The system as described in Appendix 24, further comprising a laser for irradiating a flowstream through an aperture. 26. The particle sorting module is A housing comprising a proximal end, a distal end, and a wall between them, wherein the opening is located within this wall of the housing, Droplet deflector and A system comprising the features described in Appendix 24 or 25. 27. The system according to Appendix 26, further comprising one or more ports at the distal end of a housing configured to be coupled to one or more sample collection containers.

[0164] 28. A processor comprising memory operably coupled to the processor, further comprising a processor When this memory is executed by the processor, the processor will Capture an image of the aperture of the particle sorting module and obtain the imaged aperture, Along the horizontal axis, calculate the pixel position within the imaged aperture of the boundary for each port at the distal end of the housing. The system described in Appendix 27, which includes instructions stored in memory, that causes the system to perform the following actions. 29. Calculating the pixel position is The total number of pixels across the entire imaged aperture is calculated along the horizontal axis, and Multiplying the total number of pixels by a predetermined ratio and The system described in Appendix 28, including the system described in Appendix 28. 30. The system described in Appendix 29, wherein the predetermined ratio is calculated by dividing the number of pixels within the imaged aperture at the boundary of each port by the total number of pixels in the entire imaged aperture along the horizontal axis.

[0165] 31. A system as described in any one of Appendix 27-30, in which, when executed by the processor, the memory contains instructions stored in memory that cause the processor to align a flow stream with one of the ports at the distal end of the housing. 32. The system as described in Appendix 31, wherein when the memory is executed by the processor, the processor contains instructions stored in the memory that cause the flow stream to align with the center of one of the ports at the distal end of the housing. 33. A system as described in any one of the appendices 30 to 32, in which, when the memory is executed by the processor, the processor receives instructions stored in the memory that cause the flow stream to align with the center of the distal end of the housing. 34. The system according to any one of the appendices 27 to 33, wherein the opening is located between the droplet deflector and the distal end of the housing. 35. The system described in Appendix 34, wherein the opening is located 0.5 cm to 5 cm from the distal end of the housing.

[0166] 36. The system described in Appendix 34, wherein the opening is located 0.5 cm to 5 cm from the droplet deflector. 37. The system described in any one of the appendices 24 to 36, wherein the aperture is equipped with an optical adjustment component. 38. The system as described in Appendix 37, wherein the optical adjustment component comprises a focusing lens configured to focus light onto droplets from a deflected flowstream. 39. The optical adjustment component is a system described in any one of the appendices 24 to 38, comprising a transparent optical window. 40. The optical window is a system as described in Appendix 39, comprising one or more reference identifiers.

[0167] 41. The system described in Appendix 40, wherein the reference identifier comprises markings, each marking associated with a boundary of deflection by a droplet. 42. The particle sorting module further comprises a waste liquid collection container and two sample collection containers coupled to the distal end of the housing, as described in any one of the appendices 24 to 41. 43. The system as described in Appendix 42, wherein the opening is an optical window having a reference identifier associated with the deflection boundary of each of the waste liquid collection container and the two sample collection containers. 44. The reference identifier is a system described in any one of the appendices 40 to 43, which includes a marking indicating the center of the distal end of the housing. 45. The particle sorting module is A flow cell nozzle, located at the proximal end of the housing and equipped with an orifice, This flow cell nozzle orifice and the sample inspection area that communicates with the fluid A system further comprising any one of the appendices 24-44.

[0168] 46. ​​The particle sorting module further comprises a cuvette positioned within the sample inspection area, as described in Appendix 45. 47. The system as described in Appendix 46, wherein the cuvette is co-molded with the housing. 48. The cuvette is made of glass, as described in Appendix 46 or 47. 49. The cuvette is made of plastic and is part of the system described in Appendix 46 or 47. 50. The system according to any one of the appendices 24 to 49, further comprising a sample delivery subsystem that is in fluid communication with an inlet at the proximal end of the housing of the particle sorting module.

[0169] 51. The system according to any one of the appendices 24 to 50, further comprising a sheath fluid delivery subsystem that fluidly communicates with an inlet at the proximal end of the housing of the particle sorting module. 52. The system described in any one of the appendices 24 to 51, further comprising a fluid-coupled waste liquid tank at the outlet from the particle sorting module.

[0170] 53. A method for aligning a flowstream with one or more ports at the distal end of a particle sorting module, This involves capturing images through an opening in a particle sorting module configured to visualize droplets in a flowstream, Along the horizontal axis, the pixel position within the imaged aperture of the boundary for each port at the distal end is calculated, Based on the calculated pixel positions of the boundaries of each port within the imaged aperture, the flow stream is aligned with the ports. Methods that include... 54. Calculating the pixel position is The total number of pixels across the entire imaged aperture is calculated along the horizontal axis, and Multiplying the total number of pixels by a predetermined ratio and The method described in Appendix 53, including the method described in Appendix 53. 55. The method according to Appendix 54, further comprising calculating a predetermined ratio by dividing the number of pixels within the imaged aperture at the boundary of each port by the total number of pixels in the entire imaged aperture along the horizontal axis.

[0171] 56. The particle sorting module comprises a waste liquid collection container and two sample collection containers, according to any one of the methods described in Appendix 53 to 55. 57. The method according to any one of the appendices 53 to 56, further comprising irradiating the flowstream with a laser through the aperture. 58. The particle sorting module is A sealed housing comprising a proximal end, a distal end, and a wall between them, with an opening located within this wall of the housing, Droplet deflector and A method according to any one of the appendices 53 to 57, comprising: 59. The method according to Appendix 58, wherein the opening is located between the droplet deflector and the distal end of the housing. 60. The method described in Appendix 59, wherein the opening is located 0.5 cm to 5 cm from the distal end of the housing.

[0172] 61. The method as described in Appendix 59, wherein the opening is located 0.5 cm to 5 cm from the droplet deflector. 62. The method according to any one of the appendices 53 to 61, wherein the opening is equipped with an optical adjustment component. 63. The optical adjustment component comprises a focusing lens, as described in Appendix 62. 64. The optical adjustment component comprising a transparent optical window, as described in Appendix 62.

[0173] 65. It is a kit, A particle sorting module having an opening configured to visualize droplets in a deflected flowstream, One or more sample collection containers for coupling with the particle sorting module A kit that includes the following: 66. The kit described in Appendix 65, further comprising a droplet deflector. 67. The particle sorting module is the kit described in Appendix 65 or 66, comprising a sample inlet and a sheath fluid inlet. 68. The kit as described in Appendix 67, further comprising one or more connectors for coupling the particle sorting module to a sample fluid delivery system and a sheath fluid delivery system.

[0174] 69. A kit described in any one of the appendices 65-68, wherein one or more of the particle sorting module, sample collection container, and connector are present inside the container. 70. The kit described in Appendix 69, wherein one or more of the particle sorting module, sample collection container, and connector are located inside the pouch. 71. A kit whose components are sterilized, as described in any one of the notes 65-70.

[0175] In at least some of the embodiments described above, one or more elements used in one embodiment may be used interchangeably in another embodiment, provided that such substitution is not technically impossible. It will be understood by those skilled in the art that various other omissions, additions, and modifications may be made to the above methods and structures without departing from the scope of the claimed subject matter. All such modifications and changes are intended to be within the scope of the subject matter defined by the appended claims.

[0176] In general, it will be understood by those skilled in the art that the terms used herein, and in particular in the appended claims (e.g., the text of the appended claims), are generally intended to be “open” terms (for example, “including” should be interpreted as “including but not limited to,” “having” as “at least having,” and “includes” as “including but not limited to,” etc.). It will also be understood by those skilled in the art that if a certain number of introduced claims are intended, such intention will be explicitly stated in the claims, and if such statement is not present, such intention does not exist. For example, for the sake of understanding, the following appended claims may include the use of the introductory phrases “at least one” and “one or more” to introduce the claims. However, even if the same patent claim includes introductory phrases such as "one or more" or "at least one," and indefinite articles such as "a" or "an," the use of such phrases should not be interpreted as meaning that the introduction of a claim by the indefinite article "a" or "an" limits any particular patent claim containing such introduced claim to embodiments containing only one such description (for example, "a" and / or "an" should be interpreted as meaning "at least one" or "one or more"), and the same applies to the use of definite articles used to introduce claim descriptions. Furthermore, even if a specific number of introduced claims are explicitly stated, a person skilled in the art will recognize that such a statement should be interpreted as meaning at least the number stated (for example, the statement "two descriptions" without other modifiers means at least two descriptions or two or more descriptions).Furthermore, when a convention similar to "at least one of A, B, and C, etc." is used, such a construction is generally intended to be understood by those skilled in the art (for example, "a system having at least one of A, B, and C" includes, but is not limited to, systems having A alone, B alone, C alone, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). When a convention similar to "at least one of A, B, or C, etc." is used, such a construction is generally intended to be understood by those skilled in the art (for example, "a system having at least one of A, B, or C" includes, but is not limited to, systems having A alone, B alone, C alone, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). It will be further understood by those skilled in the art that substantially any disjunctive word and / or disjunctive phrase presenting two or more alternative terms, whether in the specification, claims, or drawings, should be understood as intended to include the possibility of including one of the terms, either of the terms, or both of the terms. For example, the phrase “A or B” is understood to include the possibility of “A” or “B” or “A and B.”

[0177] Furthermore, if any feature or aspect of this disclosure is described from the perspective of the Marsch Group, a person skilled in the art will recognize that this disclosure is also described from the perspective of any individual member of the Marsch Group or any subgroup of a member of the Marsch Group.

[0178] For all purposes, including providing written explanations, as will be understood by those skilled in the art, all scopes disclosed herein also encompass all possible sub-scopes and combinations thereof. Any listed scope can be readily recognized as sufficiently indicating and enabling that the same scope can be broken down into at least equal 1 / 2, 1 / 3, 1 / 4, 1 / 5, 1 / 10, etc. As a non-limiting example, each scope considered herein can readily be broken down into a lower third, a middle third, an upper third, etc. As will also be understood by those skilled in the art, all terms such as “maximum,” “at least,” “greater than,” and “less than” include the number stated and refer to a scope that can be subsequently broken down into sub-scopes as described above. Finally, as will be understood by those skilled in the art, a scope includes each individual member. Thus, for example, the group having 1 to 3 articles refers to the group having 1, 2, or 3 articles. Similarly, the group having 1 to 5 articles refers to the group having 1, 2, 3, 4, or 5 articles, and so on.

[0179] Although the above invention has been described in some detail by examples and illustrations for the sake of clear understanding, it will be readily apparent to those skilled in the art that certain changes and modifications can be made in light of the teachings of the present invention without departing from the spirit or scope of the appended claims.

[0180] Therefore, the foregoing merely describes the principles of the present invention. Those skilled in the art will understand that various modifications embodying the principles of the present invention and falling within the spirit and scope of the invention can be devised, even if not expressly described or indicated herein. Furthermore, all examples and conditional terms described herein are intended primarily to help the reader understand the principles of the present invention and the concepts provided by the inventors for further advancing the art, and should be construed as not being limited to such specifically described examples and conditions. Furthermore, all descriptions herein, as well as specific examples thereof, describing the principles, aspects, and embodiments of the present invention, are intended to encompass both their structural and functional equivalents. Moreover, such equivalents are intended to include both currently known equivalents and those to be developed in the future, i.e., any elements developed to perform the same function, regardless of their structure. Furthermore, nothing disclosed herein, whether such disclosure is expressly stated in the claims or not, is intended to be publicly dedicated.

[0181] Accordingly, the scope of the present invention is not intended to be limited to the exemplary embodiments shown and described herein. Rather, the scope and spirit of the present invention are embodied in the appended claims. In the claims, § 112(f) or § 112(6) of the U.S. Patent Act is explicitly defined as applying to any limitation in a claim only if the phrase "means for" or "step for" is exactly identical to the phrase "means for" or "step for" is exactly identical to the phrase "step for" at the beginning of the limitation in the claim, and neither § 112(f) nor § 112(6) applies if such an identical phrase is not used in the limitation in the claim.

[0182] Cross-reference of related applications In accordance with Section 119(e) of the United States Patent Act, this application claims priority to the filing date of U.S. Provisional Patent Application No. 62 / 752,793, filed on 30 October 2018, the disclosure of which is incorporated herein by reference.

Claims

1. It comprises a housing having a proximal end, a distal end, and a wall between them. The aforementioned housing is A droplet deflector and Two or more sample collection ports at the distal end, each of which is configured to deliver droplets received at the sample collection port to a sample collection container coupled to the sample collection port, An opening located in the wall between the droplet deflector and the distal end of the housing It is equipped with, The opening is configured to provide a field of view through the opening for capturing images used to visualize droplets in a deflected flowstream and to determine the position of the boundaries of one or more sample collection ports and to align the deflected flowstream with one or more sample collection ports. The aforementioned opening and sample collection port are configured such that, for each sample collection port, the position of the opening along the horizontal axis corresponds to the boundary of the sample collection port along the horizontal axis, and each position is defined by a predetermined ratio of the total width of the opening along the horizontal axis. Particle sorting module.

2. The particle sorting module according to claim 1, wherein the opening is equipped with an optical adjustment component.

3. The particle sorting module according to claim 2, wherein the optical adjustment component comprises a focusing lens configured to focus light irradiation onto droplets from a deflected flowstream.

4. The particle sorting module according to any one of claims 1 to 3, further comprising a waste liquid collection container coupled to the distal end of the housing and two sample collection containers.

5. Located at the proximal end of the housing, it includes a flow cell nozzle with an orifice, The sample inspection area that is in fluid communication with the orifice of the flow cell nozzle A particle sorting module according to any one of claims 1 to 4, further comprising the above.

6. The particle sorting module according to claim 5, further comprising a cuvette positioned within the sample inspection area.

7. The particle sorting module according to claim 5 or 6, further comprising a sample inlet and a sheath fluid inlet at the proximal end of the flow cell nozzle.

8. A system comprising a particle sorting module according to any one of claims 1 to 7.

9. A method for aligning a flowstream with one or more ports at the distal end of a particle sorting module, Capturing an image through an opening in the particle sorting module according to any one of claims 1 to 7, Along the horizontal axis, the pixel position within the imaged aperture of the boundary for each of the ports at the distal end is calculated, For each port within the imaged aperture, the flow stream is aligned with the port based on the calculated pixel position of the boundary. Methods that include...

10. A kit comprising the particle sorting module according to any one of claims 1 to 7.

Images