Flow cytometry droplet dispensing system and method of using the system

Abstract

A flow cytometry droplet dispensing system and a method for dispensing droplets into zones using the system via flow cytometry are provided. Aspects of embodiments of the system include a sorting flow cytometer configured to sort particle-occupied droplets and particle-unoccupied droplets into zones. Methods for using the system are also provided. The systems and methods of the present invention can be used in a variety of applications.

Description

[0001] Cross-reference to related applications

[0002] Pursuant to 35 USC §119(e), this application claims priority to U.S. Provisional Patent Application Serial No. 63 / 043,323, filed June 24, 2020, and U.S. Provisional Patent Application Serial No. 63 / 114,402, filed November 16, 2020, the disclosures of which are incorporated herein by reference in their entirety. Background Technology

[0003] Flow cytometry particle sorting systems, such as sorting flow cytometers, are used to sort particles in a fluid sample based on at least one measurement characteristic of the particles. In a flow cytometry particle sorting system, particles (e.g., molecules, beads bound to analytes, or single cells) in a fluid suspension pass through a detection region (i.e., an interrogation point) in the flow, where a sensor detects particles contained in the flow of the type to be sorted. When the sensor detects particles of the type to be sorted, it triggers a sorting mechanism that selectively separates the particles of interest. The particles of interest are separated into partitions, such as the wells of a sample container, test tube, or multiwell plate.

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

[0005] To sort particles in a sample, a droplet charging mechanism charges droplets containing the type of particles to be sorted in the flow stream at the break point of the flow stream. The droplets are then passed through an electrostatic field and deflected into one or more zones, such as a sample collection container, based on the polarity and size of the charge on the droplet. Uncharged droplets are not deflected by the electrostatic field.

[0006] Traditionally, cell sorting flow cytometers use electrostatic deflection to sort droplets containing cells of interest, thereby separating the cells of interest from the rest of the sample. The deflection of one or more droplets always depends on the particle that triggers the instrument's electronics and a series of subsequent sorting and deflection decisions based on whether the particle falls within or outside user-defined boundaries (e.g., whether the cell includes specific surface markers). Summary of the Invention

[0007] The inventors have discovered that appropriately modified sorting flow cytometers (e.g., as described below) can be used as highly precise liquid microdispensing systems (e.g., capable of deflecting known volumes of liquid at milliliter-level precision). Embodiments of the invention include a sorting flow cytometer configured (e.g., as described below) to sort and deflect droplets without requiring particles to trigger a sorting decision electronics. Embodiments of the flow cytometer of the invention are configured to track droplets occupied by particles (e.g., droplets containing desired or unwanted particles (e.g., cells)) and droplets not occupied by particles (e.g., droplets excluding cells or beads), wherein the flow cytometer is configured to sort a known number of each type of droplet.

[0008] This invention provides a flow cytometer droplet dispensing system and a method for dispensing droplets into zones using the system via flow cytometry. Aspects of embodiments of the system include a sorting flow cytometer configured to sort particle-occupied droplets and particle-unoccupied droplets into zones. Methods for using the system are also provided. The systems and methods of this invention can be used in a variety of applications. Attached Figure Description

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

[0010] Figure 1 A functional block diagram of an example sorting control system according to certain embodiments is shown.

[0011] Figure 2A A schematic diagram of a particle sorting system according to certain embodiments is shown.

[0012] Figure 2B A schematic diagram of a particle sorting system according to certain embodiments is shown.

[0013] Figure 3 A functional block diagram of a particle analysis system for computation-based sample analysis and particle characterization, according to certain embodiments, is shown.

[0014] Figure 4 A flow cytometer according to certain embodiments is shown.

[0015] Figure 5 The results of volumetric allocation according to an embodiment of the present invention, as described in the experimental section, are shown. Detailed Implementation

[0016] This invention provides a flow cytometer droplet dispensing system and a method for dispensing droplets into partitions using the system. Aspects of embodiments of the system include a sorting flow cytometer configured to sort particle-occupied droplets and particle-unoccupied droplets into partitions. Methods for using the system are also provided. The systems and methods of this invention can be used in a variety of applications.

[0017] Before describing the invention in more detail, it should be understood that the invention is not limited to the specific embodiments described, as there will certainly be variations. It should also be understood that, since the scope of the invention will be limited only by the appended claims, the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0018] Where a range of values ​​is provided, it should be understood that, unless the context explicitly specifies otherwise, every intervention value (up to one-tenth of the lower limit unit) between the upper and lower limits of that range and any other stated or intervention value within that range is included in this invention. The upper and lower limits of these smaller ranges may be independently included within the smaller ranges and also within this invention, but are subject to any specifically excluded limitations within the range. Where the range includes one or both limitations, the range excluding one or both of those included limitations is also included in this invention.

[0019] This document provides certain ranges, with numerical values ​​preceded by the term "approximately". The term "approximately" is used in this document to provide textual support for the exact number preceding it and for numbers that are close to or approximate to the number preceding the term. In determining whether a number is close to or approximate to a specifically listed number, the unlisted number that is close to or approximates can be a number that provides a substantial equivalent to the specifically listed number in the context in which the number is presented.

[0020] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although any methods and materials similar to or equivalent to those described herein may be used in the practice or testing of this invention, representative illustrative methods and materials are described hereafter.

[0021] As each individual publication or patent is explicitly and individually indicated to be incorporated herein by reference, and by reference to disclose and describe the methods and / or materials relating to the cited publication, all publications and patents referenced in this specification are incorporated herein by reference. Any reference to any publication is for its prior disclosure prior to the filing date and should not be construed as an admission that the invention is not entitled to prior publication by virtue of prior art. Furthermore, the publication date provided may differ from the actual publication date and may require separate verification.

[0022] It should be noted that, unless the context clearly indicates otherwise, the singular forms “a,” “an,” “the,” etc., used herein and in the appended claims include multiple objects represented. It should also be noted that claims may be drafted to exclude any optional elements. Therefore, this statement is intended as a prior basis for the use of proprietary terms such as “alone,” “only,” etc., when stating claim elements or using the “negative” limitation.

[0023] As will be apparent to those skilled in the art upon reading this disclosure, each of the various embodiments described and illustrated herein has discrete components and features that can be readily separated from or combined with features of any of the other embodiments without departing from the scope or spirit of the invention. Any of the described methods can be performed in the order of the events or in any other logically possible order.

[0024] Although the apparatus and method have been or will be described for grammatical fluency and functional interpretation, it should be clearly understood that, unless expressly provided in 35 USC §112, the claims should not be construed as being limited in any way by the terms "means" or "steps," but should be given the full meaning and equivalence of the definitions provided in the claims in accordance with the doctrine of judicial equivalence, and if the claims are expressly provided in 35 USC §112, they should be given the full legal equivalence in accordance with 35 USC §112.

[0025] A system for distributing droplets using flow cytometry

[0026] As described above, aspects of this disclosure include a system for distributing droplets into partitions via flow cytometry, wherein the system is configured to sort particle-occupied droplets and particle-unoccupied droplets into partitions. Because the system is configured to sort particle-occupied droplets and particle-unoccupied droplets into partitions, it is configured to partition guide desired particle-occupied droplets and particle-unoccupied droplets. Partition guiding refers to the system being configured to distribute desired particle-occupied droplets and particle-unoccupied droplets into desired or predetermined partitions, enabling the system to send or place desired particle-occupied droplets and particle-unoccupied droplets into predetermined partitions. Thus, the system is configured so that the presence of a particle in the droplet is not required for the droplet to be sorted into a partition (i.e., a partition for particles to be partition guided). Therefore, the system of the present invention is configured to directly sort or partition droplets occupied by desired particles (e.g., cells or beads) and desired unoccupied droplets (i.e., droplets that do not contain particles (e.g., cells or beads) but are still desired to be sorted) into partitions. Thus, aspects of the present invention include a system configured to distribute precisely micro-volume particle-occupied and particle-unoccupied droplets into partitions via flow cytometry.

[0027] "Particle-occupied droplets" refers to droplets comprising at least one particle, which may include cells, solid carriers (e.g., beads, non-cellular particles, etc.), wherein the particles are present in a liquid medium. The number of particles in a given droplet can vary, ranging from 1 to 5 in some cases, for example, 1 to 3, wherein in some cases, particle-occupied droplets comprise a single particle, such as a cell or a bead. Particle-occupied droplets may or may not be required during use. "Desired" particle-occupied droplets refer to droplets comprising at least one pre-specified particle or particle type, such as a specific cell or cell type, or a bead or bead type. Desired particles can be any particle that meets predetermined criteria, such as the presence or absence of surface markings, light scattering profiles, etc. Conversely, undesirable particle-occupied droplets are droplets that include particles, but are undesirable because the particles do not meet predetermined criteria. The liquid composition of particle-occupied droplets can vary as needed. The liquid composition of particle-occupied droplets can be a rechargeable aqueous medium. In some cases, the aqueous medium may consist of a sheath fluid and a sample fluid, which are combined upon entering the flow cell to form a sheath of sheath fluid having an inner core surrounding the sample fluid. The particles present in the particle-occupied droplets can vary and include cells, solid carriers such as polymeric solid carriers, like beads, etc. Typically, embodiments can be configured such that the particle-occupied droplets can contain any particles that can be detected by the system and, in some cases, identified. For example, particles can be detected based on the detection of light from the interrogation point of the flow cell.

[0028] "Particle-unoccupied droplets" refers to droplets that do not contain particles. That is, particle-unoccupied droplets include liquid leaving the flow cell but do not include particles. Therefore, like particle-occupied droplets, particle-unoccupied droplets can consist of various aqueous media capable of being charged. In some cases, particle-unoccupied droplets may consist only of sheath fluid. In other cases, particle-unoccupied droplets may include both sheath fluid and sample fluid, even when no particles are present in the sample. Still in other cases, particle-unoccupied droplets may include other components, including but not limited to, various reagents, labels, etc. (e.g., as described in more detail below). In these embodiments where particle-unoccupied droplets include other components, the system may include a fluidly coupled source of such components, enabling the formation of droplets including such a source. For example, in the flow cytometer system of the present invention, a flow cell is included, which is operatively coupled to a sheath fluid delivery device fluidly coupled to a sheath fluid source and a sample fluid delivery device fluidly coupled to a sample fluid source. At least one of the sheath fluid delivery device and the sample fluid delivery device may include a pathway for introducing a component from a component source into the delivery device. This pathway may be controllable, for example, a valve, allowing a user to specify when a component is introduced from the source into the delivery device.

[0029] In some cases, the droplet has a predetermined (i.e., known) volume. In embodiments, the predetermined droplet volume can be any convenient volume, including nanoliters, depending on the system configuration. In some cases, the droplet has a known volume of 0.5 nanoliters to 100 nanoliters, for example, 1 nanoliter to 50 nanoliters, or 1 nanoliter to 10 nanoliters. A given system can be configured to produce only a single droplet of a known volume, so the droplet volume may not be adjustable. Alternatively, a given system can be configured to dynamically adjust the predetermined volume of the droplet, making the known droplet volume adjustable and changeable as needed. In some cases, the predetermined volume of the droplet can be changed based on how the system is configured, including, for example, by adjusting the configuration of the droplet generator (e.g., the oscillation frequency of the droplet generator), or in other cases by adjusting other system configurations (e.g., the flow rate or the diameter of the liquid exiting the flow cell).

[0030] Embodiments of the sorting flow cytometer system of the present invention include a droplet sorter configured to partition and guide particulate-occupied and particulate-unoccupied droplets in a droplet stream generated by a droplet stream generator configured to generate a stream of particulate-occupied and particulate-unoccupied droplets in the liquid leaving the flow cell, as described in more detail below. This droplet sorter of the embodiments of the present invention differs from conventional sorting flow cytometers in that it is capable of sorting particulate-unoccupied droplets into desired partitions. Therefore, compared to conventional sorting flow cytometers, the droplet sorter of the present invention does not merely sort or partition droplets containing particles.

[0031] The droplet sorters of the present invention can vary, as long as they can be configured to sort droplets that are not occupied by particles in addition to those occupied by particles. In some cases, the droplet sorter includes: a droplet charger configured to charge droplets in a droplet stream, for example, as described in more detail below; a droplet deflector configured to deflect charged droplets into partitions, for example, as described in more detail below; and a controller configured to control the droplet charger and the droplet deflector to partition and guide both particle-occupied and particle-unoccupied droplets. In some cases, the controller includes a processor and a memory operatively coupled thereto, wherein the memory includes instructions stored thereon that, when executed by the processor, cause the processor to specify the particle-occupied and particle-unoccupied droplets to be partitioned and guided by the droplet sorter. The instructions providing this functionality can vary as needed, wherein examples of such instructions will be further described below. In some cases, the system is configured to instruct the droplet sorter to partition each droplet by default. For example, in some cases, the system user may want to deposit a set volume of liquid into a given partition. In this case, the system can be configured to sort each droplet by default. In this way, the system can be configured to distribute micro-volumes of fluid into partitions based on the number of droplets guided by the partition. In some cases, the system is configured to instruct the droplet deflector not to deflect each droplet by default, but only to deflect droplets occupied by desired particles, such as droplets occupied by particles that meet predetermined criteria. For example, in response to instructions entered by the user during a specific period of system use, the system can be configured to be adjustable between these two default states. Thus, the system can be configured to dynamically instruct the droplet sorter to guide each droplet by partition by default, or to guide only droplets occupied by desired particles by default.

[0032] In some cases, the droplet sorter is configured to guide a first predetermined number of particle-occupied droplets and a second predetermined number of particle-unoccupied droplets into a partition. The predetermined number of droplets of a given type (e.g., particle-occupied or particle-unoccupied) can vary, ranging from 0 to 50 in some cases, such as 0 to 25, including 0 to 10, or 25 to 50, including 40 to 50. In some cases, the first and second predetermined numbers are determined to obtain a desired ratio between the number of particle-occupied droplets and the number of particle-unoccupied droplets in a given partition. In this case, the first and second predetermined numbers can be determined to achieve a desired ratio between the volume of occupied droplets and the volume of unoccupied droplets in the partition relative to a desired total volume of fluid deflected into the partition. In some cases, the occupied droplets comprise a predetermined volume of sheath fluid and a predetermined volume of sample fluid, such that the first predetermined number can be determined to deflect a desired total volume of sample fluid and / or sheath fluid into the partition. In some cases, the unoccupied droplets consist of a predetermined volume of sheath fluid, allowing a second predetermined quantity to be determined in order to deflect the desired total volume of sheath fluid into the partition.

[0033] In some cases, the system can be configured to deposit a specific total volume of liquid into a partition by deflecting (i.e., partitioning) both particle-occupied and particle-unoccupied droplets into the partition. That is, the volume of each of the particle-occupied and particle-unoccupied droplets can be known, such that sorting a specific number of particle-occupied or particle-unoccupied droplets into a given partition results in a known total volume of fluid being deposited into that partition. Therefore, the system can be configured to dispense known microvolumes of fluid (in some cases, including particles of the sample) into partitions. As described, such fluid can include any fluid included in the flow stream, such as sample liquid, sheath fluid, liquids comprising other components, such as solvents, buffers, reagents, labels, etc.

[0034] The system may further include an input module operatively connected to the controller, wherein the input module is configured to receive input messages specifying desired particle-occupied and particle-unoccupied droplets to the sorter. In other words, the input module is configured to receive input messages specifying particle-occupied and particle-unoccupied droplets to be partitioned and guided. Any convenient input module can be used. As described in detail below, the input module may include, for example, a keyboard, mouse, or touchscreen device. Alternatively, the input module may include an operative connection between the sorting controller and another device, such as a network connection, including wired or wireless connections (e.g., Bluetooth connections). By specifying the desired particle-occupied and particle-unoccupied droplets, in some cases, it means that the input module specifies that only the desired particle-occupied droplets, or in other cases only the particle-unoccupied droplets, are guided to a given partition—i.e., partitioning. In other cases, it means that the input module specifies specific characteristics of the droplets to be partitioned and guided, including, for example, the characteristics of the particles in the particle-occupied droplets. In other cases, this means specifying a first predetermined number of particle-occupied droplets and / or a second predetermined number of particle-unoccupied droplets via the input module. In other cases, this means specifying, via the input module, the volume, or ratio, of particle-occupied and / or particle-unoccupied droplets to be deflected into the partition in each case, or the total fluid volume. Still in other cases, the input module can be used to specify the amount of a particular type of fluid to be deposited into the partition, for example, a specific volume of reagent comprising liquid to be guided into the partition. In some embodiments, the system is configured to dynamically adjust the ratio of guided occupied droplets to unoccupied droplets over a period of time. In some cases, the system can be configured to adjust the ratio of occupied to unoccupied droplets between each partition in a set of partitions.

[0035] In some cases, the system is configured to record identifiers that identify specific zones, such as those into which droplets are directed into the wells of a porous plate. The identifiers may include any convenient information about the zones and / or the droplets deposited within them. For example, the system may be configured to record information in the form of identifiers that specify a particular well of the porous plate into which droplets occupied by a particular particle and / or unoccupied droplets have been deposited by flow cytometry, thereby recording information that precisely classifies the liquids present in a given zone, based on knowledge of the specific droplets already deposited in that zone. Thus, the identifiers may include information about the number of particle-occupied and / or unoccupied droplets deflected into the well.

[0036] The flow cytometry droplet dispensing system of this invention can be configured for specific workflow applications, such as those described in more detail below. For example, the system can be configured for applications using single-cell nucleic acid sequencing libraries. In such cases, the system can be configured to include sources of various components used in such library preparation applications, such as lysis agents, barcode solid carriers (e.g., barcode beads), polymerases, nucleotides, oligonucleotides (e.g., primers, TSO, etc.), and the like, as well as methods for preparing droplets comprising these sources, such as access points into sheaths and / or sample lines. Alternatively, the system can be configured to contact partitioned cells with reagents (e.g., cell regulators, such as those used for screening applications, as described in more detail below).

[0037] Figure 1 A functional block diagram of an example sorting control system according to an embodiment of the present invention is shown. The sorting control system 100 can be configured to control a sorting flow cytometer to partition and guide droplets occupied by particles and droplets not occupied by particles (e.g., as described above). Figure 1 In this system, the sorting control system 100 is operatively coupled to the sorting flow cytometer 102. A data communication channel may be included between the sorting flow cytometer 102 and the sorting controller 100. If desired, the sorting controller 100 may be configured to receive biological event data from the sorting flow cytometer 102. The biological event data received from the particle sorting flow cytometer 102 may include flow cytometer event data. The sorting controller 100 may be configured to provide a graphical display of the data to a display device 106. The sorting controller 100 may be configured to receive user input commands from a first input device. For example, the first input device may be implemented as a mouse 110. The mouse 110 may initiate droplet sorting selection signals to the sorting controller 100, such as sorting all droplets or sorting only droplets occupied by specific particles, and input other commands, such as displaying or manipulating the identification of a door on or via the display device 106 (e.g., clicking on or in the desired door when the cursor is positioned there). In some implementations, the first device can be implemented as a keyboard 108 or other means for providing input signals to the sorting controller 100, such as a touchscreen, stylus, optical detector, or voice recognition system. Some input devices can include multiple input functions. In such implementations, each input function can be considered an input device. For example, such as Figure 1The mouse 110 may include a right mouse button and a left mouse button, each of which can generate a trigger event. The sorting controller 100 may be connected to a storage device 104. The storage device 104 may be configured to receive and store biological event data from the sorting controller 100. The storage device 104 may also be configured to receive and store flow cytometry event data from the sorting controller 100. The storage device 104 may also be configured to allow the sorting controller 100 to retrieve biological event data (e.g., flow cytometry event data). The display device 106 may be configured to receive display data from the sorting controller 100. The display device 106 may also be configured to change the presented information based on input received from the analysis controller 100 and input from the sorter 102, storage device 104, keyboard 108, and / or mouse 110. In some embodiments, the sorting controller 100 may generate a user interface to receive example events for sorting. For example, the user interface may include controls for receiving example events or example images. Example events, images, or gates can be provided before the event data of the sample is collected or based on an initial set of events from a portion of the sample.

[0038] Figure 2A It is a sorting flow cytometer (e.g., according to one embodiment of the present invention) Figure 1 The diagram shows a schematic of a droplet sorting system 200 in a flow cytometer 102. In some embodiments, the droplet sorting system 200 is a cell sorting system. Figure 2A As shown, a droplet-forming transducer 202 (e.g., a piezoelectric oscillator) is coupled to a fluid conduit 201, which can be coupled to, includes, or is a nozzle 203. Within the fluid conduit 201, a sheath fluid 204 hydrodynamically focuses a sample fluid 206, comprising particles 209 (e.g., cells or solid carriers, such as polymer beads), into a moving fluid column 208 (e.g., a flow). Within the moving fluid column 208, the particles 209 (e.g., cells or solid carriers, such as polymer beads) are arranged in a single file to pass through a monitoring area 211 irradiated by an irradiation source 212 (e.g., a laser) (e.g., where the laser streams intersect). Vibration of the droplet-forming transducer 202 causes the moving fluid column 208 to break into multiple droplets 210, some of which contain particles 209 and are therefore particle-occupied droplets, while others do not contain particles (e.g., only liquid) and are therefore particle-unoccupied droplets.

[0039] During operation, detection station 214 (e.g., an event detector) identifies when a particle (or cell) of interest crosses monitoring area 211. Detection station 214 feeds into timing circuit 228, which in turn feeds into flash charging circuit 230. At a droplet interruption point notified by a timing droplet delay (Δt), a flash charge can be applied to the moving fluid column 208, causing the droplet of interest to carry a charge. The droplet of interest can comprise one or more particles or cells to be sorted. The charged droplet can then be sorted by activating a deflection plate (not shown) to deflect the charged droplet into a partition, such as a container (e.g., a collection tube or a porous or microporous sample plate), wherein the partition or pore or micropore can be associated with a specific droplet of interest. Figure 2A As shown, the droplets can also be collected in the discharge container 238.

[0040] Detection system 216 (e.g., a droplet boundary detector) is used to automatically determine the phase of the droplet drive signal as a particle of interest passes through monitoring region 211. An exemplary droplet boundary detector is described in US Pat. No. 7,679,039, which is incorporated herein by reference in its entirety. Detection system 216 allows the instrument to accurately calculate the position of each detected particle within the droplet. Detection system 216 can be fed into amplitude signal 220 and / or phase signal 218, which (via amplifier 222) is further fed into amplitude control circuitry 226 and / or frequency control circuitry 224. Amplitude control circuitry 226 and / or frequency control circuitry 224, in turn, control droplet forming transducer 202. Amplitude control circuitry 226 and / or frequency control circuitry 224 can be included in a control system.

[0041] In some embodiments, sorting electronics (e.g., detection system 216, detection station 214, and processor 240) can be coupled to a memory configured to store detected events and sorting decisions based thereon. The sorting decisions can be included in the event data of the particles. In some embodiments, detection system 216 and detection station 214 can be implemented as a single detection unit or communication coupling, such that event measurements can be collected by one of detection system 216 or detection station 214 and provided to non-collected events.

[0042] As described above, the sorting system is configured to partition and guide selected droplets occupied by particles and droplets not occupied by particles according to predetermined instructions. The sorting system can be configured to implement this function using any convenient protocol. An example of such a protocol is now provided. Charging the liquid flow is determined in the droplet time domain. The droplet time domain is determined by droplet-driving electronics that agitate the flow at a known frequency, causing it to break into droplets. Charging electronics charge the flow knowing the droplet-driving frequency, causing specific droplets to carry a charge and be deflected (e.g., by a deflection plate) as they pass through an electromagnetic field. In some cases, when it is decided to sort only droplets occupied by desired particles, e.g., droplets meeting predetermined criteria, the droplet time domain is predetermined not to be sorted. In the case of deciding to sort empty droplets, the droplet time domain is predetermined for sorting each droplet. Particles that intercept laser light represent the event time domain. To deflect the particles, the event time domain of the detected target particle is correlated with the droplet time domain, causing the charging electronics to charge the droplet containing the target particle. If the sorting of empty (unoccupied) droplets is desired, then all particles within the event time are temporally related to the droplets and negate the predetermined sorting decision, unless the particles themselves are to be sorted.

[0043] Figure 2B This is a schematic diagram of a droplet sorting system according to an embodiment of the present document. Figure 2B The droplet sorting system 200 shown includes deflecting plates 252 and 254. Charge can be applied via a current-charging line in the barbs. This produces a flow of droplets 210 containing particles and unoccupied droplets. The particles can be illuminated with one or more light sources (e.g., lasers) to generate light scattering and fluorescence information. This can be achieved, for example, through sorting electronics or other detection systems. Figure 2B (Not shown in the image) to analyze particle information. Deflecting plates 252 and 254 can be independently controlled to attract or repel charged droplets to guide them to a destination collection container (e.g., one of 272, 274, 276, or 278), such as a partition. Figure 2B As shown, deflection plates 252 and 254 can be controlled to guide droplets toward container 274 along a first path 262 or toward container 278 along a second path 268. If the particles are of no interest (e.g., do not exhibit scattering or illumination information within a specified sorting range), the deflection plates can allow the particles to continue along flow path 264. Such uncharged droplets can, for example, enter the waste container via a suction device 270. Sorting electronics can be included to initiate measurement collection, receive the fluorescence signal of the particles, and determine how to adjust the deflection plates to sort the particles. Figure 2B The example implementation of the illustrated embodiment includes the BD FACSAria flow cytometer, commercially available from Becton, Dickinson and Company (Franklin Lakes, NJ).TM series.

[0044] Figure 3 A functional block diagram of a sorting flow cytometer system according to the present invention is shown, which dispenses droplets into partitions for flow cytometers. Figure 3 The sorting flow cytometer system 300 shown can be configured to perform all or part of the methods described herein. The sorting flow cytometer system 300 includes a fluid system 302. The fluid system 302 can include or couple a sample tube 310 and a moving fluid column within the sample tube, wherein particles 330 (e.g., cells, beads) in the sample move along a common sample path 320. In some embodiments, the fluid system may include a sample source, a sample fluid, a lysis agent, a sheath fluid source, one or more reagent sources, a solid carrier source, such as beads (not shown in the figures in each case).

[0045] The sorting flow cytometer system 300 includes a detection system 304 configured to collect signals from each particle as it passes through one or more detection stations along a common sample path. Detection stations 308 typically refer to a monitoring area 340 of the common sample path. In some embodiments, detection can include detecting light or one or more other characteristics of the particle 330 as it crosses the monitoring area 340. Figure 3 The diagram shows a detection station 308 with a monitoring area 340. Some embodiments of the particle analysis system 300 can include multiple detection stations. Furthermore, some detection stations can monitor more than one area. A signal value is assigned to each signal to form a data point for each particle. This data can be referred to as event data. The data point can be a multidimensional data point including values ​​of various characteristics measured for the particle. The detection system 304 can be configured to collect a series of such data points within a first time interval.

[0046] The particle flow cytometer system 300 may also include a control system 306. The control system 306 may include one or more processors, amplitude control circuitry 226, and / or frequency control circuitry 224 (e.g., ...). Figure 2A (As shown). The control system 306 shown can be operatively associated with the fluid system 302. The control system 306 can be configured to generate a calculated signal frequency for at least a portion of the first time interval based on the number of data points collected by the detection system 304 during the first time interval. The control system 306 can also be configured to generate an experimental signal frequency based on the number of data points in a portion of the first time interval. Additionally, the control system 306 can compare the experimental signal frequency with the calculated signal frequency or a predetermined signal frequency. The control system can control one of the systems to guide droplets directly occupied by particles and droplets not occupied by particles in a partitioned manner.

[0047] Figure 4 A particle analysis system 400 for sorting a flow cytometer according to an exemplary embodiment of the present invention is shown. System 400 includes a flow cytometer 410, a controller / processor 490, and a memory 495. The flow cytometer 410 includes one or more excitation lasers 415a-415c, a focusing lens 420, a flow chamber 425, a forward scattering detector 430, a side scattering detector 435, a fluorescence collecting lens 440, one or more beam splitters 445a-445g, one or more bandpass filters 450a-450e, one or more long-pass (“LP”) filters 455a-455b, and one or more fluorescence detectors 460a-460f.

[0048] The excitation laser 415a-c emits light in the form of a laser beam. Figure 4 In the example system, the laser beams emitted from excitation lasers 415a-415c have wavelengths of 488 nm, 633 nm, and 325 nm, respectively. The laser beams are first guided through one or more beam splitters 445a and 445b. Beam splitter 445a transmits 488 nm light and reflects 633 nm light. Beam splitter 445b transmits UV light (light with wavelengths from 10 to 400 nm) and reflects both 488 nm and 633 nm light.

[0049] The laser beam is then guided to a focusing lens 420, which focuses the laser beam onto the portion of the fluid flow containing the sample particles within the flow chamber 425. The flow chamber is part of a fluid system that directs particles (typically one at a time) in the flow toward the focused laser beam for querying. The flow chamber can comprise a flow cell in a benchtop cytometer or a nozzle tip in an air cytometer.

[0050] Light from the laser beam interacts with particles in the sample through diffraction, refraction, reflection, scattering, and absorption, and is re-emitted at different wavelengths depending on the characteristics of the particles (e.g., their size, internal structure, and the presence of one or more fluorescent molecules attached to or within the particles). The fluorescence emission, along with the diffracted, refracted, reflected, and scattered light, can be transmitted through one or more beam splitters 445a-445g, bandpass filters 450a-450e, longpass filters 455a-455b, and fluorescence collecting lenses 440 to one or more forward scattering detectors 430, side scattering detectors 435, and one or more fluorescence detectors 460a-460f, respectively.

[0051] A fluorescence collecting lens 440 collects light emitted from the particle-laser beam interaction and directs the light to one or more beam splitters and filters. Bandpass filters, such as bandpass filters 450a-450e, allow a narrow range of wavelengths to pass through the filter. For example, bandpass filter 450a is a 510 / 20 filter. The first number indicates the center of the spectral band. The second number provides the range of the spectral band. Thus, a 510 / 20 filter extends 10 nm on each side of the center of the spectral band, or from 500 nm to 520 nm. Short-pass filters transmit wavelengths of light equal to or shorter than a specified wavelength. Long-pass filters, such as long-pass filters 455a-455b, transmit wavelengths of light equal to or longer than a specified wavelength. For example, long-pass filter 455a, a 670 nm long-pass filter, transmits light equal to or longer than 670 nm. Filters are typically selected to optimize the detector's specificity for a particular fluorescent dye. Filters can be configured such that the spectral band of the light transmitted to the detector is close to the emission peak of the fluorescent dye.

[0052] Beam splitters direct light of different wavelengths in different directions. Beam splitters can be characterized by filter properties such as short-pass and long-pass filters. For example, beam splitter 445g is a 620SP beam splitter, meaning that beam splitter 445g transmits light with wavelengths of 620 nm or shorter and reflects wavelengths longer than 620 nm in different directions. In one embodiment, beam splitters 445a-445g can include optical mirrors, such as dichroic mirrors.

[0053] A forward scattering detector 430 is positioned slightly off-axis from the direct beam passing through the flow cell and is configured to detect diffracted light, which is the excitation light that travels through or around the particle in the mostly forward direction. The intensity of the light detected by the forward scattering detector depends on the overall size of the particle. The forward scattering detector can include a photodiode. A side scattering detector 435 is configured to detect refracted and reflected light from the particle surface and internal structure, and tends to increase with increasing particle complexity. Fluorescence emission from fluorescent molecules associated with the particle can be detected by one or more fluorescence detectors 460a-460f. The side scattering detector 435 and the fluorescence detector can include photomultiplier tubes. The signals detected at the forward scattering detector 430, the side scattering detector 435, and the fluorescence detector can be converted into electronic signals (voltages) by the detectors. These data can provide information about the sample.

[0054] Those skilled in the art will recognize that the flow cytometer according to embodiments of the present invention is not limited to Figure 4 The flow cytometer shown can include any flow cytometer known in the art. For example, a flow cytometer can have any number of lasers, beam splitters, filters, and detectors of various wavelengths and various different configurations.

[0055] During operation, the cytometer is controlled by a controller / processor 490, and measurement data from the detector can be stored in memory 495 and processed by the controller / processor 490. Although not explicitly shown, the controller / processor 490 is coupled to the detector to receive output signals from it, and may also be coupled to the electrical and electromechanical components of the flow cytometer 400 to control lasers, fluid flow parameters, and the like. Input / output (I / O) capabilities 497 may also be provided in the system. Memory 495, controller / processor 490, and I / O 497 may be provided entirely as part of the flow cytometer 410. In such embodiments, a display may also form part of the I / O capability 497 for presenting experimental data to a user of the cytometer 400. Optionally, some or all of the memory 495 and controller / processor 490, as well as the I / O capabilities, may be part of one or more external devices (e.g., a general-purpose computer). In some embodiments, some or all of the memory 495 and controller / processor 490 may be able to communicate wirelessly or wired with the cytometer 410. The controller / processor 490, combined with memory 495 and I / O 497, can be configured to perform various functions related to the preparation and analysis of flow cytometry experiments.

[0056] Figure 4The system shown includes six different detectors that detect fluorescence in six different wavelength bands defined by the configuration of filters and / or separators in the beam path from flow cell 425 to each detector (here referred to as the "filter window" of a given detector). Different fluorescent molecules used in flow cytometry experiments will emit light with their own characteristic wavelength bands. Specific fluorescent labels used in the experiment and their associated fluorescence emission bands can be selected to generally coincide with the filter windows of the detectors. However, due to the availability of more detectors and the use of more labels, a perfect correspondence between filter windows and fluorescence emission spectra is not possible. Generally, although the peak of the emission spectrum of a particular fluorescent molecule may lie within the filter window of a particular detector, some emission spectra of that label will also overlap with the filter windows of one or more other detectors. This can be referred to as overflow. I / O 497 can be configured to receive data on a flow cytometry experiment having a set of fluorescent labels and multiple cell populations with multiple labels, each cell population having a subset of multiple labels. I / O 497 can also be configured to receive biological data, label density data, emission spectral data, data on label assignment to one or more cell populations, and cytometer configuration data. Flow cytometry experimental data (e.g., label spectral characteristics and flow cytometry configuration data) can also be stored in memory 495. Controller / processor 490 can be configured to evaluate label assignment to one or more labels.

[0057] Flow cytometer system

[0058] Other aspects of the flow cytometer system according to embodiments of the present invention are described in Ormerod (ed.), Flow Cytometry: A Practical Approach, Oxford Univ. Press (1997); Jaroszeski et al. (eds.), Flow Cytometry Protocols, Methods in Molecular Biology No. 91, Humana Press (1997); Practical Flow Cytometry, 3rd ed., Wiley-Liss (1995); Virgo, et al. (2012) Ann Clin Biochem. Jan; 49(pt 1):17-28; Linden, et al., Semin Throm Hemost. 2004 Oct; 30(5):502-11; Alison, et al. J Pathol, 2010 Dec; 222(4):335-344; and Herbig, et al. (2007) Crit Rev Ther Drug Carrier Syst. 24(3):203-255; the disclosure of which is incorporated herein by reference. Flow cytometry systems of interest having the components found in the flow cytometers of embodiments of the present invention include BD Biosciences FACSCanto. TM Flow cytometer, BD Biosciences FACSCanto TM II flow cytometer, BD Accuri TM Flow cytometer, BD Accuri TM C6 Plus flow cytometer, BD Biosciences FACSCelesta TM Flow cytometer, BD Biosciences FACSLyric TM Flow cytometer, BD Biosciences FACSVerse TM Flow cytometer, BD Biosciences FACSymphony TM Flow cytometer, BD Biosciences LSL Fortessa TM Flow cytometer, BD Biosciences LSRFortessa TM X-20 flow cytometer, BDBiosciences FACSPresto TMFlow cytometer, BD Biosciences FACSVia TM Flow cytometry and BDBiosciences FACSCalibur TM Cell sorter, a BD Biosciences FACSCount TM Cell sorting instrument, BDBiosciences FACSLyric TM Cell sorter, BD Biosciences Via TM Cell sorting instrument, BDBiosciences Influx TM Cell sorter, BD Biosciences Jazz TM Cell sorter, BD Biosciences Aria TM Cell sorting instrument, BD Biosciences FACSAria TM II Cell Sorter, BD Biosciences FACSAria TM III Cell Sorter, BD Biosciences FACSAria TM Fusion Cell Sorter and BDBiosciences FACSMelody TM Cell sorting instrument, BD Biosciences FACSymphony TM S6 cell sorter, etc.

[0059] In some cases, the subject particle sorter is a flow cytometer system configured to image particles in a flowing stream using fluorescence imaging with radio frequency labeled emission (FIRE) (e.g., using a beam generator configured to generate two or more frequency-shifted beams, including a laser excitation module), as in Diebold, et al. Nature Photonics Vol. 7(10); 806-810 (2013) and U.S. Patent Nos. 9,423,353; 9,784,661; 9,983,132; 10,006,852; 10,078,045; 10,036,699; 10,222,316; 10,288,546; 10,324,019; 10,40 The disclosures described in U.S. Patent Publications Nos. 8,758; 10,451,538; 10,620,111; and U.S. Patent Publications Nos. 2017 / 0133857; 2017 / 0328826; 2017 / 0350803; 2018 / 0275042; 2019 / 0376895 and 2019 / 0376894 are incorporated herein by reference.

[0060] In some embodiments, the subject system is a flow cytometer system, such as U.S. Patent Nos. 10,663,476; 10,620,111; 10,613,017; 10,605,713; 10,585,031; 10,578,542; 10,578,469; 10,481,074; 10,302,545; 10,145,793; 10,113,967; 10,006,852; 9,952,076; 9,933,341; 9,726,527; 9,453,789; 9,200,334; 9,097,64 The contents of the disclosures described in 0; 9,095,494; 9,092,034; 8,975,595; 8,753,573; 8,233,146; 8,140,300; 7,544,326; 7,201,875; 7,129,505; 6,821,740; 6,813,017; 6,809,804; 6,372,506; 5,700,692; 5,643,796; 5,627,040; 5,620,842; 5,602,039; 4,987,086; 4,498,766 are incorporated herein by reference.

[0061] As described above, in embodiments of the invention, the system includes multiple partitions configured to receive droplets, such as cell-containing droplets or unoccupied droplets from a sample sorted by a droplet sorter. A partition refers to any convenient container, such as a sample collection container, capable of receiving one or more droplets, such as unoccupied droplets or droplets containing particles (e.g., cells), sorted by a cell sorter, and maintaining the contents of the partition separate and isolated from other materials not sorted into the partition. Embodiments include more than one partition, such as two partitions, four partitions, 96 partitions, or 1536 or more partitions. A partition can be any convenient size capable of accommodating and maintaining particles (e.g., cells) separated from the flowing sample. In some cases, partitions are sized to accommodate more than one droplet, such as 10 droplets, 100 droplets, 1000 droplets, and 10000 droplets or more. In other cases, the partitions are sized to accommodate multiple cells, such as 10 cells, 100 cells, 1000 cells, 10000 cells, or more. In some embodiments, the partitions include wells. In some cases, the wells may be small test tubes. The wells can be any convenient shape. In some cases, the cross-sectional shape of the well is circular; in others, it is rectangular or square. The wells can be of any size with sufficient capacity to accommodate droplets, such as unoccupied droplets or droplets with particles (such as cells), as needed. For example, the volume of the well can be 0.001 mL or greater, such as 0.005 mL, 0.015 mL, 0.1 mL, 2 mL, or 5 mL or greater. In some embodiments, the wells can be pores of a multiwell plate. A multiwell plate can include any number of wells. In examples, a multiwell plate can include 6, 12, 24, 48, 96, 384, 1536, 3456, or 9600 or more wells. The pores of the multiwell plate can be arranged in any convenient manner. In some cases, the pores are arranged in a rectangular shape with an aspect ratio of approximately 2:3. In some cases, the porous plates of this disclosure can conform to recognized standards, such as those established by the Society for Biomolecular Sciences using ANSI standards. The porous plates can be composed of any convenient material. In some cases, the porous plates can be composed of polypropylene, polystyrene, or polycarbonate. In these embodiments, after sorting a predetermined number of droplets occupied by particles or droplets not occupied by particles into the first pore, the porous plate can be advanced to the second pore. The predetermined number of droplets can be 1 droplet, 2 droplets, 10 droplets, or 100 droplets or more.

[0062] In some embodiments, the system further includes a translational support stage configured to move multiple partitions (e.g., a perforated plate), and a processor including a memory operatively coupled to the processor, wherein the memory includes instructions stored thereon that, when executed by the processor, cause the processor to instruct the support stage to move the perforated plate to a second hole after sorting a predetermined number of particle-occupied droplets or particle-unoccupied droplets into a first hole. A translational support stage refers to any convenient stage capable of accommodating multiple partitions (e.g., a perforated plate). Any convenient displacement protocol can be used to translate the support stage, such as a motor-actuated translation stage, a lead screw translation assembly, a gear translation device, such as those employing stepper motors, servo motors, brushless motors, brushed DC motors, microstepper drive motors, high-resolution stepper motors, and other types of motors.

[0063] As described above, the system 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 can access memory on which instructions for performing steps of the subject method are stored. The processing module may include an operating system, a graphical user interface (GUI) controller, system memory, memory storage devices and input / output controllers, cache memory, data backup units, and many other devices. The processor may be a commercially available processor or one of other processors that are already available or will be available in the future. As is known in the art, a processor executes an operating system, and the operating system interacts with firmware and hardware in well-known ways and facilitates the processor in coordinating and executing the functions of various computer programs written in various programming languages, such as Java, Perl, C++, other high-level or low-level languages, and combinations thereof. The operating system typically cooperates with the processor to coordinate and execute the functions of other components of the computer. The operating system also provides scheduling, input / output control, file and data management, memory management, communication control, and related services according to known technologies. The processor may be any suitable analog or digital system. In some embodiments, the processor includes analog electronics that provide feedback control (e.g., negative feedback control).

[0064] System memory can be any of a variety of known or future memory storage devices. Examples include any common random access memory (RAM), magnetic media such as resident hard disks or magnetic tapes, optical media such as optical discs, flash memory devices, or other memory storage devices. Memory storage devices can be any of a variety of known or future devices, including optical disc drives, magnetic tape drives, removable hard disk drives, or floppy disk drives. This type of memory storage device typically reads from and / or writes to program storage media (not shown), such as optical discs, magnetic tapes, removable hard disks, or floppy disks, respectively. Any of these program storage media, or other media currently in use or that may be developed in the future, can be considered a computer program product. It should be understood that these program storage media typically store computer software programs and / or data. Computer software programs, also known as computer control logic, are typically stored in system memory and / or program storage devices used in conjunction with memory storage devices.

[0065] In some embodiments, a computer program product is described, including a computer-usable medium in which control logic (computer software program, including program code) is stored. When the control logic is executed by a computer's processor, the control logic causes the processor to perform the functions described herein. In other embodiments, some functions are implemented primarily in hardware using, for example, a hardware state machine. Implementing a hardware state machine to perform the functions described herein will be apparent to those skilled in the art.

[0066] The memory can be any suitable device in which the processor is capable of storing and retrieving data, such as magnetic, optical, or solid-state storage devices (including magnetic disks or optical discs or magnetic tapes or RAM, or any other suitable fixed or portable device). The processor can include a general-purpose digital microprocessor, which is appropriately programmed from a computer-readable medium carrying the necessary program code. The programming can be provided to the processor remotely via a communication channel, or pre-stored in a computer program product using any of those devices connected to the memory, such as memory or some other portable or fixed computer-readable storage medium. For example, a magnetic disk or optical disc can carry the programming and can be read by a disk writer / reader. The system of the present invention also includes programming (e.g., in the form of a computer program product) of an algorithm for practicing the methods described above. The programming according to the present invention can be recorded on a computer-readable medium (e.g., any medium that can be directly read and accessed by a computer). Such media include, but are not limited to: magnetic storage media, such as floppy disks, hard disk storage media, and magnetic tape; optical storage media, such as CD-ROMs; electrical storage media, such as RAM and ROM; portable flash drives; and mixtures of these categories, such as magnetic / optical storage media.

[0067] The processor can also access communication channels to communicate with users in remote locations. A remote location refers to a location where the user does not have direct contact with the system and relays input information from external devices (such as computers connected to a wide area network (WAN), telephone network, satellite network, or any other suitable communication channel, including mobile phones (i.e., smartphones)) to the input manager.

[0068] In some embodiments, the system 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 can be configured for wired or wireless communication, including but not limited to radio frequency (RF) communication (e.g., radio frequency identification (RFID)), Zigbee communication protocol, WiFi, infrared, wireless universal serial bus (USB), ultra-wideband (UWB), etc. Communication protocols and cellular communications (such as Code Division Multiple Access (CDMA) or Global System for Mobile Communications (GSM)).

[0069] In one embodiment, the communication interface is configured to include one or more communication ports, such as physical ports or interfaces like USB ports, RS-232 ports, or any other suitable electrical connection ports, to allow data communication between the subject system and other external devices, such as computer terminals (e.g., in a doctor's office or hospital environment), which are configured for similar complementary data communication.

[0070] In one embodiment, the communication interface is configured for infrared communication. Communication or any other suitable wireless communication protocol that enables the subject system to communicate with other devices, such as computer terminals and / or networks, communication-enabled mobile phones, personal digital assistants, or any other communication devices that the user can use in conjunction with them.

[0071] In one embodiment, the communication interface is configured to provide connectivity for data transmission via Internet Protocol (IP) through a cellular telephone network, Short Message Service (SMS), a wireless connection of a personal computer (PC) connected to the Internet on a local area network (LAN), or a WiFi connection to the Internet at a WiFi hotspot.

[0072] In one embodiment, the subject system is configured to communicate via a communication interface (e.g., using 802.11 or...). The server device communicates wirelessly with a common standard (RF protocol or IrDA infrared protocol). The server device can be another portable device, such as a smartphone, personal digital assistant (PDA), or laptop; or a larger device, such as a desktop computer, appliance, etc. In some embodiments, the server device has a display, such as a liquid crystal display (LCD), and input devices, such as buttons, keyboard, mouse, or touchscreen.

[0073] In some embodiments, the communication interface is configured to automatically or semi-automatically communicate with network or server devices data stored in the subject system (e.g., stored in an optional data storage unit) using one or more of the communication protocols and / or mechanisms described above.

[0074] The output controller may include any of a variety of known controllers for presenting information to users, whether human or machine, local or remote. If one of the display devices provides visual information, that information may typically be logically and / or physically organized as an array of image elements. The graphical user interface (GUI) controller may include any of a variety of known or future software programs for providing a graphical input and output interface between the system and the user, and for processing user input. The functional elements of the computer may communicate with each other via a system bus. Some of these communications may be implemented using networks or other types of remote communication in alternative embodiments. According to known techniques, the output manager may also provide information generated by the processing module to users at remote locations, for example, via the Internet, telephone, or satellite networks. The presentation of data by the output manager may be implemented according to a variety of known techniques. For example, the data may include SQL, HTML, or XML documents, emails or other files, or other forms of data. The data may include Internet URL addresses so that users can retrieve additional SQL, HTML, XML, or other documents or data from remote sources. One or more platforms present in the subject system may be any type of computer platform known or to be developed in the future, although they will generally be a class of computers (often referred to as servers). However, they can also be mainframe computers, workstations, or other computer types. They can be connected via any known or future type of cable or other communication system, including wireless systems (whether networked or otherwise). They may be located in the same place or physically separated. Depending on the type and / or brand of the computer platform chosen, various operating systems can be used on any computer platform. Suitable operating systems include Windows 10, Windows... Windows XP, Windows 7, Windows 8, iOS, Oracle Solaris, Linux, OS / 400, Compaq Tru64 Unix, SGI IRIX, SiemensReliant Unix, Ubuntu, Zorin OS, etc.

[0075] Methods of distributing droplets into partitions using flow cytometry

[0076] Embodiments of the invention also include a method for distributing droplets into partitions via flow cytometry (e.g., using a system as described above). Aspects of the method include sorting particle-occupied and particle-unoccupied droplets into partitions via flow cytometry (e.g., as described above). Therefore, in addition to conventional particle sorting, aspects of the method also include partitioning to guide particle-unoccupied droplets. Thus, the method of the invention includes flow cytometry protocols (including sorting flow cytometry assay protocols) used with conventional flow cytometers (e.g., according to known workflows), modified to include depositing particle-unoccupied droplets into one or more partitions.

[0077] For example, in applications where cells are sorted into the wells of a multi-well plate, the method of the present invention may additionally include guiding unoccupied droplets into the wells of the multi-well plate to add additional sheath fluid to these wells (e.g., to prevent evaporation and damage to cells present in the wells). In such applications, the unoccupied droplets may include sheath fluid or some other liquid, such as a buffer, etc. (as needed). The number of unoccupied droplets being sorted into the wells can be varied as needed (e.g., to achieve a desired total volume of liquid in the wells).

[0078] Embodiments of the method also include applications of contacting sorted cells with reagents (e.g., cell regulators), for example, to determine the activity of such reagents relative to sorted cells. For example, the method of the invention may include sorting cells into wells of a multi-well plate and then guiding partitions containing unoccupied droplets of reagent into these wells (e.g., to determine the activity of the reagent on the cells). In such screening applications, a collection of two or more partitions containing the same type of sorted cells (i.e., cells identical based on predetermined sorting criteria) may be contacted with different concentrations of the reagent being screened, for example, by partitioning reagent partitions containing different numbers of unoccupied droplets into different wells. In this way, a reagent gradient can be established between partitions containing multiple cells if needed (e.g., to determine the effect of different concentrations of reagent on the cells). In such applications, the reagent introduced into the partitions via unoccupied droplets can vary, wherein, in some cases, the reagent is a cell regulator (or at least a reagent suspected of having cell-regulating activity), examples of which include, but are not limited to, peptides, proteins, nucleic acids, small molecules, and the like. Instead of or in addition to these reagents, methods may include, for example, introducing a tag or component of a signal generation system into a partition via a droplet not occupied by particles in a similar manner. Examples of tags include fluorescent tags, nucleic acid tags, etc. Examples of signal generation systems include enzymes, such as those that convert substrates into detectable products. According to embodiments of the invention, any desired tag, signal generation system, or component thereof may be introduced into a partition via a droplet not occupied by particles.

[0079] In some embodiments, the system is configured for a single-cell sequencing workflow, such as sequence library preparation or a portion thereof. For example, a system in such embodiments may include a lysis agent source and an oligonucleotide barcode solid carrier source, such as oligonucleotide barcode beads (wherein the oligonucleotide barcode may include cell tags and unique molecular indicators, as well as other functional domains), for binding to cells in a partition to prepare a sequencing-ready library from the cells. Any convenient lysis agent may be present in the system of the invention, such as any lysis agent capable of inducing chemical lysis of the relevant cells and encapsulated in the flow stream, including, for example, enzymes or detergents or the like. In embodiments of such systems, the system is configured to guide occupied droplets comprising cells into a partition. The partition may include a barcode solid carrier, such as barcode beads, which may be magnetic. Alternatively, the system may also be configured to guide occupied droplets comprising oligonucleotide barcode solid carriers (e.g., beads, such as magnetic beads) into the partition. The system is also configured to guide a sufficient amount of lysis agent (e.g., in unoccupied droplets) into the partition. Therefore, the system in this embodiment may additionally include an oligonucleotide barcode solid carrier source, such as barcode oligonucleotides comprising cell tags or oligonucleotide barcode beads. In some cases, the barcode oligonucleotides also include unique molecular indicators. In some embodiments, the oligonucleotide barcode solid carrier may be configured to identify aspects of cells that deviate to the same partition as the oligonucleotide barcode solid carrier. In some embodiments, the system may additionally include one or more additional components used in the preparation of a single cell sequence library, such as nucleotides, polymerases, oligonucleotides, such as primers or TSOs. In some cases, the system also includes a processor including a memory operatively coupled to the processor, wherein the memory includes instructions stored thereon that, when executed by the processor, cause the processor to record identifiers identifying which partitions the cells and the oligonucleotide barcode solid carriers deviate to. That is, the system may be configured to establish a record of which partition each cell and oligonucleotide barcode solid carrier deviates to. Further details regarding the preparation of a nucleic acid library using barcode beads and components therein are provided in International Patent Application Serial No. PCT / US2020 / 060692, filed on November 16, 2020, the disclosure of which is incorporated herein by reference.

[0080] In some cases, the system is configured to dispense one or more reagents (e.g., in unoccupied droplets) into partitions. For example, the system of the present invention may include a sheath fluid source. The system may additionally include a first reagent source. The first reagent may vary. In some cases, the first reagent is a cell regulator, wherein examples of cell regulators are diverse and include, but are not limited to: peptides or proteins, nucleic acids; small molecules, etc. In some cases, the first reagent may include a tag or member of a signal generation system. In some cases, the system further includes a second reagent source. The first reagent may vary, and in some cases, the second reagent may be any of the above-described reagents with respect to the first reagent. In some cases, the system is configured to provide dynamically configurable amounts of a first reagent and an optional second reagent in the sheath fluid and / or sample. By means of dynamically configurable reagent amounts, this means that in some cases, the system may be configured to dynamically adjust the volume or concentration of the reagent introduced into the flow stream. For example, in one embodiment, the system may be configured to dynamically adjust the volume or concentration of the reagent introduced into the sheath fluid. In other embodiments, for example, via a sample fluid, the system may be configured to dynamically adjust the volume or concentration of the reagent introduced into the sample. Ultimately, embodiments of the system of the present invention can be configured to dynamically adjust the volume or concentration of reagents deflected into the partition, for example, by dynamically adjusting the amount of reagent introduced into the flow stream, for example, via a sheath fluid or a sample (e.g., via a sample fluid).

[0081] Computer-readable storage media

[0082] This disclosure also includes a non-transitory computer-readable storage medium thereon storing instructions for operating a flow cytometer according to embodiments of the invention, capable of sorting particle-occupied and particle-unoccupied droplets. In some embodiments, the computer-readable storage medium of interest includes a computer program stored thereon, wherein the computer program, when loaded onto a computer, includes instructions having an algorithm for guiding a droplet sorter to partition and guide desired particle-occupied and particle-unoccupied droplets (e.g., as described above). The computer-readable storage medium can be used on one or more computers to achieve full or partial automation of a system for implementing the methods described herein. In some embodiments, instructions according to the methods described herein can be encoded onto a computer-readable medium in a “programmable” form, wherein the term “computer-readable medium” as used herein refers to any non-volatile storage medium involved in providing instructions and data to a computer for execution and processing. Examples of suitable non-volatile storage media include floppy disks, hard disks, optical disks, magneto-optical disks, CD-ROMs, CD-Rs, magnetic tapes, non-volatile memory cards, ROMs, DVD-ROMs, Blu-ray discs, solid-state drives, and network-attached storage (NAS), whether these devices are inside or outside a computer. Files containing information can be "stored" on a computer-readable medium, where "stored" means recording the information so that it can be accessed and retrieved by the computer later. The computer-implemented methods described herein can be executed using programming languages ​​that can be written in one or more of any number of computer programming languages. These languages ​​include, for example, Java (Sun Microsystems, Inc., Santa Clara, CA), Visual Basic (Microsoft Corp., Redmond, WA), and C++ (AT&T Corp., Bedminster, NJ), among many others.

[0083] Computer-readable storage media can be used in one or more computer systems having a display and operator input devices. Operator input devices can be, for example, a keyboard, mouse, or the like. A processing module includes a processor that can access memory on which instructions for performing steps of the subject method are stored. The processing module may include an operating system, a graphical user interface (GUI) controller, system memory, memory storage devices and input / output controllers, cache memory, data backup units, and many other devices. The processor may be a commercially available processor or one of other processors that are already available or will be available in the future. As is known in the art, a processor executes an operating system, and the operating system interacts with firmware and hardware in well-known ways and facilitates the processor in coordinating and executing the functions of various computer programs written in various programming languages, such as Java, Perl, C++, other high-level or low-level languages, and combinations thereof. The operating system typically cooperates with the processor to coordinate and execute the functions of other components of the computer. The operating system also provides scheduling, input / output control, file and data management, memory management, communication control, and related services according to known techniques.

[0084] The following examples are provided as examples and not as limitations.

[0085] experiment

[0086] Figure 5 Details of embodiments of the present invention are provided. Figure 5 The image shows a cross-section of a perforated plate 500 having a first row of 510 and a second row of 520. Figure 5 An example of a colorimetric gradient generated by additional droplets containing the sample medium is provided. The sample medium contains beads and horseradish peroxidase (HRP). The wells contain only TMB. Figure 5The porous plate 500 shown is depicted after a specific number of droplets have been deflected into the pores of the porous plate 500 using the system described in the present invention. Initially, the wells in the multi-well plate contained only a TMB (3,3',5,5”-tetramethylbenzidine) matrix solution. To illustrate the system's effectiveness, the sample medium used included beads and horseradish peroxidase (HRP) (although other fluids may be included in the system). When droplets containing beads and HRP bind to TMB already present in the wells, the color of the solution in the wells changes accordingly. The more droplets containing beads and HRP deposited in the wells, the more drastic the color change. For example, a well with one droplet of beads and HRP will be clearer and more transparent than a well with ten droplets of beads and HRP. The system was used to deflect a single droplet containing beads and HRP into well 530 and add one droplet containing beads and HRP to each adjacent well to the left of well 530, depositing ten droplets in well 550 except for well 540. Well 540 is a control example where no droplets were deflected, and therefore no beads and HRP were added to that well. Figure 5 The diagram describes how, apart from the completely transparent control orifice 540, the solution in each orifice moving leftward from 530 to 550 becomes less clear and less transparent. Similarly, in row 520, the system is used to deflect a single droplet comprising a bead and HRP into orifice 560, with additional droplets deflected into each adjacent orifice, moving leftward from orifice 560 such that ten droplets are deflected into orifice 570. Similar to row 510, row 520, moving from right to left from orifice 560 to orifice 570, shows the solution in each orifice gradually becoming less clear and less transparent.

[0087] Notwithstanding the appended claims, this disclosure is also defined by the following terms:

[0088] 1. A system for distributing droplets into partitions using flow cytometry, the system comprising:

[0089] Droplet sorting system, including:

[0090] A droplet flow generator configured to generate a flow of both particle-occupied and particle-unoccupied droplets from a liquid exiting a flow cell; and

[0091] A droplet sorter is configured to partition droplets that are occupied by particles and droplets that are not occupied by particles.

[0092] 2. The system according to Clause 1, wherein the droplet sorter comprises:

[0093] A droplet charger configured to charge the droplets of the droplet stream;

[0094] A droplet deflector configured to deflect charged droplets into a partition; and

[0095] A controller is configured to control the droplet charger and droplet deflector to guide droplets occupied by particles and droplets not occupied by particles in a partitioned manner.

[0096] 3. The system according to Clause 2, wherein the controller is configured to partition and guide a first predetermined number of droplets occupied by particles and a second predetermined number of droplets not occupied by particles.

[0097] 4. The system according to clause 2 or 3, wherein the controller comprises:

[0098] A processor and a memory operatively coupled to the processor, wherein the memory includes instructions stored thereon that, when executed by the processor, cause the processor to specify droplets occupied by particles and droplets not occupied by particles, guided by the droplet sorter partition.

[0099] 5. The system according to any one of the preceding clauses further includes an input module operatively connected to the droplet sorter, wherein the input module is configured to receive input messages specifying droplets occupied by particles and droplets not occupied by particles to be partitioned and guided.

[0100] 6. The system according to any one of the preceding clauses, wherein the system is configured to dynamically adjust the ratio of droplets occupied by partitioned particles to droplets not occupied by particles over a period of time.

[0101] 7. The system according to any one of the preceding clauses, wherein the system is configured to dynamically instruct the droplet sorter to partition and guide each droplet by default, or to partition and guide only droplets occupied by desired particles by default.

[0102] 8. The system according to any one of the preceding clauses, wherein the droplet has a predetermined volume.

[0103] 9. The system according to Clause 8, wherein the droplet generator is configured to dynamically adjust the predetermined volume of the droplet.

[0104] 10. The system according to any one of the preceding clauses, wherein the droplet occupied by the particle comprises particles selected from cells and solid carriers.

[0105] 11. The system according to any one of the preceding clauses, wherein the partition is a hole in a perforated plate.

[0106] 12. The system according to Clause 11, wherein the system is configured to record identifiers of the holes in a porous plate into which droplets are guided.

[0107] 13. The system according to Clause 12, wherein the identifier identifies the number of droplets deflected into the orifice and occupied by particles and / or the number of droplets not occupied by particles.

[0108] 14. The system according to any one of the preceding clauses, wherein the system is configured to prepare a single-cell nucleic acid library in a partition.

[0109] 15. The system according to Clause 14, wherein the system includes a cell sample source, a barcode solid carrier source, and a cell lysis agent source.

[0110] 16. The system according to Clause 15, wherein the system is configured to direct droplets occupied by particles comprising cells, droplets occupied by particles comprising barcode solid carriers, and droplets not occupied by particles comprising lysis agents into the same partition.

[0111] 17. The system according to Clause 16, wherein the system is further configured to record identifiers for identifying partitions, including droplets occupied by particles of the cell, droplets occupied by particles of the barcode solid carrier, and droplets not occupied by particles of the lysis agent, which are directed into the partitions.

[0112] 18. The system according to any one of clauses 1 to 13, wherein the system is configured to direct unoccupied droplets comprising reagent particles into a partition comprising cells.

[0113] 19. The system according to Clause 18, wherein the reagent comprises a cell regulator.

[0114] 20. The system according to Clause 19, wherein the cell regulator comprises peptides, proteins, nucleic acids, or small molecules.

[0115] 21. The system according to any one of clauses 1 to 13, wherein the system is configured to direct unoccupied droplets of particles comprising a tag or member of a signal generating system into a partition comprising a cell.

[0116] 22. The system according to any one of clauses 1 to 21, wherein the system further comprises:

[0117] A flow cell, configured to allow a flow of liquid containing particles to pass through an inquiry point;

[0118] A light source, used to illuminate the inquiry point; and

[0119] A detector used to detect light from the point of inquiry.

[0120] 23. A method for distributing droplets into partitions by flow cytometry, the method comprising:

[0121] A droplet flow is generated from the liquid leaving the flow cell, comprising droplets occupied by particles and droplets not occupied by particles; and

[0122] A droplet sorter configured to partition droplets occupied by particles and droplets not occupied by particles guides the flow of droplets.

[0123] 24. The method according to Clause 23, wherein the droplet sorter comprises:

[0124] A droplet charger configured to charge the droplets of the droplet stream;

[0125] A droplet deflector configured to deflect charged droplets into a partition; and

[0126] A controller is configured to control the droplet charger and droplet deflector to guide droplets occupied by particles and droplets not occupied by particles in a partitioned manner.

[0127] 25. The method according to Clause 24, wherein the controller is configured to partition and guide a first predetermined number of droplets occupied by particles and a second predetermined number of droplets not occupied by particles.

[0128] 26. The method according to clause 24 or 25, wherein the controller comprises:

[0129] A processor and a memory operatively coupled to the processor, wherein the memory includes instructions stored thereon that, when executed by the processor, cause the processor to designate to the droplet sorter droplets occupied by particles to be partitioned and droplets not occupied by particles.

[0130] 27. The method according to any one of clauses 23 to 26, wherein the method further comprises inputting an input message to an input module operatively connected to the droplet sorter, the input message specifying droplets occupied by particles and droplets not occupied by particles to be partitioned and guided.

[0131] 28. The method according to any one of clauses 23 to 27, wherein the method includes adjusting the ratio of droplets occupied by partitioned particles to droplets not occupied by particles over a period of time.

[0132] 29. The method according to any one of clauses 24 to 28, wherein the method includes instructing the droplet sorter to partition and guide each droplet by default, or to partition and guide only droplets occupied by desired particles by default.

[0133] 30. The method according to any one of clauses 24 to 29, wherein the droplet has a predetermined volume.

[0134] 31. The method according to Clause 30, wherein the method includes adjusting a predetermined volume of the droplet.

[0135] 32. The method according to any one of clauses 23 to 31, wherein the droplet occupied by the particle comprises particles selected from cells and solid carriers.

[0136] 33. The method according to any one of clauses 22 to 32, wherein the partition is a hole in a perforated plate.

[0137] 34. The method according to clause 33, wherein the method includes recording an identifier that identifies the orifice of the porous plate to which the droplet is guided.

[0138] 35. The method according to Clause 34, wherein the identifier identifies the number of droplets occupied by particles deflected into the orifice and / or the number of droplets not occupied by particles.

[0139] 36. The method according to any one of clauses 23 to 35, wherein the method comprises preparing a single-cell nucleic acid library in a partition.

[0140] 37. The method according to Clause 36, wherein the system comprises a cell sample source, a barcode solid carrier source, and a cell lysis agent source.

[0141] 38. The method according to Clause 37, wherein the method includes directing droplets occupied by particles comprising cells, droplets occupied by particles comprising barcode solid carriers, and droplets not occupied by particles comprising lysis agents into the same partition.

[0142] 39. The method according to Clause 38, wherein the system is further configured to record identifiers of the partitions, including droplets occupied by particles of the cell, droplets occupied by particles of the barcode solid carrier, and droplets not occupied by particles of the lysis agent, which are directed into the partitions.

[0143] 40. The method according to any one of clauses 23 to 35, wherein the method comprises directing unoccupied droplets comprising reagent particles into a partition comprising cells.

[0144] 41. The method according to clause 40, wherein the reagent comprises a cell regulator.

[0145] 42. The method according to Clause 41, wherein the cell regulator comprises peptides, proteins, nucleic acids, or small molecules.

[0146] 43. The method according to any one of clauses 23 to 35, wherein the method comprises directing droplets of particles not occupied by a tag or member of a signal generation system into a partition comprising a cell.

[0147] Although the invention has been described in detail by way of illustrations and examples for the purpose of clarity, it will be readily understood by those skilled in the art, based on the teachings of the invention, that certain changes and modifications may be made without departing from the spirit or scope of the appended claims.

[0148] Therefore, the foregoing has only illustrated the principles of the invention. It should be understood that, although not explicitly described or shown herein, those skilled in the art will be able to design various devices that embody the principles of the invention and are included within its spirit and scope. Furthermore, all examples and conditional language used herein are primarily intended to assist the reader in understanding the principles of the invention and the concepts contributed by the inventors to further developments in the field, and should be understood as not being limited to these specifically enumerated examples and conditions. Moreover, all statements herein describing the principles, aspects, and embodiments of the invention, as well as specific examples thereof, are intended to include their structural and functional equivalents. Furthermore, such equivalents are contemplated to include both currently known equivalents and future-developed equivalents (i.e., any element developed that performs the same function, regardless of its structure). Furthermore, nothing disclosed herein is intended to be made public, whether or not such disclosure is expressly set forth in the claims.

[0149] Therefore, the scope of the invention is not limited to the exemplary embodiments shown and described herein. Rather, the scope and spirit of the invention are embodied in the appended claims. In the claims, 35 USC §112(f) or 35 USC §112(6) is expressly defined as referring to a limitation in a claim only if the exact phrase “means” or the exact phrase “step is” is used in the limitation in the claim; if the exact phrase is not used in the limitation in the claim, then 35 USC §112(f) or 35 USC §112(6) is not referred to.

Claims

1. A system for distributing droplets into partitions using flow cytometry, the system comprising: Droplet sorting system, including: A droplet-forming transducer configured to generate a flow of both particle-occupied and particle-unoccupied droplets from a liquid exiting a flow cell via a vibrating or oscillating nozzle; and A droplet sorter configured to guide droplets occupied by particles and droplets not occupied by particles in separate zones, wherein the droplet sorter comprises: A droplet charger configured to charge droplets of a droplet stream; A droplet deflector configured to deflect charged droplets into a partition; and A controller is configured to control the droplet charger and droplet deflector to guide droplets occupied by particles and droplets not occupied by particles in a partitioned manner.

2. The system of claim 1, wherein, The controller is configured to partition droplets occupied by a first predetermined number of particles and droplets not occupied by a second predetermined number of particles.

3. The system of claim 1 or 2, wherein, The controller includes: A processor and a memory operatively coupled to the processor, wherein the memory includes instructions stored thereon that, when executed by the processor, cause the processor to specify droplets occupied by particles and droplets not occupied by particles, guided by the droplet sorter partition.

4. The system of claim 1 or 2, further comprising an input module operably connected to the droplet sorter, wherein, The input module is configured to receive input messages specifying droplets occupied by particles to be partitioned and droplets not occupied by particles.

5. The system according to claim 1 or 2, wherein, The system is configured to dynamically adjust the ratio of droplets occupied by partitioned particles to droplets not occupied by particles over a period of time.

6. The system according to claim 1 or 2, wherein, The system is configured to dynamically instruct the droplet sorter to partition each droplet by default, or to partition only droplets occupied by desired particles and droplets not occupied by desired particles by default.

7. The system according to claim 1 or 2, wherein, The droplet has a predetermined volume.

8. The system according to claim 7, wherein, The droplet generator is configured to dynamically adjust the predetermined volume of the droplet.

9. The system according to claim 1 or 2, wherein, The droplets occupied by the particles include particles selected from cells and solid carriers.

10. The system according to claim 1 or 2, wherein, The partition is a hole in a perforated plate.

11. The system according to claim 1 or 2, wherein, The system also includes: An inquiry point, wherein the flow cell is configured to allow a flow of liquid containing particles to pass through the inquiry point; A light source, used to illuminate the inquiry point; and A detector configured to detect the interaction between illumination light and particles in a flowing stream, wherein the detector is configured to communicate with the controller.

12. A method for distributing droplets into partitions by flow cytometry, the method comprising: A droplet flow, comprising both particle-occupied and particle-unoccupied droplets, is generated from the liquid leaving the flow cell by a vibrating or oscillating nozzle. and A droplet sorter configured to partition droplets occupied by particles and droplets not occupied by particles guides the droplet flow in a partitioned manner, wherein the droplet sorter comprises: A droplet charger configured to charge the droplets of the droplet stream; A droplet deflector configured to deflect charged droplets into a partition; and A controller is configured to control the droplet charger and droplet deflector to guide droplets occupied by particles and droplets not occupied by particles in a partitioned manner.

13. The method according to claim 12, wherein, The controller is configured to partition droplets occupied by a first predetermined number of particles and droplets not occupied by a second predetermined number of particles.

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