Cell viability compensation (CVC) beads for flow cytometry applications and methods of using the same

Abstract

To provide an improved and useful control composition for cell viability assays.SOLUTION: The present disclosure provides control compositions, as well as methods of making and using the same. Aspects of the control compositions include a control particle characterized by: a core; an aminated polymer comprising an amine configured to react with an amine-reactive dye; and a nucleic acid configured to bind to a DNA-binding dye, wherein the aminated polymer and the nucleic acid are covalently associated with the core. Aspects of the invention further include methods of synthesizing the control particles of the compositions. The synthesis methods comprise: obtaining a core; and covalently associating a polymer comprising an amine and a nucleic acid with the core to produce a control particle. Also provided are methods for using the control compositions to perform compensation on cell viability data obtained using, for example, flow cytometry, as well as kits for practicing the subject methods.SELECTED DRAWING: None

Description

[Technical Field]

[0001] Optical detection is often used to characterize the components of biological samples. When a sample is illuminated, light can be scattered by the sample, transmitted through the sample, and emitted by the sample (e.g., by fluorescence). Variations in sample components, such as morphology, absorbance, and the presence of fluorescent labels, can cause variations in the light scattered, transmitted, or emitted by the sample. To quantify these variations, light is collected and directed toward a detector surface. One technique that utilizes optical detection to characterize the components of a sample is flow cytometry. [Background technology]

[0002] In flow cytometry applications, viability stains are used to distinguish between viable and nonviable mammalian cells based on fluorescence intensity. Viability stains work by exploiting differences in the cell membranes of necrotic (i.e., nonviable) and healthy (i.e., viable) cells. In necrotic cells, the damaged cell membrane allows dyes to enter the cell interior and interact with various intracellular components, whereas the intact cell membrane of healthy cells prevents most dyes from gaining such access.

[0003] Currently, there are two common techniques primarily employed for viability staining: amine-reactive fluorescent dye-based staining assays and DNA-intercalating dye-based staining assays. In amine-reactive dye-based assays, the reactive dye can penetrate the membranes of nonviable cells and react with the abundant free amines on internal proteins, resulting in a strong fluorescent labeling of nonviable cells (i.e., compared with viable cells) in the sample. Similarly, in DNA-intercalating dye-based assays, the DNA-binding dye can penetrate the membranes of nonviable cells and react with DNA in the nucleus, resulting in a relatively strong fluorescent labeling of nonviable cells.

[0004] Like other sample characterization techniques that use flow cytometry, viability staining assays require controls to ensure accurate and reproducible results, for example, by setting voltage and compensation parameters. Unfortunately, the majority of current viability staining protocols require depleting a portion of the experimenter's often limited cell sample to create the necessary viability controls. Furthermore, creating such controls is time-consuming, unreliable, and often fails to achieve the resolution between live and dead cells necessary to obtain meaningful results. Summary of the Invention [Problem to be solved by the invention]

[0005] Therefore, there is a need for improved and useful control compositions for cell viability assays. [Means for solving the problem]

[0006] Embodiments of the present invention provide novel and useful control compositions and methods for their use that address the above-mentioned limitations. To achieve this, embodiments of the present invention leverage recent advances in materials science and biochemistry and novel particle coating technologies to efficiently synthesize a new class of convenient and effective control particles. The disclosed coating technology utilizes a unique layer-by-layer approach to enable the creation of highly versatile compensation particles capable of binding both amine-reactive and DNA-intercalating dyes. This versatility, combined with the highly tunable nature of the disclosed particle synthesis method, can simplify current laboratory workflows while providing users with additional flexibility in developing future experimental designs. Accordingly, embodiments of the disclosed compositions and methods find use in a variety of applications where it is desirable to perform accurate and adaptable cell viability assays at low cost and with increased efficiency, for example, as described in more detail below.

[0007] In one embodiment, a control particle for performing a cell viability assay is provided. The control particle embodiment includes a core, an amine-containing polymer (i.e., an aminated polymer) configured to react with an amine-reactive dye, and a nucleic acid configured to bind to a DNA-binding dye, wherein the aminated polymer and nucleic acid are covalently associated with the core.

[0008] In certain embodiments, the core of the control particle has substantially no autofluorescence. In some embodiments, the core has low nonspecific binding. In some embodiments, the core comprises a polymer, such as poly(methyl methacrylate) (PMMA) or a polyacrylamide hydrogel. In some embodiments, the core comprises an inorganic material, such as silicon dioxide (i.e., silica). In some embodiments, the diameter of the core can range from 1 μm to 10 μm. In some embodiments, the nucleic acid is covalently bound to the surface of the core, either directly or via a linker, and the aminated polymer is covalently bound to the nucleic acid. In some embodiments, the aminated polymer and nucleic acid are layered on the core. In some embodiments, multiple aminated polymer coating layers and / or nucleic acid coating layers are covalently associated with the core. In some embodiments, the particle comprises five or more aminated polymer layers and / or five or more nucleic acid layers. In some embodiments, aminated polymer layers of the coating alternate with nucleic acid layers of the coating. In some embodiments, the control particle is a bead.

[0009] In certain embodiments, the one or more aminated polymers of the control particle each contain multiple amines. In some embodiments, each aminated polymer contains sufficient spacing between each amine to prevent self-quenching of the amine-reactive dye. In some embodiments, the one or more aminated polymers comprise a polypeptide, such as a protein. In some embodiments, the protein contains arginine and / or lysine residues. In some embodiments, the protein is a histone or myelin basic protein (MBP). In some embodiments, the one or more aminated polymers comprise a polysaccharide, such as a cationic polymer. In some embodiments, the cationic polymer comprises one or more of branched polyethyleneimine, polyallylamine, polylysine, or chitosan. In some embodiments, the one or more amines of each aminated polymer are configured to react with an amine-reactive dye that is free-flowing in solution. In some embodiments, the amine-reactive dye has an emission maximum wavelength in the range of 400 nm to 850 nm. In some embodiments, the amine-reactive dye has an absorption maximum (excitation maximum) wavelength in the range of 300 nm to 800 nm. In some embodiments, the amine-reactive dye includes a BD Horizon™ reagent, such as the BD Horizon™ Fixable Viability Stain (FVS) reagent.

[0010] In certain embodiments, the one or more nucleic acids of the control particle have a size ranging from 300 base pairs to 700 base pairs. In some embodiments, each of the one or more nucleic acids of the control particle comprises deoxyribonucleic acid (DNA). In some embodiments, the DNA comprises double-stranded DNA (dsDNA). In some embodiments, the DNA comprises a naturally occurring DNA sequence, such as a DNA sequence obtained from salmon. In some embodiments, the DNA comprises a synthetic DNA sequence. In some embodiments, the one or more nucleic acids are configured to bind to a DNA-binding dye that flows freely in solution. In some embodiments, the DNA-binding dye has an emission maximum wavelength ranging from 400 nm to 850 nm. In some embodiments, the DNA-binding dye has an absorption maximum (excitation maximum) wavelength ranging from 300 nm to 800 nm. In some embodiments, the DNA-binding dye comprises propidium iodide (PI), 7-amino-actinomycin D (7-AAD), and / or 4',6-diaminidino-2-phenylindole (DAPI).

[0011] In certain embodiments, the control particles are positive control particles. In some embodiments, the positive control particles are labeled with multiple amine-reactive dyes covalently bound to amines of one or more aminated polymers and / or multiple DNA-binding dyes bound to one or more nucleic acids. In certain embodiments, the control particles are negative control particles. In some embodiments, the negative control particles include one or more polyethylene glycol (PEG) compounds configured to prevent amine-reactive dyes from reacting with the amines of the negative control particles. In some embodiments, the negative control particles do not include aminated polymers or nucleic acids, e.g., the negative control particles consist of a core.

[0012] In certain embodiments, a control composition is provided that includes a plurality of control particles. In some embodiments, the control composition includes a plurality of unlabeled positive control particles. In some embodiments, the unlabeled control composition further includes a plurality of negative control particles. In some embodiments, the control composition includes a plurality of labeled positive control particles. In some embodiments, the labeled control composition further includes a plurality of negative control particles. In some embodiments, the control composition includes a liquid solution, such as, for example, a buffer. In some embodiments, the control composition is lyophilized.

[0013] In another aspect, a method of synthesizing a control particle of the present invention is provided, the method aspect including obtaining a core and covalently associating a polymer comprising an amine and a nucleic acid with the core to produce the control particle.

[0014] In certain embodiments, the surface of the core includes a functional group, such as an amide, maleimide, or thiol group. In some embodiments, the method further includes surface functionalization of the core with a functional group. For example, the resulting core can include silica, and the functionalization can include amination. In some embodiments, the method further includes modifying the functional group of the core to include a reactive functional group, e.g., using a reagent. For example, an aminated silica core can be modified to include a thiol group, e.g., using a thiolating reagent such as 2-iminothiolane. In some embodiments, the method further includes positively charging the core to increase binding efficiency with a nucleic acid, e.g., before associating an aminated polymer with the core. In some embodiments, the method further includes negatively charging the core to increase binding efficiency with an aminated polymer, e.g., before associating a nucleic acid with the core.

[0015] In certain embodiments, the nucleic acid includes deoxyribonucleic acid (DNA), such as double-stranded DNA (dsDNA). In some embodiments, the particle synthesis method further includes modifying the DNA to include a functional group reactive with the functional group of the core and / or the functional group of the aminated polymer. For example, the core and / or the aminated polymer can be modified to include a thiol group, and the DNA can be modified to include a maleimide group. In some embodiments, the nucleic acid is covalently associated with the core by reacting a functional group of the DNA with a reactive functional group of the core. In some embodiments, the nucleic acid is covalently associated with the core by reacting a functional group of the DNA with a functional group of the aminated polymer, for example, when the aminated polymer is already covalently associated with the core. In some embodiments, the 5' and / or 3' ends of the DNA are functionalized. In some embodiments, the DNA is dsDNA, and both strands of the dsDNA are functionalized. In some embodiments, the dsDNA is covalently attached to the core by reacting a functional group on a first strand of the dsDNA with a reactive functional group on the core, and the dsDNA is covalently attached to the aminated polymer by reacting a functional group on a second strand of the dsDNA with a functional group on the aminated polymer, hi some embodiments, both strands of the dsDNA are covalently attached to the aminated polymer.

[0016] In certain embodiments, the particle synthesis method further includes modifying the aminated polymer to include a functional group reactive with the functional group of the core and / or the functional group of the nucleic acid. For example, the core and / or the nucleic acid can be modified to include a maleimide group, and the aminated polymer can be modified to include a thiol group. In some embodiments, the aminated polymer is covalently associated with the core by reacting the functional group of the aminated polymer with a reactive functional group of the core. In some embodiments, for example, if the nucleic acid is already covalently associated with the core, the aminated polymer is covalently associated with the core by reacting the functional group of the aminated polymer with a functional group of the nucleic acid. In some embodiments, the aminated polymer is functionalized with two or more functional groups. For example, the aminated polymer can be a protein, and two or more lysine residues of the protein can be thiolated using, for example, 2-iminothiolane. In some embodiments, the aminated polymer is covalently attached to the core by reacting a first functional group of the aminated polymer with a reactive functional group of the core, and the aminated polymer is covalently attached to the nucleic acid by reacting a second functional group of the aminated polymer with a functional group of the nucleic acid, hi some embodiments, more than one functional group of the aminated polymer is covalently attached to the nucleic acid.

[0017] In certain embodiments, the aminated polymer and nucleic acid are covalently associated with the core by alternately layering the aminated polymer and nucleic acid coating. For example, the nucleic acid may comprise dsDNA, and two or more nucleic acids may be modified to include a maleimide group at the 5' end of each strand of the dsDNA; the aminated polymer may comprise a protein; the two or more aminated polymers may be modified to include two or more thiol groups; and the core may include multiple thiol groups. The layering may then include reacting the maleimide at the first 5' end of the first nucleic acid with the thiol group of the core, reacting the maleimide at the second 5' end of the first nucleic acid with the first thiol group of the first aminated polymer, reacting the second thiol group of the first aminated polymer with the maleimide at the first 5' end of the second nucleic acid, and reacting the maleimide at the second 5' end of the second nucleic acid with the first thiol group of the second aminated polymer, etc. In some embodiments, five or more rounds of layering are performed, such that the control particle includes five or more layers of nucleic acids and five or more layers of aminated polymers, hi some embodiments, each round of layering includes covalently associating multiple nucleic acids and multiple aminated polymers with the core.

[0018] In certain embodiments, the control particles are positive control particles. In some embodiments, the particle synthesis method further comprises incubating the positive control particles in a solution containing multiple amine-reactive dyes and / or DNA-binding dyes to create labeled positive control particles. In certain embodiments, the control particles are negative control particles. In some embodiments, the particle synthesis method further comprises conjugating one or more polyethylene glycol (PEG) compounds to amines of the negative control particles. In some embodiments, the negative control particles do not contain aminated polymers or nucleic acids. In some embodiments, synthesizing the negative control particles comprises conjugating one or more polyethylene glycol (PEG) compounds to one or more amine groups of the core to prevent amine-reactive dyes from reacting with the core.

[0019] In another aspect, a method is provided for determining a compensation value for cell viability data obtained from a particle analyzer (e.g., a flow cytometer) using the control particles of the present invention. The method aspect includes analyzing the labeled compensation control composition using the flow cytometer to obtain flow cytometry data, and calculating the compensation value based on the flow cytometry data.

[0020] In certain embodiments, the compensation value determination method further includes preparing a labeled compensation control composition. In some embodiments, the preparation includes contacting the unlabeled compensation control composition with an amine-reactive dye and / or a DNA-binding dye. In certain embodiments, the determined compensation value is used to perform compensation of the cell viability data. In some embodiments, compensation is performed by contacting a cell sample with the same dye bound to labeled positive control particles, analyzing the cell sample using a particle analyzer (e.g., a flow cytometer) to generate cell viability data, and correcting the cell viability data using the determined compensation value.

[0021] In another aspect, kits are provided that include the compensation control compositions described herein. In some embodiments, the kits further include an amine-reactive dye and / or a DNA-binding dye for labeling positive control particles of the compensation control composition. In some embodiments, the kits further include instructions for using the compensation control composition to determine a compensation value for cell viability data obtained from flow cytometry analysis. In some embodiments, the kits further include instructions for performing flow cytometry analysis and using the determined compensation value to perform compensation on the obtained cell viability data. [Brief explanation of the drawings]

[0022] [Figure 1] 1 is a diagram of the mechanism of two viability stains according to an embodiment of the present invention. [Figure 2]1 is a representation of a cell viability compensation (CVC) bead according to an embodiment of the present invention. [Figure 3] 1 shows an exemplary method for synthesizing CVC beads according to an embodiment of the present invention. [Figure 4] 1 provides staining results for conventional compensated beads and CVC beads synthesized according to an embodiment of the present invention with BD Horizon™ FVS520 amine-reactive dye. [Figure 5] 1 provides staining results for conventional compensated beads and CVC beads synthesized according to an embodiment of the present invention with BD Horizon™ FVS570 amine-reactive dye. [Figure 6] 1 provides staining results for conventional compensated beads and CVC beads synthesized according to an embodiment of the present invention with BD Horizon™ FVS700 amine-reactive dye. [Figure 7] 1 provides staining results for two DNA intercalating dyes, BD Pharmingen™ 7-AAD and BD Pharmingen™ DAPI, for conventional compensation beads and CVC beads synthesized according to embodiments of the present invention. [Figure 8] 1 provides the results of a stability study performed on CVC beads synthesized according to an embodiment of the present invention. [Figure 9A] FIG. 1 shows a schematic diagram of an exemplary particle sorting system for use with CVC beads of the present invention, according to certain embodiments. [Figure 9B] FIG. 1 shows a schematic diagram of an exemplary particle sorting system for use with CVC beads of the present invention, according to certain embodiments. [Figure 10] FIG. 1 shows a functional block diagram of an exemplary particle analyzer control system for use with the CVC beads of the present invention, according to certain embodiments. [Figure 11A] 1 illustrates an exemplary particle analysis system for use with CVC beads of the present invention, according to certain embodiments, and shows a functional block diagram of the particle analysis system. [Figure 11B]1 illustrates an exemplary particle analysis system for use with CVC beads of the present invention, according to certain embodiments, and shows a flow cytometer of the particle analysis system. [Figure 12A] 1 illustrates an exemplary particle sorter for fluorescent imaging using radio frequency tagged emission for use in performing a viability staining assay according to certain embodiments, and provides a schematic diagram of an image-enabled particle sorter. [Figure 12B] 1 illustrates a data processing technique for fluorescent imaging using radio frequency tagged luminescence for use in performing a viability staining assay according to certain embodiments, and provides a flow chart illustrating the data processing technique. [Figure 13] FIG. 1 shows a block diagram of a computing system for use with CVC beads of the present invention in accordance with certain embodiments. DETAILED DESCRIPTION OF THE INVENTION

[0023] The present disclosure provides control compositions and methods for making and using the same. Embodiments of the control compositions include control particles characterized by a core, a polymer containing an amine configured to react with an amine-reactive dye, and a nucleic acid configured to bind to a DNA-binding dye, wherein the aminated polymer and nucleic acid are covalently associated with the core. Embodiments of the invention further include methods of synthesizing control particles of the compositions, the synthesis method comprising obtaining a core and covalently associating a polymer containing an amine and nucleic acid with the core to produce the control particle. Also provided are methods of using the control compositions to perform compensation for cell viability data obtained using a particle analyzer (e.g., a flow cytometer), as well as kits for carrying out the subject methods.

[0024] Before describing the present invention in more detail, it is to be understood that this invention is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

[0025] Where a range of values is provided, unless the context clearly dictates otherwise, it is understood that each intervening value, to the tenth of the unit of the lower limit, between the upper and lower limits of that range, and any otherwise stated or intervening value in that stated range, is included in the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also included in the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both limits, ranges excluding either or both of those included limits are also included in the invention.

[0026] In this specification, certain ranges are set forth, and the numerical values are preceded by the term "about." The term "about" is used herein to provide literal support for the exact number it precedes, as well as a number that is close to or approximately the number it precedes. In determining whether a number is close to or approximately equal to a specifically stated number, the unstated number that is close or approximate may be a number that, in the context in which it is presented, results in a substantial equivalence to the specifically stated number.

[0027] 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 belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, representative exemplary methods and materials are described herein.

[0028] All publications and patents cited in this specification are incorporated herein by reference as if each individual publication or patent was specifically and individually indicated to be incorporated by reference, and are incorporated herein by reference to describe and describe the methods and / or materials in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the publication dates provided may be different from the actual publication dates which may need to be independently confirmed.

[0029] It should be noted that, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. It should be further noted that the claims may be drafted to exclude any optional element. Accordingly, this statement is intended to serve as a prior basis for using exclusive terminology such as "solely," "only," and the like, or for using "negative" limitations in connection with the recitation of claim elements.

[0030] As will be apparent to those skilled in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has distinct components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the invention. Any recited method can be carried out in the order of events recited or in any other order which is logically possible.

[0031] Although apparatus and methods have been or will be described for grammatical fluidity with functional descriptions, unless expressly formulated under 35 U.S.C. § 112, the claims should not be construed as necessarily limited in any way by means- or step-limitation constructions, but should be given the full scope of the meaning and equivalents of the definitions provided by the claims under the doctrine of equivalents, and if the claims are expressly formulated under 35 U.S.C. § 112, they should be expressly understood to be entitled to the full legal equivalents under 35 U.S.C. § 112.

[0032] As summarized above, control compositions are provided. In further describing various embodiments of the present invention, the subject compositions, including, for example, control particles of the composition, are first described in more detail. Methods of synthesizing the control particles of the composition are then described. Additionally, methods of using the control compositions to compensate cell viability data, and kits for practicing the subject methods are also provided.

[0033] Control Composition As summarized above, control compositions, such as flow cytometry compensation control compositions, are provided. The control compositions can be used to verify the functionality or performance of particle analysis systems (e.g., flow cytometry systems), protocols, and reagents used in flow cytometry assays. In certain embodiments, the control compositions can be used to interpret flow cytometry data.

[0034] In some embodiments, the control composition is used to perform fluorescence compensation of flow cytometry data, including, for example, correcting for fluorescence spillover by removing the signal of a given fluorescent particle (e.g., a given fluorophore or fluorescent dye) from each secondary channel or detector. In other words, the signal of a given fluorescent particle is removed from all detectors or channels of the flow cytometer except for the channel / detector specifically designated to measure the fluorescent particle. In some cases, fluorescence compensation can be performed using a subject control composition (e.g., cell viability compensation [CVC] beads) for flow cytometry cell viability assays, for example, to distinguish between live and dead cell populations in flow cytometry data. In some embodiments, the flow cytometry cell viability assay uses an amine-reactive dye to distinguish between live and dead cells. In certain embodiments, the flow cytometry cell viability assay uses a DNA-binding dye to distinguish between live and dead cells.

[0035] In some embodiments, the control composition of the present invention can be a positive control composition. The positive control composition can include positive control particles configured to bind to fluorescent particles (e.g., fluorophores) of a given assay. In some embodiments, the positive control particles are configured to bind to fluorophores of both an amine-reactive fluorescent dye-based cell viability assay and a DNA-intercalating dye-based cell viability assay (i.e., the positive control particles are configured to bind to both an amine-reactive fluorescent dye and a DNA-intercalating dye). In some cases, the positive control particles can be configured to bind to other fluorescent particles in addition to the amine-reactive fluorescent dye and the DNA-intercalating dye. For example, the positive control particles can be configured to bind to one or more different fluorescently labeled antibodies and / or one or more different fluorescently labeled nucleic acid probes in addition to the amine-reactive fluorescent dye and the DNA-intercalating dye.

[0036] As described above, embodiments of the control composition include positive control particles. The positive control particles may include or be characterized by a core, an amine-containing polymer (e.g., an aminated polymer) configured to react with an amine-reactive dye, and a nucleic acid configured to bind to a DNA-binding dye, with the aminated polymer and nucleic acid being covalently associated with the core. In some embodiments, the core of the positive control particle includes a material with low autofluorescence and / or low nonspecific binding. In some embodiments, the core of the positive control particle, e.g., when considered as a whole, exhibits substantially no autofluorescence and / or low nonspecific binding. In some embodiments, the aminated polymer and nucleic acid molecules are layered on the core. In other words, multiple aminated polymer molecules and / or nucleic acid molecules covalently associated with the core may be dispersed relatively uniformly across the surface of the core to form a coating on the core. In some cases, the nucleic acid molecules and aminated polymer molecules form separate, alternating coating layers on the surface of the core. In these examples, the positive control particle may include multiple layers of aminated polymer and nucleic acid molecule, e.g., two or more layers, or three or more layers, or four or more layers, or five or more layers, or ten or more layers, etc. In some cases, the positive control particle includes a sufficient number of layers to bind an amount of amine-reactive dye and / or DNA-binding dye (e.g., of a given cell viability assay) such that the positive control particle is at least as bright as cells stained with the same dye. In these examples, the positive control particle may include a sufficient number of layers to bind an amount of amine-reactive dye and / or DNA-binding dye equal to or greater than the amount of nonviable cells (e.g., nonviable mammalian tissue cells grown under specified conditions) in a particular cell viability assay. In some embodiments, the positive control particle may be configured to have substantially the same autofluorescence as cells in a given flow cytometry assay (e.g., a cell viability assay). In these cases, the cells of interest may be mammalian cells. In some cases, the positive control particle is a bead.

[0037] In certain embodiments, the control composition of the present disclosure may include negative control particles. In some cases, the negative control particles include a core. In some cases, the core of the negative control particles may be substantially the same as the core of the positive control particles. For example, the core of the negative control particles may include the same material(s), be the same size and / or shape, have the same properties (e.g., autofluorescence properties), etc. as the core of the positive control particles. In some embodiments, the core of the negative control particles includes a material with low autofluorescence and / or low nonspecific binding. In some embodiments, the core of the negative control particles is substantially non-autofluorescent and / or has low nonspecific binding, e.g., when considered as a whole. In certain embodiments, the negative control particles do not include aminated polymers or nucleic acids (i.e., do not include aminated polymer molecules or nucleic acid molecules). In these examples, the core of the negative control particles may be conjugated with a molecule (e.g., a hydrophilic polymer such as polyethylene glycol [PEG]) to consume surface groups that can react with amine-reactive dyes or DNA-binding dyes. In some embodiments, a low non-specific binding polymer layer or coating, such as a silane layer or a hydrophilic polymer layer (e.g., PEG, poly(vinylpyrrolidone) (PVP), poly(acrylic acid) (PAA), etc.), can be deposited on the surface of the negative control particle core. In some embodiments, the negative control particle is configured to have substantially the same autofluorescence as the positive control particle. In some embodiments, the negative control particle is configured to have substantially the same autofluorescence as the cells of a given flow cytometry assay (e.g., a cell viability assay). In these cases, the cells of interest can be mammalian cells. In some cases, the negative control particle is a bead.

[0038] A control composition (e.g., a CVC composition) can include a plurality of positive control particles. In some embodiments, a control composition can include a plurality of negative control particles. In some cases, a control composition can include a plurality of both positive and negative control particles. In certain embodiments, a control composition can include positive control particles labeled with (e.g., bound / associated with) an amine-reactive dye and / or a DNA-binding dye. In some embodiments, a control composition can be suspended in a solution (e.g., a liquid buffer). In other cases, a control composition can be substantially free of liquid (e.g., the control composition can be dehydrated or lyophilized).

[0039] In certain embodiments, the control composition of the present disclosure is a storage-stable control composition, meaning that the control composition is substantially stable over an extended period of time. "Stable" or "storage-stable" or "substantially stable" refers to a control composition containing control particles that retain their properties (e.g., autofluorescence, binding properties, etc.) and do not significantly decrease or change in reactivity over an extended period of time. For example, stable positive control particles may retain their ability to react with amine-reactive dyes and / or their ability to bind to DNA-binding dyes, and negative control particles may maintain their inactivity with respect to amine-reactive dyes and / or DNA-binding dyes, regardless of whether the negative and positive control particles are stored together. For example, a storage-stable control composition may not exhibit significant changes in dye-binding activity (of either the negative or positive control particles of the composition) due to deterioration of the control composition over an extended period of time. This stability may be reflected by no or negligible change in the fluorescence of the positive and / or negative control particles of the control composition as a result of staining with a particular fluorescent particle (e.g., an amine-reactive dye or a DNA-binding dye), e.g., a change in fluorescent activity of 10% or less, or 9% or less, or 8% or less, or 7% or less, or 6% or less, or 5% or less, or 4% or less, or 3% or less, or 2% or less, or 1% or less over time. In certain cases, a storage-stable control composition exhibits a change in fluorescent activity (e.g., dye-binding activity) of 5% or less over time.

[0040] In some cases, the storage-stable control composition substantially retains its fluorescent particle (e.g., amine-reactive dye and / or DNA-binding dye) binding activity over an extended period of time, e.g., retains 100%, or 99% or more, or 98% or more, or 97% or more, or 96% or more, or 95% or more, or 94% or more, or 93% or more, or 92% or more, or 91% or more, or 90% or more, or 85% or more, or 80% or more, or 75% or more of its activity over an extended period of time. For example, the storage-stable control composition may retain 90% or more of its binding activity over an extended period of time. In some cases, the storage-stable composition retains 95% or more of its binding activity over an extended period of time. A long-term period is a period of 1 week or more, or 2 weeks or more, or 3 weeks or more, or 1 month or more, or 2 months or more, or 3 months or more, or 4 months or more, or 6 months or more, or 9 months or more, or 1 year or more, or 1.5 years (e.g., 18 months) or more, or 2 years or more, or 2.5 years (e.g., 30 months) or more, or 3 years or more, or 3.5 years (e.g., 42 months) or more, or 4 years or more, or 4.5 years (e.g., 54 months) or more, or 5 years or more, etc. For example, a long-term period may be 6 months or more. In some cases, a long-term period is 9 months or more. In some cases, a long-term period is 1 year (e.g., 12 months) or more. In some cases, a long-term period is 1.5 years (e.g., 18 months) or more. In some cases, a long-term period is 2 years (e.g., 24 months) or more. In some examples, a long-term period is 10 years or less, e.g., 7.5 years or less, 5 years or less, e.g., 2 years or less.

[0041] Positive control particles An embodiment of the control composition includes one or more positive control particles. "Positive control particle" refers to a particle, e.g., a bead, configured to bind to amine-linked fluorescent particles and / or nucleic acid-linked fluorescent particles. "Fluorescent particle" refers to a particle, e.g., a fluorophore or quantum dot, that contains a component that absorbs light or other electromagnetic radiation and subsequently emits light (e.g., a component that absorbs light of a given wavelength and subsequently emits light of a longer wavelength). The amine-linked fluorescent particle and the nucleic acid-linked fluorescent particle can be an amine-reactive dye and a DNA-intercalating dye, respectively, used in flow cytometry cell viability assays.

[0042] As previously mentioned, flow cytometry cell viability assays involve fluorescent amine-reactive dyes and / or DNA-intercalating dyes used to stain samples containing cells. In nonviable cells (i.e., dead or dying cells), the cell membrane is damaged, allowing the dyes to enter the cell and interact with cellular components, resulting in a strong fluorescent label. Amine-reactive dyes interact with free amines inside the cell, while DNA-binding dyes penetrate the nucleus and bind to double-stranded DNA (dsDNA) (e.g., between adjacent base pairs or within the minor or major groove). In contrast, the intact cell membrane of viable cells (i.e., living / healthy cells) prevents the dyes from accessing the cell interior, resulting in a relatively weak staining of viable cells. In some cases, fluorescence intensity is measured using a particle analyzer (e.g., a flow cytometer).

[0043] In some embodiments, a control composition (e.g., a CVC composition) containing positive control particles of the present disclosure is used to determine compensation or spillover values for flow cytometry cell viability assays. In flow cytometry, compensation is performed to correct for fluorescence spillover, e.g., additional detection of fluorescent signals (e.g., emission of a particular fluorophore) in detection channels other than those specifically designated for measuring the fluorescent signal. The wavelength range of a given detection channel can be determined by the emission wavelength(s) of the fluorophore being detected, e.g., the fluorophore of an amine-reactive dye or DNA-binding dye. To correct for fluorescence spillover, positive control particles of the present disclosure can be used to determine the amount of fluorescence spillover generated by a given fluorescent particle, e.g., a given cell viability dye. In these examples, the positive control particles of the control composition can be contacted with one or more cell viability dyes (i.e., one or more amine-reactive dyes and / or DNA-binding dyes) to label the positive control particles. The labeled control composition (i.e., containing labeled positive control particles) can then be introduced into a flow cytometer to measure its fluorescence at one or more excitation wavelengths. Fluorescence measurements generated from the labeled control composition can then be used to calculate a spillover or compensation value for each fluorescent particle (e.g., cell viability dye) in the cell viability assay. In some cases, the spillover or compensation value can be calculated by determining the amount of fluorescence detected in a secondary channel (i.e., a channel other than the detection channel specifically designated for measuring a given fluorescent particle) for each fluorescent particle. The compensation / spillover value(s) can then be used to adjust the flow cytometry data collected from the labeled sample cells in the cell viability assay.

[0044] As described above, the positive control particles of the control composition include a core, an amine-containing polymer configured to react with an amine-reactive dye, and a nucleic acid configured to bind to a DNA-binding dye, wherein the aminated polymer and nucleic acid are covalently associated with the core. By "covalently associated," we mean that the aminated polymer and nucleic acid are covalently attached to the surface of the core, either directly or via a linker. In some embodiments, the linker may include one or more aminated polymer molecules and / or one or more nucleic acid molecules. In some embodiments, the aminated polymer and nucleic acid molecules are layered on the core. In other words, multiple aminated polymer molecules and / or nucleic acid molecules covalently associated with the core may be dispersed relatively uniformly across the entire surface of the core, forming a coating on the core. In these examples, the positive control particles may include multiple layers of aminated polymer and nucleic acid, for example, two or more layers, three or more layers, four or more layers, five or more layers, or ten or more layers of each.

[0045] In some examples, the aminated polymer and nucleic acid molecules are layered alternately on the core. In other words, each layer contains either one or more aminated polymer molecules or one or more nucleic acid molecules, and there are no two consecutive aminated polymer or nucleic acid layers. In other cases, the aminated polymer and nucleic acid are uniformly mixed and dispersed across the entire surface of the core. In some cases, the positive control particle includes a sufficient number of layers to bind a certain amount of amine-reactive dye and / or DNA-binding dye (e.g., for a given cell viability assay), such that the positive control particle is at least as bright as cells stained with the same dye (e.g., during a cell viability assay). In these examples, the cells may be, for example, mammalian cells. In these examples, the positive control particle may include a sufficient number of layers to bind an amount of amine-reactive dye and / or DNA-binding dye equivalent to or greater than that of nonviable cells of a particular cell viability assay (e.g., nonviable mammalian tissue cells grown under specific conditions). In some embodiments, the positive control particle may be configured to have substantially the same autofluorescence as the cells of a given flow cytometry assay.

[0046] As described above, embodiments of the control particles of the present disclosure include a core. In some cases, the core of the control particle is substantially non-autofluorescent when exposed to light having a wavelength, for example, in the range of about 350 nm to about 1000 nm. "Substantially non-autofluorescent" means that when illuminated with light sufficient to cause fluorescent emission from the fluorescent particle (e.g., a cell viability dye), the core emits less fluorescence than cells labeled with the fluorescent particle or control particles. In other words, the core of the present disclosure is configured such that fluorescent events arising from the fluorescent particle (e.g., a labeled positive control particle or sample cell) can be easily and reliably distinguished from background fluorescence emanating from the core. Therefore, fluorescence inherently emanating from the core (i.e., core autofluorescence) does not interfere with accurately determining spillover values of fluorescent particles used in flow cytometry assays (e.g., spillover values of cell viability dyes in flow cytometry cell viability assays).

[0047] In some embodiments, the control composition of the present disclosure is configured such that, when used to determine compensation or spillover value(s) for a flow cytometry cell viability assay, the intrinsic fluorescence of the positive control particle (e.g., including the intrinsic fluorescence of the substantially non-autofluorescent core) does not substantially contribute to fluorescence spillover. In some cases, the core comprises a low-autofluorescent material that does not fluoresce or substantially does not fluoresce within a predetermined wavelength range used to calculate spillover value(s) for a fluorescent particle of interest (e.g., a cell viability dye in a cell viability assay). In certain embodiments, the core comprises a low-autofluorescent material that does not fluoresce or substantially does not fluoresce when excited with ultraviolet (UV) light (e.g., about 320 nm to 380 nm) or violet light (e.g., about 390 nm to 420 nm).

[0048] In some embodiments, the cores of the present disclosure exhibit low nonspecific binding. "Low nonspecific binding" means that the core exhibits low affinity (e.g., a relatively high dissociation constant when interacting with) undesirable biological materials (e.g., proteins, polysaccharides, nucleic acids, cells, etc.) and non-biological materials (e.g., silica, cationic polymers, plastics, etc.), such that undesirable materials rarely or never bind to, interact with, or hybridize with the core. In some embodiments, the cores of the present disclosure exhibit low nonspecific binding and substantially no interaction with fluorescent particles (e.g., stains, dyes, labeled antibodies, etc.) in flow cytometry assays (e.g., low nonspecific binding to cell viability dyes in cell viability assays). For example, the dissociation constant of the interaction between the core and an amine-reactive dye can be at least one order of magnitude greater than the dissociation constant of the interaction between an aminated polymer (e.g., those described herein) and an amine-reactive dye. In some embodiments, the dissociation constant of the interaction between the core and the DNA-binding dye can be at least one order of magnitude greater than the dissociation constant of the interaction between a nucleic acid (e.g., those described herein) and the DNA-binding dye. In some embodiments, the core has low non-specific binding to, and does not substantially interact with, any materials used to prepare samples for flow cytometry analysis.

[0049] Cores of the present disclosure may include, but are not limited to, microparticles, beads (e.g., microbeads, magnetic beads, ion torrent beads, flow cytometry beads), or microspheres. Cores of the present disclosure may include any material that allows the core to be substantially non-autofluorescent (e.g., at the excitation wavelength of the fluorescent particles of the flow cytometry assay of interest) and have low nonspecific binding (e.g., to the fluorescent particles, cells, reagents, and other control particles used in the flow cytometry assay of interest). In some embodiments, the core may include a polymer (e.g., plastic, polydimethylsiloxane [PDMS], polystyrene, polypropylene, agarose, gelatin, hydrogel, methylstyrene, acrylic polymer, latex, Sepharose, cellulose, nylon, silicone, poly[methyl methacrylate] [PMMA], hydrogel, polyacrylamide hydrogel, polysulfone, polyethylen, etc.), ceramic, glass, metal (e.g., titanium, gold, etc.), silica, or any combination thereof. In certain embodiments, the core can consist essentially of silica, PMMA, and / or polyacrylamide hydrogel. In some embodiments, the core can be silica beads. In other embodiments, the core can be PMMA beads or polyacrylamide hydrogel beads.

[0050] The core of the present disclosure can be of any suitable size or shape. In some cases, the size of the core is selected to approximately match the size of the cells to be analyzed, for example, using a flow cytometry cell viability assay. In some embodiments, the diameter of the core is about 0.1 to about 150 microns, e.g., about 0.1 to 0.5 microns, or about 0.5 to 1 micron, or about 1 to 1.5 microns, or about 1.5 to 5 microns, or about 5 to 7 microns, or about 7 to 10 microns, or about 10 to 25 microns, or about 25 to 50 microns, or about 50 to 100 microns, or about 100 to 150 microns. In certain embodiments, the core is about 6 microns. The shape of the core can be, but is not limited to, a sphere, a disk, an ellipse, a cube, a cylinder, a pyramid, an irregular shape, or any other suitable shape. In some embodiments, the core is solid. In other embodiments, the core is hollow.

[0051] As described above, fluorescent particle-binding molecules (e.g., aminated polymer molecules and / or nucleic acid molecules) can be conjugated to the surface of the core of the present disclosure. In some embodiments, the fluorescent particle-binding molecules are directly and covalently bound to the core. In some cases, the fluorescent particle-binding molecules are indirectly covalently bound to the core, for example, via a linker. In some embodiments, the linker can include one or more fluorescent particle-binding molecules (e.g., aminated polymer molecules and / or nucleic acid molecules). For example, aminated polymer molecules can be covalently bound to nucleic acid molecules, which are covalently bound to the surface of the core, either directly or via a linker. In some embodiments, the core can be modified to include chemical groups to facilitate conjugation of fluorescent particle-binding molecules. Suitable modifications and linkers are further described below. In certain embodiments, the fluorescent particle-binding molecules conjugated to the surface of the core are configured to bind to cell viability assay dyes, such as amine-reactive dyes or DNA-binding dyes, and can include aminated polymers and / or nucleic acids, as discussed above and further described below. In some embodiments, molecules configured to reduce binding of undesired substances (e.g., undesired biological or non-biological compounds, stains, dyes, antibodies, or other fluorescent or labeling molecules) are conjugated to the surface of the core. Such molecules can include, but are not limited to, polyethylene glycols (PEGs) of various molecular weights and branching structures, zwitterionic polymers, poly(hydroxy-functional acrylates), poly(2-oxazolines), poly(vinylpyrrolidone), poly(glycerol), peptides, proteins, and polysaccharides, polyacrylamides, polyesters, or any combination thereof.

[0052] As described above, embodiments of the positive control particle of the present disclosure include a core covalently associated with a molecule containing an amine configured to react with an amine-reactive dye. Any molecule or combination of molecules containing an amine (e.g., a primary, secondary, or tertiary amine) capable of binding with an amine-reactive dye may be used in the positive control particle. In some cases, the molecule is an aminated polymer. "Aminized polymer" simply means a polymer having one or more amines. In certain embodiments, one or more aminated polymers of the positive control particle each contain multiple amines. In these cases, the aminated polymer(s) may be configured to prevent self-quenching of the amine-reactive dye. For example, the aminated polymer may be selected so that its structure does not contribute to self-quenching; for example, the aminated polymer may include sufficient spacing between each amine to prevent self-quenching of the amine-reactive dye.

[0053] In some embodiments, the one or more aminated polymers of the positive control particle include a cationic polymer, such as a polysaccharide. In some cases, the cationic polymer includes one or more of branched polyethyleneimine, polyallylamine, polylysine, or chitosan. In some embodiments, the aminated polymer includes a polypeptide, such as a protein. In these cases, the protein may include arginine and / or lysine residues. In some embodiments, the protein is a histone or myelin basic protein (MBP). In some cases, the protein may be configured to bind to other fluorescent particles in addition to the amine-reactive dye. For example, the protein may be configured to bind to a specific binding member conjugated to a fluorophore, and the specific binding member may include, for example, an antibody. As used herein, the term "antibody" refers to a protein consisting of one or more polypeptides substantially encoded by all or part of a recognized immunoglobulin gene. In some cases, the protein may be an epitope of an antigen to which the fluorescent particle comprising the antibody is configured to bind. In some embodiments, the positive control particle may include two or more different aminated polymers, such as two or more different proteins. For example, a positive control particle may contain five layers of aminated polymer molecules, four of the aminated polymer layers containing histone proteins, and the outermost aminated polymer layer containing CD proteins.

[0054] In some embodiments, one or more amines of each aminated polymer are configured to react with an amine-reactive dye that flows freely in solution. In some embodiments, the amine-reactive dye has an emission maximum wavelength in the range of 350 nm to 900 nm. For example, in some embodiments, the amine-reactive dye has an emission maximum wavelength in the range of 400 nm to 900 nm, or 400 nm to 850 nm. In some embodiments, the amine-reactive dye has an absorption maximum (excitation maximum) wavelength in the range of 300 nm to 1000 nm. For example, in some embodiments, the amine-reactive dye has an absorption maximum wavelength in the range of 300 nm to 900 nm, or 300 nm to 800 nm. In some embodiments, the amine-reactive dye includes a BD Horizon™ reagent, such as the BD Horizon™ Fixable Viability Stain (FVS) reagent. In some cases, the amine-reactive dye includes at least one dye selected from the group consisting of BD Horizon™ Fixable Viability Stain, Biolegend Zombie™ dye, Thermo Fischer eFluor™ dye, Thermo Fischer LIVE / DEAD™ dye, Proteintech Phantom Dye, and Tombo Biosceinces Ghost Dye™.

[0055] As previously described, embodiments of the positive control particles of the present disclosure include a core covalently associated with a nucleic acid configured to bind to a DNA-binding dye. Any nucleic acid or combination of nucleic acids (e.g., DNA, ribonucleic acid [RNA], or peptide nucleic acid [PNA]) of any size that can bind to a DNA-binding dye (e.g., a DNA-intercalating dye) can be used in the positive control particles. In certain embodiments, the one or more nucleic acids of the positive control particles have a size ranging from 200 base pairs to 1000 base pairs. In certain embodiments, the one or more nucleic acids of the positive control particles have a size ranging from 300 base pairs to 700 base pairs, e.g., from 400 base pairs to 600 base pairs, e.g., from 450 base pairs to 550 base pairs. In some embodiments, the one or more nucleic acids of the positive control particles are single-stranded. In certain embodiments, the one or more nucleic acids of the positive control particles are double-stranded.

[0056] In some embodiments, the one or more nucleic acids of the control particle each consist essentially of DNA. In some embodiments, the DNA comprises double-stranded DNA (dsDNA). In some embodiments, the DNA comprises a naturally occurring DNA sequence, such as a DNA sequence obtained from salmon. In some embodiments, the DNA comprises a synthetic DNA sequence. In some cases, the nucleic acid (e.g., a dsDNA molecule) can be configured to bind to a DNA-intercalating dye. In some embodiments, the nucleic acid can be configured to bind to other fluorescent particles in addition to the DNA-intercalating dye. For example, the nucleic acid (e.g., a dsDNA molecule) can be configured to bind to a specific binding member conjugated to a fluorophore, which can include, for example, a nucleic acid-binding probe. In other words, the nucleic acid can include a dsDNA having a sequence configured to bind to a specific genetic probe (e.g., a single-stranded DNA or RNA fragment conjugated to a fluorophore). In some embodiments, the positive control particle can include two or more different nucleic acids, such as, for example, two or more dsDNA molecules each containing a different sequence. For example, a positive control particle may contain five layers of nucleic acid molecules, four of the nucleic acid layers containing dsDNA sequences derived from salmon, and the outermost nucleic acid layer containing a dsDNA sequence substantially identical to all or a portion of a gene of interest.

[0057] In some embodiments, one or more nucleic acids are configured to bind to a DNA-binding dye that flows freely in solution. In some embodiments, the DNA-binding dye has an emission maximum wavelength in the range of 350 nm to 900 nm. For example, in some embodiments, the DNA-binding dye has an emission maximum wavelength in the range of 400 nm to 900 nm, or 400 nm to 850 nm. In some embodiments, the DNA-binding dye has an absorption maximum (excitation maximum) wavelength in the range of 300 nm to 1000 nm. For example, in some embodiments, the DNA-binding dye has an absorption maximum wavelength in the range of 300 nm to 900 nm, or 300 nm to 800 nm. In some embodiments, the DNA-binding dye includes propidium iodide (PI), 7-amino-actinomycin D (7-AAD), and / or 4',6-diaminidino-2-phenylindole (DAPI). In some cases, the DNA binding dye includes at least one dye selected from the group consisting of 7-AAD, DAPI, propidium iodide, Hoechst dye, ethidium bromide, LDS751, Thermo Fischer Sytox™ dye, Thermo Fischer T-PRO™ dye, Thermo Fischer TOTO™ dye, Thermo Fischer YO-PRO™ dye, Biolegend Helix-NP™ dye, Biotium RedDot™ dye, and Biostatus Limited DRAQ™ dye.

[0058] As noted above, embodiments of the positive control particles of the present disclosure include a core (e.g., as described herein) covalently associated with a nucleic acid and an aminated polymer. In some embodiments, the nucleic acid and / or aminated polymer are directly covalently attached to the surface of the core. In some cases, the nucleic acid and / or aminated polymer are indirectly covalently attached to the surface of the core, e.g., via a linker. In some embodiments, the linker can include one or more fluorescent particle-binding molecules (e.g., aminated polymer molecules and / or nucleic acid molecules). For example, the aminated polymer molecule can be covalently attached to a nucleic acid molecule, and the nucleic acid molecule is covalently attached to the surface of the core, either directly or via a linker.

[0059] The linker employed may include, for example, one or more reactive groups configured to stably associate two components of the positive control particle together. For example, suitable linkers may include, but are not limited to, carboxyl-to-amine reactive groups, amine reactive groups, sulfhydryl reactive groups, aldehyde reactive groups, photoreactive groups, or hydroxyl reactive groups. In some embodiments, the linker may be a reactive linker pair (e.g., provided by the linker and the chemical groups of, for example, the core, aminated polymer, or nucleic acid described herein) such as maleimide / thiol, thiol / thiol, pyridyldithiol / thiol, succinimidyl iodoacetate / thiol, N-succinimidyl ester (NHS ester), sulfodichlorophenol ester (SDP ester), or pentafluorophenyl ester (PFP ester) / amine, bissuccinimidyl ester / amine, imidoester / amine, hydrazine or amine / aldehyde, dialdehyde or benzaldehyde, isocyanate / hydrogen. Employ reactive linking chemistries including, but not limited to, alkoxyl or amine, carbohydrate-periodate / hydrazine or amine, diazirine / aryl azide chemistry, pyridyldithiol / aryl azide chemistry, alkyne / azide, carboxy-carbodiimide / amine, amine / sulfo-SMCC (sulfosuccinimidyl 4-[N-maleimidomethyl]cyclohexane-1-carboxylate) / thiol and amine / BMPH (N-[β-maleimidopropionic acid]hydrazide.TFA) / thiol, azide / triarylphosphine, nitrone / cyclooctyne, azide / tetrazine and formylbenzamide / hydrazino-nicotinamide. In certain embodiments, the linker employs a cycloaddition reaction such as a [1+2] cycloaddition, a [2+2] cycloaddition, a [3+2] cycloaddition, a [2+4] cycloaddition, a [4+6] cycloaddition, or a cheletropic reaction, including linkers that undergo a 1,3-dipolar cycloaddition (e.g., an azide-alkyne Huisgen cycloaddition), a Diels-Alder reaction, an inverse electron demand Diels-Alder cycloaddition, an ene reaction, or a [2+2] photochemical cycloaddition reaction.In some embodiments, the linker can comprise an alkyl chain, an alkoxy chain, an alkenyl chain, or an alkynyl chain, and the number of carbon atoms in the chain can vary, in some instances, from 2 to 25, e.g., from 5 to 20, and one or more carbon atoms are replaced with, e.g., NH or CH3-N, as reactive functional groups for covalent bonding.

[0060] In some embodiments, the aminated polymer and nucleic acid are layered on the core. In some embodiments, multiple aminated polymer and / or nucleic acid coating layers are covalently associated with the core. For example, the particle may contain five or more aminated polymer layers and / or five or more nucleic acid layers. In some embodiments, the aminated polymer coating layers alternate with nucleic acid coating layers. In other words, each layer contains either one or more aminated polymer molecules or one or more nucleic acid molecules, and there are no two consecutive aminated polymer or nucleic acid layers. In these examples, the positive control particle may contain multiple layers of aminated polymer and nucleic acid, for example, two or more, three or more, four or more, five or more, or ten or more layers of each. In other cases, the aminated polymer and nucleic acid are uniformly mixed and dispersed over the entire surface of the core. In some cases, the positive control particles comprise a sufficient number of layers to bind a certain amount of amine-reactive dye and / or DNA-binding dye (e.g., for a given cell viability assay), such that the positive control particles are at least as bright as cells stained with the same dye (e.g., during a cell viability assay). In these examples, the positive control particles may comprise a sufficient number of layers to bind at least the same amount of amine-reactive dye and / or DNA-binding dye as nonviable cells of a particular cell viability assay (e.g., nonviable mammalian tissue cells grown under particular conditions).

[0061] Negative control particles In certain embodiments, the control composition of the present disclosure may include one or more negative control particles. "Negative control particles" refers to particles, e.g., beads, configured to have a lower affinity (e.g., a higher dissociation constant when interacting with) for the amine-linked fluorescent particles and / or nucleic acid-linked fluorescent particles to which corresponding positive control particles (e.g., as described above) are configured to bind, compared to the positive control particles. In some embodiments, the negative control particles are configured to have substantially the same autofluorescence (i.e., the same intrinsic fluorescence) as the positive control particles. In some embodiments, the negative control particles are configured to have substantially the same autofluorescence as the cells of a given flow cytometry assay (e.g., a cell viability assay). In these cases, the cells of interest may be mammalian cells. In some cases, the negative control particles are beads.

[0062] In some examples, the negative control particle comprises a core. In some cases, the core of the negative control particle may be substantially the same as the core of the positive control particle. For example, the core of the negative control particle may comprise the same material(s), be the same size and / or shape, have the same properties (e.g., autofluorescence properties), etc. as the core of the positive control particle. In some embodiments, the core of the negative control particle may be essentially the same as the core of the positive control particle. In some embodiments, the core of the negative control particle comprises a material that has low autofluorescence and / or low nonspecific binding. In some embodiments, the core of the negative control particle is substantially non-autofluorescent and / or has low nonspecific binding, e.g., when considered as a whole.

[0063] In certain embodiments, the negative control particles do not contain aminated polymers or nucleic acids (i.e., do not contain aminated polymer or nucleic acid molecules described herein). In some cases, the negative control particles contain a polymer (e.g., as described above) that has substantially the same autofluorescence as the aminated polymer of the positive control, and the negative control polymer does not react with or bind to an amine-reactive dye. For example, the negative control particles may contain a protein with substantially fewer lysine and arginine residues than the aminated polymer of the positive control particles. In some cases, the negative control particles contain a nucleic acid (e.g., as described above) that has substantially the same autofluorescence as the nucleic acid of the positive control, and the negative control nucleic acid does not react with or bind to a DNA-binding dye. For example, the negative control particles may contain a nucleic acid that is substantially free of GC base pairs, and the DNA-binding dye may be 7-AAD. In other embodiments, the negative control nucleic acid is modified to prevent binding of the DNA-binding dye or is a different type of nucleic acid than the positive control nucleic acid. For example, the negative control nucleic acid may be a PNA, and the positive control nucleic acid may be DNA.

[0064] As described above, in some embodiments, negative control particles contain a core but do not contain polymers or nucleic acids. In these examples, the core of the negative control particle can be conjugated with a molecule to consume surface groups that can react with amine-reactive dyes or DNA-binding dyes. Such molecules can include, but are not limited to, polyethylene glycol (PEG) of various molecular weights and branched structures, zwitterionic polymers, poly(hydroxy-functional acrylates), poly(2-oxazolines), poly(vinylpyrrolidone), poly(glycerol), peptides, proteins, and polysaccharides, or any combination thereof. In some cases, a low nonspecific binding polymer layer or coating, such as a silane layer or a hydrophilic polymer layer (e.g., PEG, poly(vinylpyrrolidone) (PVP), poly(acrylic acid) (PAA), poly(carboxybetaine acrylamide) (PCBAA), etc.), can be deposited on the surface of the negative control particle core.

[0065] Figure 1 illustrates the mechanisms of two types of viability staining according to embodiments of the present invention. At the top, two cells are depicted before incubation with cell viability dyes. On the left, a viable cell with an intact membrane (i.e., a living, healthy cell) is shown, while on the right, a nonviable cell with an intact membrane (i.e., a dead or dying cell) is shown. Next, both the viable and nonviable cells are incubated with an amine-reactive dye (center left) and a DNA-intercalating dye (center right) to distinguish between viable and nonviable cells. At the bottom, viable and nonviable cells are shown after incubation with either the amine-reactive dye or the DNA-intercalating dye. In the amine-reactive dye-based assay (bottom left), the amine-reactive dye binds to amines on proteins in the intact membrane of viable cells but cannot enter viable cells and bind to free amines inside the cells. In contrast, in the case of nonviable cells, the amine-reactive dye binds to both amines in the damaged membrane and amines inside the cells, resulting in higher fluorescence intensity from the amine-reactive dye in nonviable cells. In the DNA-intercalating dye-based assay (bottom right), the DNA-intercalating dye cannot enter viable cells and does not bind to dsDNA in the cell nucleus. In contrast, in nonviable cells, the DNA-intercalating dye penetrates all the way to the cell nucleus and binds directly to the dsDNA molecules, resulting in higher fluorescence intensity from the DNA-intercalating dye in nonviable cells. Therefore, both the amine-reactive dye-based assay and the DNA-intercalating dye-based assay can distinguish between viable and nonviable cells.

[0066] FIG. 2 provides a representation of a positive CVC bead according to an embodiment of the present invention. Positive CVC bead 200 includes core 201 consisting essentially of silica, coating layer 202 of DNA molecules configured to bind to a DNA-binding dye, and coating layer 203 of aminated polymer molecules configured to bind to an amine-reactive dye. DNA coating 202 includes multiple dsDNA sequences, each approximately 500 base pairs in length, spread relatively uniformly across the surface of core 201. DNA coating 202 is covalently attached to the surface of core 201, either directly or via a linker, while aminated polymer coating 203 is covalently associated with core 201 through covalent bonds formed with DNA coating 202. Aminated polymer coating 203 includes multiple amine-rich histone proteins spread relatively uniformly across DNA coating 202.

[0067] For illustrative purposes, dsDNA molecules 204 used to attach DNA-binding dyes and amine groups 205 used to attach amine-reactive dyes are depicted on the outside of the particle. Both the dsDNA molecules of DNA coating 202 and the histone proteins of aminated polymer coating 203 are positioned such that a freely flowing dye in liquid solution can diffuse close enough for the dsDNA molecules and histone proteins (and their amines) to interact with the freely flowing dye. This allows the amines (e.g., 205) of aminated polymer coating 203 to bind to the amine-reactive dye in liquid solution and the dsDNA molecules (e.g., 204) of DNA coating 202 to bind to the DNA-binding dye in liquid solution.

[0068] Manufacturing method Aspects of the present disclosure also include methods of manufacturing (i.e., synthesizing or producing) the control particles described herein. In certain embodiments, the manufacturing methods can be used to manufacture positive control particles. In certain embodiments, the manufacturing methods can be used to manufacture negative control particles. In some embodiments, one or more steps of the methods can be used to produce both the positive control particles and negative control particles of the present disclosure. In certain embodiments, the particle synthesis method includes obtaining a core and covalently associating a polymer comprising an amine and a nucleic acid with the core to, for example, produce a positive control particle.

[0069] In certain embodiments, the surface of the core includes a functional group, such as, for example, an amide, maleimide, or thiol group. In some embodiments, the method further includes surface functionalizing the core with a functional group. For example, the resulting core can include silica, and the functionalization can include amination. In some embodiments, the method further includes modifying the functional group of the core to include a reactive functional group, e.g., using a reagent. For example, an aminated silica core can be modified to include a thiol group, e.g., using a thiolating reagent such as 2-iminothiolane. In some embodiments, the method further includes positively charging the core, e.g., when the core is covalently attached to a nucleic acid molecule (prior to association with an aminated polymer as described herein). In some embodiments, the method further includes negatively charging the core, e.g., when the core is covalently attached to an aminated polymer (prior to association with a nucleic acid as described herein).

[0070] In certain embodiments, the nucleic acid includes deoxyribonucleic acid (DNA), such as double-stranded DNA (dsDNA). In some embodiments, the particle synthesis method further includes modifying the DNA to include a functional group reactive with the functional group of the core and / or the functional group of the aminated polymer. For example, the core and / or the aminated polymer can be modified to include a thiol group, and the DNA can be modified to include a maleimide group. In some embodiments, the particle synthesis method further includes modifying the DNA to include a functional group reactive with the reactive chemistry of the linker (i.e., the linker is further configured to bind to the core and / or the aminated polymer). In some embodiments, the nucleic acid is covalently associated with the core by reacting a functional group of the DNA with a reactive functional group of the core and / or a linker associated with the core. In some embodiments, the nucleic acid is covalently associated with the core by reacting a functional group of the DNA with a functional group of the aminated polymer and / or a linker associated with the aminated polymer. In some embodiments, the 5' and / or 3' ends of the DNA are functionalized. In some embodiments, the DNA is dsDNA, and both strands of the dsDNA are functionalized. In some embodiments, the dsDNA is covalently attached to the core by reacting a functional group on the first strand of the dsDNA with a reactive functional group on the core (or its linker), and the dsDNA is covalently attached to the aminated polymer by reacting a functional group on the second strand of the dsDNA with a functional group on the aminated polymer (or its linker). In some embodiments, both strands of the dsDNA are covalently attached to the aminated polymer.

[0071] In certain embodiments, the particle synthesis method further includes modifying the aminated polymer to include a functional group reactive with a functional group of the core and / or a functional group of the nucleic acid. For example, the core and / or nucleic acid can be modified to include a maleimide group, and the aminated polymer can be modified to include a thiol group. In some embodiments, the particle synthesis method further includes modifying the aminated polymer to include a functional group reactive with the reactive chemistry of the linker (i.e., the linker is further configured to bind to the core and / or nucleic acid). In some embodiments, the aminated polymer is covalently associated with the core by reacting the functional group of the aminated polymer with a reactive functional group of the core and / or a linker associated with the core. In some embodiments, the aminated polymer is covalently associated with the core by reacting the functional group of the aminated polymer with a functional group of the nucleic acid and / or a linker associated with the nucleic acid. In some embodiments, the aminated polymer is functionalized with two or more functional groups. For example, the aminated polymer can be a protein, and two or more lysine residues of the protein can be thiolated, for example, using 2-iminothiolane. In some embodiments, the aminated polymer is covalently attached to a core by reacting a first functional group of the aminated polymer with a reactive functional group of the core (or a linker thereof), and the aminated polymer is covalently attached to a nucleic acid by reacting a second functional group of the aminated polymer with a functional group of the nucleic acid (or a linker thereof). In some embodiments, two or more functional groups of the aminated polymer are covalently attached to a nucleic acid.

[0072] In certain embodiments, the aminated polymer and nucleic acid are covalently associated with the core by alternately layering the aminated polymer and nucleic acid coatings. For example, the nucleic acid may comprise dsDNA and be modified to include a maleimide group at the 5' end of each strand of the dsDNA; the aminated polymer may comprise a protein and be modified to include two or more thiol groups; and the core may comprise multiple thiol groups (e.g., after modification). Layering may then include reacting the maleimide at the first 5' end of the first nucleic acid with the thiol group of the core, then reacting the maleimide at the second 5' end of the first nucleic acid with the first thiol group of the first aminated polymer, then reacting the second thiol group of the first aminated polymer with the maleimide at the first 5' end of the second nucleic acid, then reacting the maleimide at the second 5' end of the second nucleic acid with the first thiol group of the second aminated polymer, etc. In some embodiments, five or more rounds of layering are performed, such that the control particle includes five or more layers of nucleic acids and five or more layers of aminated polymers, hi some embodiments, each round of layering includes covalently associating multiple nucleic acids and multiple aminated polymers to the core.

[0073] In certain embodiments, the control particles are positive control particles. In some embodiments, the particle synthesis method further comprises incubating the positive control particles in a solution containing multiple amine-reactive dyes and / or DNA-binding dyes to create labeled positive control particles. In certain embodiments, the control particles are negative control particles. In some embodiments, the particle synthesis method further comprises conjugating one or more polyethylene glycol (PEG) compounds (or other hydrophilic polymers described above) to amines of the negative control particles. In some embodiments, the negative control particles do not contain aminated polymers or nucleic acids. In some embodiments, the synthesis of the negative control particles comprises conjugating one or more polyethylene glycol (PEG) compounds (or other hydrophilic polymers) to one or more amine groups of the core to prevent the amine-reactive dyes from reacting with the core.

[0074] FIG. 3 illustrates a method for synthesizing the positive control beads of FIG. 2 according to an embodiment of the present invention. In step 300, for example, silica beads are produced and surface functionalized to stably associate amines with the surface of the silica beads, resulting in an aminated silica core. In step 310, the amines of the silica beads are modified with a thiolation reagent 311 (i.e., 2-iminothiolane), thereby causing the beads to contain thiol groups. The thiolated silica beads are then positively charged, resulting in a thiolated, positively charged core 312.

[0075] In step 320, dsDNA molecules are generated using DNA sequences obtained, for example, from salmon. The dsDNA molecules are then modified to include maleimide functional groups that react with the thiol groups of the core at the 5' or 3' ends of each individual strand of the dsDNA molecule. Multiple maleimide-functionalized dsDNA molecules 321 are then covalently attached to the thiolated, positively charged core, producing a silica core with a single coating layer 322 of dsDNA molecules.

[0076] In step 330, histone proteins 331 are obtained, for example, by purifying a mammalian cell lysate. The histone proteins are then modified with a thiolation reagent 332 (i.e., 2-iminothiolane), thereby including thiol groups on the proteins. A plurality of thiolated histone proteins 333 are then covalently attached to the remaining maleimide groups of the plurality of dsDNA molecules bound to the silica core, producing a silica core having a single coating layer of dsDNA molecules and a single layer of histone molecules 334. In step 340, step 320 is repeated to attach additional dsDNA coating layers to the remaining thiol groups of the plurality of histone proteins in the outermost histone layer, and step 330 is repeated to attach additional histone coating layers to the remaining maleimide groups of the plurality of dsDNA molecules in the outermost histone layer, producing positive control beads having five aminated polymer (i.e., histone) coating layers and five nucleic acid (i.e., dsDNA) coating layers 341.

[0077] How to use Aspects of the present disclosure also include methods of using the subject control compositions. As described above, the control composition can include a plurality of positive control particles, a plurality of negative control particles, or a plurality of both positive and negative control particles. Thus, methods of using the control composition can include using the composition to verify the functionality or confirm the performance of particle analysis systems (e.g., flow cytometry systems), protocols, and reagents used in flow cytometry assays. In some embodiments, the methods include using the control composition to interpret flow cytometry data. In these examples, the methods can include using the control composition to perform fluorescence compensation of the flow cytometry data.

[0078] In some embodiments, the control composition is used for fluorescence compensation, e.g., by correcting for fluorescence spillover by removing the signal of a given fluorescent particle (e.g., a given fluorophore or fluorescent dye) from each secondary channel or detector. In other words, the control composition is used to remove the signal of a given fluorescent particle from all detectors or channels of a particle analyzer (e.g., a flow cytometer) except for the channel / detector specifically designated to measure the fluorescent particle. In some cases, fluorescence compensation may be performed on a flow cytometry cell viability assay, e.g., to distinguish between live and dead cell populations in flow cytometry data. In some embodiments, the flow cytometry cell viability assay is performed using an amine-reactive dye to distinguish between live and dead cells. In certain embodiments, the flow cytometry cell viability assay is performed using a DNA-binding dye (e.g., a DNA-intercalating dye) to distinguish between live and dead cells.

[0079] In some embodiments, a control composition including the positive control particles of the present disclosure is used to determine a compensation or spillover value for a flow cytometry cell viability assay. In some cases, the wavelength range that a given detection channel is configured to detect may be determined by the emission wavelength(s) of the fluorophore being detected, e.g., the fluorophore of an amine-reactive dye or DNA-binding dye. To correct for fluorescence spillover, the positive control particles of the present disclosure may be used to determine the amount of fluorescence spillover generated by a given fluorescent particle, e.g., a given cell viability dye. In these examples, the positive control particles of the control composition may be contacted with one or more cell viability dyes (i.e., one or more amine-reactive dyes and / or DNA-binding dyes) to label the positive control particles. The labeled control composition (i.e., including the labeled positive control particles) may then be introduced into a flow cytometer to measure its fluorescence at one or more excitation wavelengths. Fluorescence measurements generated from the labeled control composition may then be used to calculate a spillover or compensation value for each fluorescent particle (e.g., cell viability dye) in the cell viability assay. In some cases, spillover or compensation values may be calculated by determining the amount of fluorescence detected in a secondary channel (i.e., a channel other than the detection channel specifically designated for measuring a given fluorescent particle) for each fluorescent particle. The compensation / spillover value(s) may then be used to adjust flow cytometry data collected from labeled sample cells in a cell viability assay. In some embodiments, the labeled control composition includes, for example, negative control particles having substantially the same autofluorescence as positive control particles and / or cells in a given flow cytometry assay (e.g., a cell viability assay). In these cases, fluorescence measurements generated from the negative control particles may be used to adjust or correct fluorescence measurements generated from the positive control particles before calculating the compensation / spillover value(s).

[0080] In certain embodiments, the subject control compositions (e.g., the negative and positive control particles of the present invention) can be used to perform fluorescence minus one (FMO) controls. For example, a flow cytometry assay can include three or more fluorescent particles, i.e., one or more cell viability dyes (i.e., amine-reactive dyes or DNA-binding dyes) and one or more other fluorescent particles (e.g., antibody-fluorophore conjugates, nucleic acid probes, etc.). In these cases, a population of positive control particles can be generated for each fluorescent particle, such that the control composition includes positive control particles configured to bind to all fluorescent particles in the assay except one of each fluorescent particle. The control composition can then be labeled (i.e., by incubating with all dyes in the assay), and the labeled control composition can then be introduced into a flow cytometer to measure its fluorescence at one or more excitation wavelengths. Fluorescence measurements generated from the labeled control composition can then be used to calculate spillover or compensation values for each fluorescent particle (e.g., cell viability dye) in the cell viability assay.

[0081] Sample analysis As described above, the subject control compositions can be used to analyze cell samples, such as, for example, performing a flow cytometry cell viability assay to determine viable and nonviable cells in a sample. Cells that may be present in a sample include eukaryotic cells (e.g., mammalian cells) and / or prokaryotic cells (e.g., bacterial or archaeal cells). The sample can be obtained from an in vitro source (e.g., a cell suspension from laboratory cells grown in culture) or an in vivo source (e.g., a mammalian subject, a human subject, etc.). In some embodiments, the cell sample is obtained from an in vitro source. In vitro sources include, but are not limited to, prokaryotic (e.g., bacterial, archaeal) cell cultures, environmental samples containing prokaryotic and / or eukaryotic (e.g., mammalian, proteus, fungal, etc.) cells, eukaryotic cell cultures (e.g., established cell line cultures, known or purchased cell line cultures, immortalized cell line cultures, primary cell cultures, laboratory yeast cultures, etc.), tissue cultures, etc. In vitro sources may further include biological tissues, including both healthy and diseased tissues (e.g., cancerous, malignant, necrotic, etc.), such as portions of the skin, respiratory system, gastrointestinal system, cardiovascular system, genitourinary tract, tumors, organs, etc. In other examples, cells of a cell sample may be obtained from biologically produced bodily fluids, such as blood, mucus, lymphatic fluid, synovial fluid, cerebrospinal fluid, saliva, bronchoalveolar lavage fluid, amniotic fluid, amniotic cord blood, urine, vaginal fluid, and / or semen.

[0082] In certain embodiments, the source of the sample is a "mammal" or "mammalian," which terms are used broadly to refer to organisms belonging to the mammalian class, including carnivores (e.g., dogs and cats), rodents (e.g., mice, guinea pigs, and rats), and primates (e.g., humans, chimpanzees, and monkeys). In some examples, the subject is a human. The methods may be applied to samples obtained from human subjects of either sex and at any stage of development (i.e., newborn, infant, juvenile, adolescent, adult), and in certain embodiments, the human subject is a juvenile, adolescent, or adult. While the present invention may be applied to samples from human subjects, it will be understood that the methods may also be performed on samples from other animal subjects (i.e., "non-human subjects"), such as, but not limited to, birds, mice, rats, dogs, cats, livestock, and horses.

[0083] In certain embodiments, the control composition employed in the methods of the present invention can be employed in a flow cytometry protocol (e.g., for analyzing a sample as described above). When performing such a method, the sample and control composition (e.g., in the flow stream of a flow cytometer) are illuminated with light from a light source. In some embodiments, the light source is a broadband light source that emits light having a wide range of wavelengths, e.g., ranging from 50 nm or greater, e.g., 100 nm or greater, e.g., 150 nm or greater, e.g., 200 nm or greater, e.g., 250 nm or greater, e.g., 300 nm or greater, e.g., 350 nm or greater, e.g., 400 nm or greater, and 500 nm or greater. For example, one suitable broadband light source emits light having a wavelength between 200 nm and 1500 nm. Other examples of suitable broadband light sources include light sources that emit light having a wavelength between 400 nm and 1000 nm. Where the method includes irradiation with a broadband light source, broadband light source protocols of interest may include, but are not limited to, halogen lamps, deuterium arc lamps, xenon arc lamps, stabilized fiber-coupled broadband light sources, continuous spectrum broadband LEDs, superluminescent light emitting diodes, semiconductor light emitting diodes, broadband LED white light sources, multi-LED integrated white light sources, other broadband light sources, or combinations thereof.

[0084] In other embodiments, the methods of the present invention include irradiating with a narrowband light source that emits light at a specific wavelength or narrow range of wavelengths, for example, a light source that emits light at a narrow range of wavelengths, such as 50 nm or less, for example 40 nm or less, for example 30 nm or less, for example 25 nm or less, for example 20 nm or less, for example 15 nm or less, for example 10 nm or less, for example 5 nm or less, for example 2 nm or less, and a light source that emits light at a specific wavelength (i.e., monochromatic light). When the method includes irradiating with a narrowband light source, narrowband light source protocols of interest can include, but are not limited to, a narrow wavelength LED, a laser diode, or a broadband light source coupled to one or more optical bandpass filters, a diffraction grating, a monochromator, or any combination thereof.

[0085] In certain embodiments, the method includes irradiating the sample and control compositions with one or more lasers. As previously mentioned, the type and number of lasers will vary depending on the sample and control compositions and the desired light to be collected, and can be gas lasers such as helium-neon lasers, argon lasers, krypton lasers, xenon lasers, nitrogen lasers, CO lasers, CO lasers, argon-fluorine (ArF) excimer lasers, krypton-fluorine (KrF) excimer lasers, xenon-chlorine (XeCl) excimer lasers, or xenon-fluorine (XeF) excimer lasers, or combinations thereof. In other examples, the method includes irradiating the flowstream with a dye laser, such as a stilbene, coumarin, or rhodamine laser. In yet another example, the method includes irradiating the flowstream with a metal vapor laser, such as a helium-cadmium (HeCd) laser, a helium-mercury (HeHg) laser, a helium-selenium (HeSe) laser, a helium-silver (HeAg) laser, a strontium laser, a neon-copper (NeCu) laser, a copper laser, or a gold laser, and combinations thereof. In yet other cases, the method includes irradiating the flowstream with a solid-state laser, such as a ruby laser, a Nd:YAG laser, a NdCrYAG laser, an Er:YAG laser, a Nd:YLF laser, a Nd:YVO4 laser, a Nd:YCa4O(BO3)3 laser, a Nd:YCOB laser, a titanium sapphire laser, a thulium YAG laser, a ytterbium YAG laser, a ytterbium 2O3 laser, or a cerium-doped laser, and combinations thereof.

[0086] The sample and control compositions can be illuminated with one or more of the above-mentioned light sources, for example, two or more light sources, for example, three or more light sources, for example, four or more light sources, for example, five or more light sources, and ten or more light sources. The light source can include any combination of light sources. For example, in some embodiments, the method includes illuminating the sample and control compositions in the flow stream with a laser array, such as an array having one or more gas lasers, one or more dye lasers, and one or more solid-state lasers.

[0087] The sample and control compositions may be irradiated with wavelengths ranging from 200 nm to 1500 nm, e.g., 250 nm to 1250 nm, e.g., 300 nm to 1000 nm, e.g., 350 nm to 900 nm, and 400 nm to 800 nm. For example, when the light source is a broadband light source, the sample and control compositions may be irradiated with wavelengths ranging from 200 nm to 900 nm. In other examples, when the light source includes multiple narrowband light sources, the sample and control compositions may be irradiated with specific wavelengths ranging from 200 nm to 900 nm. For example, the light source may be multiple narrowband LEDs (1 nm to 25 nm), each independently emitting light having a wavelength ranging from 200 nm to 900 nm. In other embodiments, the narrowband light source includes one or more lasers (e.g., a laser array), and the sample and control compositions may be irradiated with specific wavelengths ranging from 200 nm to 700 nm using a laser array having a gas laser, excimer laser, dye laser, metal vapor laser, and solid-state laser, as described above.

[0088] When more than one light source is employed, the sample and control composition can be illuminated by the light sources simultaneously, sequentially, or a combination thereof. For example, the sample and control composition can be illuminated by each light source simultaneously. In other embodiments, the flow stream is illuminated sequentially by each light source. When multiple light sources are employed to illuminate the sample and control composition sequentially, the time for which each light source illuminates the sample and control composition can independently be 0.001 microseconds or more, e.g., 0.01 microseconds or more, e.g., 0.1 microseconds or more, e.g., 1 microsecond or more, e.g., 5 microseconds or more, e.g., 10 microseconds or more, e.g., 30 microseconds or more, and 60 microseconds or more. For example, the method can include illuminating the sample and control composition with a light source (e.g., a laser) for a period ranging from 0.001 microseconds to 100 microseconds, e.g., 0.01 microseconds to 75 microseconds, e.g., 0.1 microseconds to 50 microseconds, e.g., 1 microsecond to 25 microseconds, and 5 microseconds to 10 microseconds. In embodiments in which the sample and control compositions are sequentially illuminated with two or more light sources, the duration for which the sample and control compositions are illuminated by each light source may be the same or different.

[0089] The time interval between illumination by each light source can also be varied, as desired, such that illumination by each light source is independently separated by a delay of 0.001 microseconds or more, e.g., 0.01 microseconds or more, e.g., 0.1 microseconds or more, e.g., 1 microsecond or more, e.g., 5 microseconds or more, e.g., 10 microseconds or more, e.g., 15 microseconds or more, e.g., 30 microseconds or more, and 60 microseconds or more. For example, the time interval between illumination by each light source can range from 0.001 microseconds to 60 microseconds, e.g., 0.01 microseconds to 50 microseconds, e.g., 0.1 microseconds to 35 microseconds, e.g., 1 microsecond to 25 microseconds, and 5 microseconds to 10 microseconds. In certain embodiments, the time interval between illumination by each light source is 10 microseconds. In embodiments in which the sample and control compositions are illuminated sequentially by more than two (i.e., three or more) light sources, the delay between illumination by each light source can be the same or different.

[0090] The sample and control compositions may be illuminated continuously or at discrete intervals. In some examples, the method includes illuminating the sample and control compositions continuously with a light source. In other examples, the sample and control compositions are illuminated with a light source at discrete intervals, such as every 0.001 millisecond, every 0.01 millisecond, every 0.1 millisecond, every 1 millisecond, every 10 milliseconds, every 100 milliseconds, every 1000 milliseconds, or other intervals.

[0091] Depending on the light source, the sample and control compositions may be illuminated from a variety of distances, such as 0.01 mm or more, for example 0.05 mm or more, for example 0.1 mm or more, for example 0.5 mm or more, for example 1 mm or more, for example 2.5 mm or more, for example 5 mm or more, for example 10 mm or more, for example 15 mm or more, for example 25 mm or more, and 50 mm or more. The illumination angle may also vary, such as from 10° to 90°, for example 15° to 85°, for example 20° to 80°, for example 25° to 75°, and 30° to 60°, for example at an angle of 90°.

[0092] In a particular example, the sample and control compositions are irradiated with multiple angularly polarized beams of frequency-shifted light to detect cells in the flow stream, as described in Diebold et al., Nature Photonics Vol. 7(10);806-810 (2013), the disclosures of which are incorporated herein by reference, and in 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, 10,408,758, 10,451,53 ... ,620,111, and U.S. Patent Publication Nos. 2017 / 0133857, 2017 / 0328826, 2017 / 0350803, 2018 / 0275042, 2019 / 0376895, and 2019 / 0376894, and imaging by fluorescence imaging using radio frequency tagged emission (FIRE) to generate a frequency encoded image.

[0093] In embodiments, light from the illuminated sample and the illuminated control composition is transmitted to a light detection system and measured by one or more photodetectors. In practicing the subject methods, light from the sample and the control composition is transmitted to three or more wavelength separators, each configured to pass light having a predetermined spectral range. The spectral range of light from each wavelength separator is transmitted to one or more light detection modules having optical components configured to transmit light having a predetermined subspectral range to a photodetector.

[0094] The light can be measured by the light detection system continuously or at discrete intervals. In some examples, the method includes measuring the light continuously. In other examples, the light is measured at discrete intervals, such as every 0.001 milliseconds, every 0.01 milliseconds, every 0.1 milliseconds, every 1 millisecond, every 10 milliseconds, every 100 milliseconds, every 1000 milliseconds, or other intervals.

[0095] Measurements of collected light can be made one or more times during the subject methods, such as two or more times, such as three or more times, such as five or more times, and ten or more times. In certain embodiments, light propagation is measured two or more times, and in certain instances the data is averaged.

[0096] In some embodiments, the methods include conditioning the light before detecting it with the subject light detection system. For example, light from the sample and control composition may pass through one or more lenses, mirrors, pinholes, slits, gratings, optical refractors, and any combination thereof. In some examples, the collected light passes through one or more focusing lenses to reduce the profile of the light directed to the light detection system or optical collection system, for example, as described above. In other examples, light emitted from the sample and control composition passes through one or more collimators to reduce the divergence of the light beam transmitted to the light detection system.

[0097] system Aspects of the present invention further include systems configured to perform the above-described methods. Systems of interest include a processor configured to display results according to embodiments of the present invention. In embodiments, the subject processor operates in conjunction with programmable logic, which may be implemented in hardware, software, firmware, or any combination thereof, to display the results. In some such embodiments, the system includes a display configured to render a visualization. Any suitable display may be employed. The subject display may include, but is not limited to, a monitor, a tablet computer, a smartphone, or other electronic device configured to display a graphical interface. In this section, the word "sample" may refer to both the cell sample and the control composition of the present disclosure, or to just the cell sample, depending on the context.

[0098] The subject programmable logic may be implemented in any of a variety of devices, such as a specially programmed event processing computer, a wireless communication device, an integrated circuit device, etc. In some embodiments, the programmable logic may be executed by a specially programmed processor, which may include one or more processors, such as one or more digital signal processors (DSPs), configurable microprocessors, application specific integrated circuits (ASICs), field programmable gate arrays (FPGAs), or other equivalent integrated or discrete logic circuitry. Combinations of computing devices, e.g., a combination of a DSP and a microprocessor, multiple microprocessors, one or more microprocessors in conjunction with a DSP core, or other such configurations in at least partial data connection, may implement one or more of the functions described herein.

[0099] In certain examples, the system is or includes a particle analyzer. Particle analyzers of interest may include a flow cell for transporting particles in a flow stream, a light source for illuminating particles in the flow stream at an inspection point, and a particle-modulated light detector for detecting the particle-modulated light. In certain embodiments, the particle analyzer is a flow cytometer. In some cases where the particle analyzer is a flow cytometer, the flow cytometer is a full-spectrum flow cytometer.

[0100] As discussed herein, "flow cell" is described in its conventional sense as referring to a component, such as a cuvette, that includes a flow channel having a liquid flow stream for transporting particles within a sheath fluid. Cuvettes of interest include containers having a passageway therethrough. The flow stream may include a liquid sample injected from a sample tube. Flow cells of interest include flow channels that are accessible to light. In some examples, the flow cell includes a transparent material (e.g., quartz) through which light can pass. In some embodiments, the flow cell is a stream-in-air flow cell, where optical interrogation of particles occurs outside the flow cell (i.e., in free space).

[0101] In some cases, the flow stream is configured for illumination with light from a light source at an inspection point. The flow stream comprising the flow channel may include a liquid sample injected from a sample tube. In certain embodiments, the flow stream may include a narrow, rapidly flowing stream of liquid arranged so that linearly separated particles transported therein are aligned and separated from one another. As used herein, the term "inspection point" refers to a region within the flow cell where light from a light source illuminates particles, e.g., for analysis. The size of the inspection point may vary as needed. For example, if 0 μm represents the axis of light emitted by the light source, the inspection point may range from -100 μm to 100 μm, e.g., -50 μm to 50 μm, e.g., -25 μm to 40 μm, and -15 μm to 30 μm.

[0102] After particles are illuminated in the flow cell, particle-modulated light can be observed. "Particle-modulated light" refers to light received from particles in a flow stream after the particles are illuminated with light from a light source. In some cases, the particle-modulated light is side-scattered light. As discussed herein, side-scattered light refers to light refracted and reflected from the surface and internal structure of a particle. In additional embodiments, the particle-modulated light includes forward-scattered light (i.e., light that passes through the particle or travels around the particle in a predominantly forward direction). In still other cases, the particle-modulated light includes fluorescent light (i.e., light emitted from a fluorescent dye after illumination with excitation wavelength light).

[0103] As noted above, aspects of the present invention also include a light source configured to illuminate particles passing through the flow cell at an inspection point. Any convenient light source may be employed as the light source described herein. In some embodiments, the light source is a laser. In embodiments, the laser may be any convenient laser, such as a continuous wave laser. For example, the laser may be a diode laser, such as an ultraviolet diode laser, a visible diode laser, or a near-infrared diode laser. In other embodiments, the laser may be a helium-neon (HeNe) laser. In some examples, the laser is a gas laser, such as a helium-neon laser, an argon laser, a krypton laser, a xenon laser, a nitrogen laser, a CO laser, a CO laser, an argon-fluorine (ArF) excimer laser, a krypton-fluorine (KrF) excimer laser, a xenon-chlorine (XeCl) excimer laser, a xenon-fluorine (XeF) excimer laser, or a combination thereof. In other examples, the subject flow cytometer includes a dye laser, such as a stilbene, coumarin, or rhodamine laser. In yet another example, lasers of interest include metal vapor lasers such as helium cadmium (HeCd), helium mercury (HeHg), helium selenium (HeSe), helium silver (HeAg), strontium, neon copper (NeCu), copper, or gold lasers, and combinations thereof. In yet another example, the subject flow cytometers include solid-state lasers such as ruby, Nd:YAG, NdCrYAG, Er:YAG, Nd:YLF, Nd:YVO, Nd:YCaO(BO), Nd:YCOB, titanium sapphire, thulium YAG, ytterbium YAG, ytterbium O, or cerium-doped lasers, and combinations thereof.

[0104] The laser light source according to certain embodiments may also include one or more optical conditioning components. In certain embodiments, the optical conditioning components may be disposed between the light source and the flow cell and may include any device capable of changing the spatial width of the illumination or some other characteristic of the illumination from the light source, such as the illumination direction, wavelength, beam width, beam intensity, and focal spot. The optical conditioning protocol may include any convenient device for adjusting one or more characteristics of the light source, including, but not limited to, lenses, mirrors, filters, optical fibers, wavelength separators, pinholes, slits, collimation protocols, and combinations thereof. In certain embodiments, the flow cytometer of interest includes one or more focusing lenses. The focusing lens may be, in one example, a demagnification lens. In yet other embodiments, the flow cytometer of interest includes an optical fiber.

[0105] Where the optical adjustment component is configured to move, it may be configured to move continuously or in discrete intervals such as, for example, in increments of 0.01 μm or more, for example 0.05 μm or more, for example 0.1 μm or more, for example 0.5 μm or more, for example 1 μm or more, for example 10 μm or more, for example 100 μm or more, for example 500 μm or more, for example 1 mm or more, for example 5 mm or more, for example 10 mm or more, and 25 mm or more.

[0106] Any displacement protocol may be employed to move the optical adjustment component structure, for example, coupled to a movable support stage or directly coupled to a motorized translation stage, a lead screw translation assembly, a geared translation device (such as those employing stepper motors, servo motors, brushless electric motors, brushed DC motors, microstep drive motors, high resolution stepper motors, and other types of motors).

[0107] The light source can be positioned at any suitable distance from the flow cell, for example, the light source and the flow cell are separated by a distance of 0.005 mm or more, for example, 0.01 mm or more, for example, 0.05 mm or more, for example, 0.1 mm or more, for example, 0.5 mm or more, for example, 1 mm or more, for example, 5 mm or more, for example, 10 mm or more, for example, 25 mm or more, and 100 mm or more. Furthermore, the light source can be positioned at any suitable angle relative to the flow cell, for example, an angle in the range of 10 to 90 degrees, for example, 15 to 85 degrees, for example, 20 to 80 degrees, for example, 25 to 75 degrees, and 30 to 60 degrees, for example, 90 degrees.

[0108] In some embodiments, the light source of interest includes multiple lasers configured to provide laser light for individual illumination of the flow stream, e.g., two or more lasers, e.g., three or more lasers, e.g., four or more lasers, e.g., five or more lasers, e.g., ten or more lasers, and fifteen or more lasers configured to provide laser light for individual illumination of the flow stream. Depending on the desired wavelength of light for illuminating the flow stream, each laser may have a specific wavelength ranging from 200 nm to 1500 nm, e.g., 250 nm to 1250 nm, e.g., 300 nm to 1000 nm, e.g., 350 nm to 900 nm, and 400 nm to 800 nm. In certain embodiments, the lasers of interest may include one or more of a 405 nm laser, a 488 nm laser, a 561 nm laser, and a 635 nm laser.

[0109] As described above, a particle analyzer of interest may further include one or more particle-modulated light detectors for detecting particle-modulated light intensity data. In some embodiments, the particle-modulated light detector(s) include one or more forward-scattered light detectors configured to detect forward-scattered light. For example, a subject particle analyzer may include one forward-scattered light detector, or a plurality of forward-scattered light detectors, such as two or more, e.g., three or more, e.g., four or more, and five or more. In certain embodiments, the particle analyzer includes one forward-scattered light detector. In other embodiments, the particle analyzer includes two forward-scattered light detectors.

[0110] Any convenient detector for detecting collected light may be used in the forward scattered light detectors described herein. Detectors of interest may include, but are not limited to, optical sensors or detectors such as active pixel sensors (APS), avalanche photodiodes, image sensors, charge-coupled devices (CCDs), intensified charge-coupled devices (ICCDs), light-emitting diodes, photon counters, bolometers, pyroelectric detectors, photoresistors, photovoltaic cells, photodiodes, photomultiplier tubes (PMTs), phototransistors, quantum dot photoconductors or photodiodes, and combinations thereof, as well as other detectors. In certain embodiments, the collected light is measured with a charge-coupled device (CCD), a semiconductor charge-coupled device (CCD), an active pixel sensor (APS), a complementary metal-oxide-semiconductor (CMOS) image sensor, or an N-type metal-oxide-semiconductor (NMOS) image sensor. In certain embodiments, the detector is a photomultiplier tube, e.g., with an effective detection surface area of each region of 0.01 cm. 2 ~10cm 2 , e.g. 0.05cm 2 ~9cm 2 , e.g. 0.1cm 2 ~8cm 2 , e.g. 0.5cm 2 ~7cm 2 , and 1 cm 2 ~5cm 2 It is a photomultiplier tube with a range of

[0111] In embodiments, the forward scattered light detector is configured to measure light continuously or at discrete intervals. In some examples, the detector of interest is configured to measure collected light continuously. In other examples, the detector of interest is configured to measure at discrete intervals, such as every 0.001 milliseconds, 0.01 milliseconds, 0.1 milliseconds, 1 millisecond, 10 milliseconds, 100 milliseconds, 1000 milliseconds, or other intervals.

[0112] In additional embodiments, the one or more particle modulation light detector(s) may include one or more side scattered light detectors for detecting side scattered wavelengths of light (i.e., light refracted and reflected from the surface and internal structure of the particle). In some embodiments, the particle analyzer includes a single side scattered light detector. In other embodiments, the particle analyzer includes a plurality of side scattered light detectors, such as two or more, e.g., three or more, e.g., four or more, and five or more.

[0113] Any convenient detector for detecting the collected light can be used in the side-scattered light detector described herein. Detectors of interest can include, but are not limited to, optical sensors or detectors such as active pixel sensors (APS), avalanche photodiodes, image sensors, charge-coupled devices (CCDs), intensified charge-coupled devices (ICCDs), light-emitting diodes, photon counters, bolometers, pyroelectric detectors, photoresistors, photovoltaic cells, photodiodes, photomultiplier tubes (PMTs), phototransistors, quantum dot photoconductors or photodiodes, and combinations thereof, as well as other detectors. In certain embodiments, the collected light is measured with a charge-coupled device (CCD), a semiconductor charge-coupled device (CCD), an active pixel sensor (APS), a complementary metal-oxide semiconductor (CMOS) image sensor, or an N-type metal-oxide semiconductor (NMOS) image sensor. In certain embodiments, the detector is a photomultiplier tube, e.g., each region having an active detection surface area of 0.01 cm. 2 ~10cm 2 , e.g. 0.05cm 2 ~9cm 2 , e.g. 0.1cm 2 ~8cm 2 , e.g. 0.5cm 2 ~7cm 2 , and 1 cm 2 ~5cm 2 It is a photomultiplier tube with a range of

[0114] In embodiments, the subject particle analyzers also include a fluorescence detector configured to detect light at one or more fluorescent wavelengths, hi other embodiments, the particle analyzers include a plurality of fluorescence detectors, such as 2 or more, 3 or more, 4 or more, 5 or more, 10 or more, 15 or more, and 20 or more.

[0115] Any detector convenient for detecting collected light can be used in the fluorescence detectors described herein. Detectors of interest can include, but are not limited to, optical sensors or detectors such as active pixel sensors (APS), avalanche photodiodes, image sensors, charge-coupled devices (CCDs), intensified charge-coupled devices (ICCDs), light-emitting diodes, photon counters, bolometers, pyroelectric detectors, photoresistors, photovoltaic cells, photodiodes, photomultiplier tubes (PMTs), phototransistors, quantum dot photoconductors or photodiodes, and combinations thereof, as well as other detectors. In certain embodiments, the collected light is measured with a charge-coupled device (CCD), a semiconductor charge-coupled device (CCD), an active pixel sensor (APS), a complementary metal-oxide semiconductor (CMOS) image sensor, or an N-type metal-oxide semiconductor (NMOS) image sensor. In certain embodiments, the detector is a photomultiplier tube, e.g., with an effective detection surface area of each region of 0.01 cm. 2 ~10cm 2 , e.g. 0.05cm 2 ~9cm 2 , e.g. 0.1cm 2 ~8cm 2 , e.g. 0.5cm 2 ~7cm 2 , and 1 cm 2 ~5cm 2 It is a photomultiplier tube with a range of

[0116] When a subject particle analyzer includes multiple fluorescence detectors, each fluorescence detector can be the same, or the collection of fluorescence detectors can be a combination of different types of detectors. For example, when a subject particle analyzer includes two fluorescence detectors, in some embodiments, the first fluorescence detector is a CCD-type device and the second fluorescence detector (or imaging sensor) is a CMOS-type device. In other embodiments, both the first and second fluorescence detectors are CCD-type devices. In still other embodiments, both the first and second fluorescence detectors are CMOS-type devices. In still other embodiments, the first fluorescence detector is a CCD-type device and the second fluorescence detector is a photomultiplier tube (PMT). In still other embodiments, the first fluorescence detector is a CMOS-type device and the second fluorescence detector is a photomultiplier tube. In still other embodiments, both the first and second fluorescence detectors are photomultiplier tubes.

[0117] In embodiments of the present disclosure, a fluorescence detector of interest is configured to measure collected light at one or more wavelengths, e.g., two or more wavelengths, e.g., five or more different wavelengths, e.g., ten or more different wavelengths, e.g., twenty-five or more different wavelengths, e.g., fifty or more different wavelengths, e.g., one hundred or more different wavelengths, e.g., two ... three hundred or more different wavelengths, and measure light emitted by a sample in the flow stream at four hundred or more different wavelengths. In some embodiments, two or more detectors in a particle analyzer described herein are configured to measure the same or overlapping wavelengths of collected light.

[0118] In some embodiments, the fluorescence detector of interest is configured to measure light collected over a wavelength range (e.g., 200 nm to 1000 nm). In certain embodiments, the detector of interest is configured to collect a spectrum of light over a wavelength range. For example, a particle analyzer may include one or more detectors configured to collect a spectrum of light over one or more wavelength ranges from 200 nm to 1000 nm. In still other embodiments, the detector of interest is configured to measure light emitted by a sample in the flow stream at one or more specific wavelengths. For example, a particle analyzer may include one or more detectors configured to measure light at one or more of the following wavelengths: 450 nm, 518 nm, 519 nm, 561 nm, 578 nm, 605 nm, 607 nm, 625 nm, 650 nm, 660 nm, 667 nm, 670 nm, 668 nm, 695 nm, 710 nm, 723 nm, 780 nm, 785 nm, 647 nm, 617 nm, and any combination thereof. In certain embodiments, one or more detectors may be configured to pair with a particular fluorophore, such as one used in a sample in a fluorescence assay.

[0119] In some embodiments, the particle analyzer includes one or more wavelength separators disposed between the flow cell and the particle-modulation light detector(s). As used herein, the term "wavelength separator" is used in its conventional sense to refer to an optical component configured to separate light collected from a sample into predetermined spectral ranges. In some embodiments, the particle analyzer includes a single wavelength separator. In other embodiments, the particle analyzer includes multiple wavelength separators, e.g., two or more wavelength separators, e.g., three or more, e.g., four or more, e.g., five or more, e.g., six or more, e.g., seven or more, e.g., eight or more, e.g., nine or more, e.g., ten or more, e.g., fifteen or more, e.g., twenty-five or more, e.g., fifty or more, e.g., seventy-five or more, and one hundred or more wavelength separators. In some embodiments, the wavelength separators are configured to separate the light collected from the sample into predetermined spectral ranges by passing light having a predetermined spectral range and reflecting light in one or more remaining spectral ranges. In other embodiments, the wavelength separators are configured to separate the light collected from the sample into predetermined spectral ranges by passing light having a predetermined spectral range and absorbing light in one or more remaining spectral ranges. In yet other embodiments, the wavelength separators are configured to spatially diffract light collected from the sample into predetermined spectral ranges. Each wavelength separator can be any convenient light separation protocol, such as one or more dichroic mirrors, bandpass filters, diffraction gratings, beam splitters, or prisms. In some embodiments, the wavelength separator is a prism. In other embodiments, the wavelength separator is a diffraction grating. In certain embodiments, the wavelength separator in the subject optical detection system is a dichroic mirror.

[0120] Suitable flow cytometry systems include those described in Ormerod (ed.), Flow Cytometry: A Practical Approach, Oxford Univ. Press (1997); Jaroszeski et al. (eds.), Flow Cytometry Protocols, Methods in Molecular Biology No. 91, Humana Press (1997); Practical Flow Cytometry, 3rd Edition, Wiley-Liss (1995); Virgo et al., (2012) Ann Clin Biochem. January; 49(pt1):17-28; Linden et al., Semin Thromb Hemost. October 2004; 30(5):502-11; Alison et al., J Pathol. December 2010; 222(4):335-344; and Herbig et al., (2007) Crit Rev Ther Drug Carrier Syst. 24(3):203-255.In particular examples, flow cytometry systems of interest include a BD Biosciences FACSCanto™ flow cytometer, a BD Biosciences FACSCanto™ II flow cytometer, a BD Accuri™ flow cytometer, a BD Accuri™ C6 Plus flow cytometer, a BD Biosciences FACSCelesta™ flow cytometer, a BD Biosciences FACSLyric™ flow cytometer, a BD Biosciences FACSVerse™ flow cytometer, a BD Biosciences FACSymphony™ flow cytometer, a BD Biosciences LSRFortessa™ flow cytometer, a BD Biosciences LSRFortessa™ X-20 flow cytometer, a BD Biosciences FACSPresto™ flow cytometer, a BD Biosciences FACSVia™ flow cytometer, and a BD Biosciences FACSCalibur™ cell sorter, a BD Biosciences FACSCount™ cell sorter, a BD Biosciences FACSLyric™ cell sorter, a BD Biosciences FACSCelesta™ flow cytometer, a BD Biosciences FACSLyric™ flow cytometer, a BD Biosciences FACSVerse™ flow cytometer, a BD Biosciences FACSymphony™ flow cytometer, a BD Biosciences LSRFortessa™ flow cytometer, a BD Biosciences LSRFortessa™ X-20 flow cytometer, a BD Biosciences FACSPresto™ flow cytometer, a BD Biosciences FACSVia™ flow cytometer, and a BD Biosciences FACSCalibur™ cell sorter. These include BD Biosciences Via™ cell sorter, BD Biosciences Influx™ cell sorter, BD Biosciences Jazz™ cell sorter, BD Biosciences Aria™ cell sorter, BD Biosciences FACSAria™ II cell sorter, BD Biosciences FACSAria™ III cell sorter, BD Biosciences FACSAria™ Fusion cell sorter, and BD Biosciences FACSMelody™ cell sorter, BD Biosciences FACSymphony™ S6BD, FACSDiscover™ S8 cell sorter, etc.

[0121] In some embodiments, the subject systems may be modified in accordance with 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, 9,090,091, the disclosures of which are incorporated herein by reference in their entireties. No. 7,640, No. 9,095,494, No. 9,092,034, No. 8,975,595, No. 8,753,573, No. 8,233,146, No. No. 8,140,300, No. 7,544,326, No. 7,201,875, No. 7,129,505, No. 6,821,740, No. 6,813,017 Nos. 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.

[0122] In certain examples, the flow cytometry system of the present invention may be any of the flow cytometry systems described in Diebold et al., Nature Photonics Vol. 7(10); 806-810 (2013), the disclosures of which are incorporated herein by reference, as well as in 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, 10,408,758, 10,451,538, 10,622,624, and the like. 0,111, and U.S. Patent Publication Nos. 2017 / 0133857, 2017 / 0328826, 2017 / 0350803, 2018 / 0275042, 2019 / 0376895, and 2019 / 0376894. In such examples, the flow cytometry data may include image data of particles, such as cells, present in the sample. See, for example, Schraivogel et al., Science Vol. 375(6578); 315-320(2022), the disclosures of which are incorporated herein in their entirety, and U.S. Provisional Patent Application No. 63 / 256,974, the disclosures of which are incorporated herein in their entirety. An example of such a system is the FACSDiscover™ S8 Cell Sorter cell sorter.

[0123] In some embodiments, the system is a particle analyzer, and particle analysis system 300 (FIG. 9A) can be used to analyze and characterize particles with or without physically sorting the particles into a collection vessel. FIG. 9A shows a functional block diagram of a particle analysis system for computation-based sample analysis and particle characterization. In some embodiments, particle analysis system 300 is a flow system. The particle analysis system 300 shown in FIG. 9A can be configured to perform all or part of the methods described herein. Particle analysis system 300 includes a fluidic system 302. Fluidic system 302 can include or be coupled to a sample tube 310 and a moving fluid column within the sample tube, where particles 330 (e.g., cells) of the sample move along a common sample path 320.

[0124] The particle analysis system 300 includes a detection system 304 configured to collect a signal from each particle as it passes through one or more detection stations along a common sample path. The detection stations 308 generally refer to monitoring regions 340 of the common sample path. Detection, in some implementations, may include detecting light or one or more other properties of the particle 330 as it passes through the monitoring region 340. FIG. 9A shows one detection station 308 with one monitoring region 340. Some implementations of the particle analysis system 300 may include multiple detection stations. Additionally, some detection stations may monitor more than one region.

[0125] Each signal is assigned a signal value that forms a data point for each particle. As previously mentioned, this data may be referred to as event data. The data points may be multidimensional data points that include values for each property measured for the particle. The detection system 304 is configured to collect such data points consecutively over a first time interval.

[0126] The particle analysis system 300 may also include a control system 306. The control system 306 may include one or more processors, amplitude control circuitry, and / or frequency control circuitry. The illustrated control system may be operatively associated with the fluid system 302. The control system may be configured to generate a calculated signal frequency for at least a portion of the first time interval based on the Poisson distribution and the number of data points collected by the detection system 304 during the first time interval. The control system 306 may further be configured to generate an experimental signal frequency based on the number of data points for the portion of the first time interval. The control system 306 may further compare the experimental signal frequency to the calculated signal frequency or a predetermined signal frequency.

[0127] 9B shows a system 400 for flow cytometry according to an exemplary embodiment of the invention. System 400 includes a flow cytometer 410, a controller / processor 490, and a memory 495. Flow cytometer 410 includes one or more excitation lasers 415a-415c, a focusing lens 420, a flow chamber 425, a forward scatter detector 430, a side scatter detector 435, a fluorescence collection lens 440, one or more beam splitters 445a-445g, one or more bandpass filters 450a-450e, one or more longpass ("LP") filters 455a-455b, and one or more fluorescence detectors 460a-460f.

[0128] Pump lasers 415a-415c emit light in the form of laser beams. In the exemplary system of FIG. 9B, the wavelengths of the laser beams emitted from pump lasers 415a-415c are 488 nm, 633 nm, and 325 nm, respectively. The laser beams are first directed 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 ranging from 10 to 400 nm) and reflects 488 nm and 633 nm light.

[0129] The laser beam is then directed onto a focusing lens 420, which focuses the beam onto the portion of the fluid stream where the sample particles are located within a flow chamber 425. The flow chamber is part of a fluid system in which particles within the stream are typically directed, one at a time, towards the focused laser beam for examination. The flow chamber can comprise the flow cell of a benchtop cytometer or the nozzle tip of a stream-in air cytometer.

[0130] Light from the laser beam(s) interacts with particles in the sample by diffraction, refraction, reflection, scattering, and absorption, and is re-emitted at various wavelengths, depending on the particle's properties, such as its size, internal structure, and the presence of one or more fluorescent molecules attached to or naturally present on or within the particle. Fluorescence emission, diffracted light, refracted light, reflected light, and scattered light can be routed through one or more of beam splitters 445a-445g, bandpass filters 450a-450e, longpass filters 455a-455b, and fluorescence collection lens 440 to one or more of forward scatter detector 430, side scatter detector 435, and one or more fluorescence detectors 460a-460f.

[0131] The fluorescence collection lens 440 collects light emitted from the particle's interaction with the laser beam and routes 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 represents the center of the spectral band. The second number provides the range of the spectral band. Thus, a 510 / 20 filter extends 10 nm on either side of the center of the spectral band, from 500 nm to 520 nm. Shortpass filters transmit wavelengths of light below a specified wavelength. Longpass filters (such as longpass filters 455a-455b) transmit wavelengths of light above a specified wavelength. For example, longpass filter 455a is a 670 nm longpass filter, transmitting light above 670 nm. Filters are often selected to optimize the detector's specificity for a particular fluorescent dye. The filter can be configured so that the spectral band of light transmitted to the detector is close to the emission peak of the fluorescent dye.

[0132] 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. For example, beam splitter 445g is a 620SP beam splitter, meaning that beam splitter 445g transmits light with wavelengths of 620 nm or less and reflects light with wavelengths longer than 620 nm in different directions. In one embodiment, beam splitters 445a-445g can comprise optical mirrors, such as dichroic mirrors.

[0133] The forward scatter detector 430 is positioned slightly off-axis from the direct beam through the flow cell and is configured to detect diffracted light, which is excitation light that travels primarily forward through or around the particle. The intensity of the light detected by the forward scatter detector depends on the overall size of the particle. The forward scatter detector may include a photodiode. The side scatter detector 435 is configured to detect refracted and reflected light from the particle's surface and internal structure, which tends to increase as the particle's structural complexity increases. Fluorescence emission from fluorescent molecules associated with the particle can be detected by one or more fluorescence detectors 460a-460f. The side scatter detector 435 and the fluorescence detector may include photomultiplier tubes. The signals detected by the forward scatter detector 430, side scatter detector 435, and fluorescence detector can be converted to electronic signals (voltage) by the detectors. This data can provide information about the sample.

[0134] Those skilled in the art will recognize that flow cytometers according to embodiments of the present invention are not limited to the flow cytometer shown in Figure 9B, but 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 in a variety of different configurations.

[0135] During operation, the operation of the cytometer is controlled by the controller / processor 490, and measurement data from the detectors can be stored in memory 495 and processed by the controller / processor 490. While not explicitly shown, the controller / processor 490 is coupled to the detectors to receive output signals therefrom and may also be coupled to electrical and electromechanical components of the flow cytometer 400 to control lasers, fluid flow parameters, etc. Input / output (I / O) functionality 497 may also be provided in the system. The memory 495, controller / processor 490, and I / O 497 may be provided entirely as an integral part of the flow cytometer 410. In such an embodiment, a display may form part of the I / O functionality 497 for presenting experimental data to a user of the cytometer 400. Alternatively, the memory 495 and the controller / processor 490 and some or all of the I / O functionality may be part of one or more external devices, such as a general-purpose computer. In some embodiments, some or all of the memory 495 and the controller / processor 490 may be in wireless or wired communication with the cytometer 410. Controller / processor 490, in conjunction with memory 495 and I / O 497, can be configured to perform a variety of functions associated with the preparation and analysis of flow cytometer experiments.

[0136] The system shown in FIG. 9B includes six different detectors that detect fluorescence in six different wavelength bands (which may be referred to herein as the "filter windows" of a given detector) defined by the configuration of filters and / or splitters in the beam path from the flow cell 425 to each detector. Different fluorescent molecules used in a flow cytometer experiment emit light in their own unique wavelength bands. The particular fluorescent labels used in the experiment and their associated fluorescence emission bands may be selected to generally coincide with the filter windows of the detectors. However, as the number of detectors and labels used increases, perfect correspondence between filter windows and fluorescence emission spectra becomes impossible. Even if the peak of the emission spectrum of a particular fluorescent molecule may fall within the filter window of one particular detector, it is generally true that a portion of that label's emission spectrum may also overlap the filter windows of one or more other detectors. This may be referred to as spillover. The I / O 497 can be configured to receive data for a flow cytometer experiment having a panel of fluorescent labels and multiple cell populations with multiple markers, each cell population having a subset of the multiple markers. I / O 497 can also be configured to receive biological data assigning one or more markers to one or more cell populations, marker density data, emission spectrum data, data assigning labels to one or more markers, and cytometer configuration data. Flow cytometer experimental data, such as spectral properties of the labels and flow cytometer configuration data, can also be stored in memory 495. Controller / processor 490 can be configured to evaluate one or more assignments of labels to markers.

[0137] 10 shows a functional block diagram of an example particle analyzer control system for analyzing and displaying biological events, such as an analysis controller 1002. The analysis controller 1002 can be configured to implement various processes for controlling the graphical display of biological events.

[0138] The particle analyzer or sorting system 1001 can be configured to acquire biological event data. For example, a flow cytometer can generate flow cytometry event data. The particle analyzer 1001 can be configured to provide the biological event data to an analysis controller 1002. A data communication channel can be included between the particle analyzer or sorting system 1001 and the analysis controller 1002. The biological event data can be provided to the analysis controller 1002 via the data communication channel.

[0139] The analysis controller 1002 can be configured to receive biological event data from the particle analyzer or sorting system 1001. The biological event data received from the particle analyzer or sorting system 1001 can include flow cytometry event data. The analysis controller 1002 can be configured to provide a graphical display including a first plot of the biological event data on the display device 1004. The analysis controller 1002 can further be configured to render a region of interest as a gate around a population of the biological event data shown by the display device 1004, e.g., overlaid on the first plot. In some embodiments, the gate can be a logical combination of one or more graphical regions of interest plotted on a single parameter histogram or bivariate plot. In some embodiments, the display can be used to display particle parameters or saturation detector data.

[0140] Analysis controller 1002 can further be configured to display the biological event data within the gate differently from other events in the biological event data outside the gate on display device 1004. For example, analysis controller 1002 can be configured to render the color of the biological event data contained within the gate distinct from the color of the biological event data outside the gate. Display device 1004 can be implemented as a monitor, tablet computer, smartphone, or other electronic device configured to display a graphical interface.

[0141] The analysis controller 1002 can be configured to receive a gate selection signal identifying a gate from a first input device. For example, the first input device can be implemented as a mouse 1005. The mouse 1005 can send a gate selection signal to the analysis controller 1002 to identify a gate displayed on or manipulated via the display device 1004 (e.g., by clicking on or in the desired gate while a cursor is positioned there). In some implementations, the first device can be implemented as a keyboard 1006 or other means of providing input signals to the analysis controller 1002 (such as a touch screen, 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, as shown in FIG. 10, the mouse 1005 can include a right mouse button and a left mouse button, each of which can generate a trigger event. A trigger event can cause the analysis controller 1002 to change how the data is displayed, what portions of the data are actually displayed on the display device 1004, and / or provide input to further processing, such as selecting a population of interest for particle sorting.

[0142] In some embodiments, the analysis controller 1002 can be configured to detect when a gate selection is initiated by the mouse 1005. The analysis controller 1002 can further be configured to automatically modify the visualization of the plot to facilitate the gating process. The modifications can be made based on a particular distribution of the biological event data received by the analysis controller 1002.

[0143] The analysis controller 1002 can be connected to a storage device 1003. The storage device 1003 can be configured to receive and store biological event data from the analysis controller 1002. The storage device 1003 can also be configured to receive and store flow cytometry event data from the analysis controller 1002. The storage device 1003 can further be configured to enable acquisition of biological event data, such as flow cytometry event data, by the analysis controller 1002.

[0144] The display device 1004 can be configured to receive display data from the analysis controller 1002. The display data can comprise a plot of the biological event data and a gate outlining a section of the plot. The display device 1004 can be further configured to change the information displayed according to a combination of input received from the analysis controller 1002 and input from the particle analyzer 1001, the storage device 1003, the keyboard 1006, and / or the mouse 1005.

[0145] In some implementations, the analysis controller 1002 can generate a user interface for receiving sample events for selection. For example, the user interface can include controls for receiving example events or example images. The example events or images or example gates can be provided before collecting event data for the sample, or can be provided based on an initial set of events for a portion of the sample.

[0146] FIG. 11A is a schematic diagram of a particle sorting system 200 (e.g., a particle analyzer or sorting system 202) according to one embodiment presented herein. In some embodiments, the particle sorting system 200 is a cell sorting system. As shown in FIG. 11A, a droplet-forming transducer 202 (e.g., a piezoelectric oscillator) is coupled to a fluid conduit 201, which may be coupled to, include, or be a nozzle 203. Within the fluid conduit 201, a sheath fluid 204 hydrodynamically focuses a sample fluid 206 comprising particles 209 into a moving fluid column 208 (e.g., a stream). Within the moving fluid column 208, the particles 209 (e.g., cells) move in a single file across a monitoring region 211 (e.g., where laser streams intersect) and are illuminated by an illumination source 212 (e.g., a laser). Vibration of droplet-forming transducer 202 causes moving fluid column 208 to break up into multiple droplets 210, some of which contain particles 209.

[0147] During operation, the detection station 214 (e.g., an event detector) determines when a particle (or cell) of interest crosses the monitoring region 211. The detection station 214 feeds a timing circuit 228, which in turn feeds a flash charging circuit 230. At the droplet separation point, signaled by a timed droplet delay (Δt), a flash charge can be applied to the moving fluid column 208 so that the droplet of interest carries a charge. The droplet of interest can contain one or more particles or cells to be sorted. The charged droplets can then be sorted by activating a deflection plate (not shown) to deflect the droplets into a container, such as a collection tube or a multi-well or microwell sample plate, where a well or microwell can be associated with the droplet of particular interest. As shown in FIG. 11A, the droplets can be collected in a drain receptacle 238.

[0148] Detection system 216 (e.g., a droplet boundary detector) serves 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 U.S. Patent No. 7,679,039, which is incorporated herein by reference in its entirety. Detection system 216 enables the instrument to accurately calculate the position of each detected particle within the droplet. Detection system 216 can feed amplitude signal 220 and / or phase signal 218, which in turn feeds amplitude control circuit 226 and / or frequency control circuit 224 (via amplifier 222). Amplitude control circuit 226 and / or frequency control circuit 224 then control droplet forming transducer 202. Amplitude control circuit 226 and / or frequency control circuit 224 can be included in a control system.

[0149] In some implementations, the sorting electronics (e.g., detection system 216, detection station 214, and processor 240) can be coupled with a memory configured to store the detected events and sorting decisions based thereon. The sorting decisions can be included in the particle's event data. In some implementations, the detection system 216 and detection station 214 can be implemented as a single detection unit or communicatively coupled such that event measurements can be collected and provided to a non-collecting element by either the detection system 216 or the detection station 214.

[0150] FIG. 11B is a schematic diagram of a particle sorting system according to one embodiment presented herein. The particle sorting system 100 shown in FIG. 11B includes deflection plates 152 and 154. An electric charge can be applied via a stream of charging wires within the barbs, which generates a stream of droplets 110 containing the particles 110 for analysis. The particles can be illuminated with one or more light sources (e.g., lasers) to generate light scattering and fluorescence information. The particle information is analyzed, such as by sorting electronics or another detection system (not shown in FIG. 11B). Deflection plates 152 and 154 can be independently controlled to attract or repel the charged droplets, directing them to a desired collection receptacle (e.g., 172, 174, 176, or 178). 11B, deflection plates 152 and 154 can be controlled to direct particles along a first path 162 to a receptacle 174 or along a second path 168 to a receptacle 178. If the particle is not of interest (e.g., does not exhibit scattering or illumination information within a specified sorting range), the deflection plates may allow the particle to continue along flow path 164. Such uncharged droplets may flow into a waste receptacle, such as via an aspirator 170.

[0151] Sorting electronics can be included to initiate measurement collection, receive the particle's fluorescent signal, and determine how to adjust the deflection plates to sort the particle. Exemplary implementations of the embodiment shown in Figure 11B include the BD FACSAria™ series of flow cytometers available from Becton, Dickinson and Company (Franklin Lakes, NJ).

[0152] In some embodiments, particle sorting systems of interest are configured to sort particles in an enclosed particle sorting module such as described in U.S. Patent Publication No. 2017 / 0299493, filed March 28, 2017, the disclosure of which is incorporated herein by reference. In certain embodiments, particles (e.g., cells) of a sample are sorted using a sorting determination module having multiple sorting determination units such as described in U.S. Patent Publication No. 2020 / 0256781, the disclosure of which is incorporated herein by reference. In some embodiments, the subject systems include a particle sorting module having deflection plates such as described in U.S. Patent Publication No. 2017 / 0299493, filed March 28, 2017, the disclosure of which is incorporated herein by reference.

[0153] In certain embodiments, the system is a fluorescence imaging system using a radio frequency tagged luminescence image-enabled particle sorter, as shown in FIG. 12A. The particle sorter 1200 includes an optical illumination component 1200a including a light source 1201 (e.g., a 488 nm laser), which generates an optical output beam 1201a that is split into beams 1202a and 1202b by a beam splitter 1202. The optical beam 1202a is propagated through an acousto-optic device (e.g., an acousto-optic deflector, AOD) 1203 to generate an output beam 1203a having one or more angularly deflected optical beams. In some examples, the output beam 1203a generated from the acousto-optic device 1203 includes a local oscillator beam and multiple radio frequency comb beams. The optical beam 1202b is propagated through an acousto-optic device (e.g., an acousto-optic deflector, AOD) 1204 to generate an output beam 1204a having one or more angularly deflected optical beams. In some examples, output beam 1204a generated from acousto-optic device 1204 includes a local oscillator beam and multiple radio frequency comb beams. Output beams 1203a and 1204a generated from acousto-optic devices 1203 and 1204, respectively, are combined with beam splitter 1205 to generate output beam 1205a, which is conveyed through optical component 1206 (e.g., an objective lens) to illuminate particles in flow cell 1207. In certain embodiments, acousto-optic device 1203 (AOD) splits a single laser beam into an array of beamlets, each with a different optical frequency and angle. A second AOD 1204 adjusts the optical frequency of a reference beam, which is then superimposed with the array of beamlets in beam combiner 1205. In certain embodiments, the light illumination system having a light source and an acousto-optical device may also include those described in Schraivogel et al. ("High-speed fluorescence image-enabled cell sorting", Science (2022), 375(6578):305-320) and U.S. Patent Publication No. 2021 / 0404943, the disclosures of which are incorporated herein by reference.

[0154] Output beam 1205a illuminates sample particles 1208 propagating through flow cell 1207 (e.g., with sheath fluid 1209) at illumination region 1210. As shown in illumination region 1210, multiple beams (e.g., angularly deflected radio frequency shifted optical beams shown as dots across illumination region 1210) overlap with a reference local oscillator beam (shown as a shaded line across illumination region 1210). Due to their different optical frequencies, the overlapping beams exhibit beating behavior, whereby each beamlet emits at a different frequency f 1-n carries a sinusoidal modulation.

[0155] Light from the illuminated sample is transmitted to a light detection system 1200b, which includes multiple light detectors. The light detection system 1200b includes a forward-scattered light photodetector 1211 for generating a forward-scattered light image 1211a and a side-scattered light photodetector 1212 for generating a side-scattered light image 1212a. The light detection system 1200b also includes a bright-field light detector 1213 for generating a light loss image 1213a. In some embodiments, the forward-scattered light detector 1211 and the side-scattered light detector 1212 are photodiodes (e.g., avalanche photodiodes, APDs). In some examples, the bright-field light detector 1213 is a photomultiplier tube (PMT). Fluorescence from the illuminated sample is also detected by fluorescence detectors 1214-1217. In some examples, the light detectors 1214-1217 are photomultiplier tubes. Light from the illuminated sample is directed via beam splitter 1220 to side scatter detection channel 1212 and fluorescence detection channels 1214-1217. Light detection system 1200b includes bandpass optical components 1221, 1222, 1223, and 1224 (e.g., dichroic mirrors) for transmitting light of predetermined wavelengths to photodetectors 1214-1217. In some examples, optical component 1221 is a 534 nm / 40 nm bandpass. In some examples, optical component 1222 is a 586 nm / 42 nm bandpass. In some examples, optical component 1223 is a 700 nm / 54 nm bandpass. In some examples, optical component 1224 is a 783 nm / 56 nm bandpass. The first number represents the center of the spectral band. The second number represents the range of the spectral band. Thus, a 510 / 20 filter extends 10 nm on either side of the center of the spectral band, from 500 nm to 520 nm.

[0156] Data signals generated in response to light detected in scattered light detection channels 1211 and 1212, brightfield light detection channel 1213, and fluorescence detection channels 1214-1217 are processed by real-time digital processing by processors 1250 and 1251. Images 1211a-1217a are generated in each light detection channel based on the data signals generated by processors 1250 and 1251. Image-corresponding sorting is performed in response to a sorting signal generated by sorting trigger 1252. Sorting component 1200c includes deflection plates 1231 for deflecting particles into a sample container 1232 or a waste stream 1233. In some examples, sorting component 1200c is configured to sort particles using an enclosed particle sorting module such as that described in U.S. Patent Publication No. 2017 / 0299493, filed March 28, 2017, the disclosure of which is incorporated herein by reference. In certain embodiments, the sorting component 1200c includes a sorting determination module having multiple sorting determination units, such as those described in U.S. Patent Publication No. 2020 / 0256781, the disclosure of which is incorporated herein by reference.

[0157] FIG. 12B illustrates image-enabled particle sorting data processing according to certain embodiments. In some examples, the image-enabled particle sorting data processing is a low-latency data processing pipeline. Each photodetector generates high-frequency modulated pulses that encode an image (waveform). Fourier analysis is performed to reconstruct the image from the modulated pulses. The image processing pipeline generates a set of image features (image analysis), which are combined with features derived from the pulse processing pipeline (event packets). Real-time sorting electronics then classify particles based on the image features and generate sort decisions that are used to selectively charge droplets.

[0158] Computer Control System Aspects of the present disclosure also include computer control systems, including one or more computers for fully or partially automating the subject methods and systems of particle analysis. In some embodiments, the system includes a computer having a computer program stored thereon, the computer program including instructions, when loaded into the computer, for displaying data according to embodiments of the present invention. In some examples, the system is or comprises a flow cytometer.

[0159] The system may include a display and an operator input device. The operator input device may be, for example, a keyboard, a mouse, etc. The processing module includes a processor that can access memory on which instructions for executing the steps of the subject method are stored. The processing module may include an operating system, a graphical user interface (GUI) controller, system memory, memory storage devices, and input / output controllers, cache memory, data backup units, and many other devices. The processor may be a commercially available processor, or it may be one of other processors that are available or become available. The processor runs an operating system, which interfaces with firmware and hardware in a well-known manner and facilitates the processor's coordination and execution of the functions of various computer programs, which may be written in various programming languages known in the art, such as Java, Perl, C++, Python, other high-level or low-level languages, and combinations thereof. The operating system typically works in conjunction with the processor to coordinate and execute the functions of the 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, all in accordance with known techniques. In some embodiments, the processor includes analog electronics to provide feedback control, such as negative feedback control.

[0160] The system memory may be any of a variety of known or future memory storage devices. Examples include commonly available random access memory (RAM), magnetic media such as a resident hard disk or tape, optical media such as a read-and-write compact disk, a flash memory device, or other memory storage device. The memory storage device may be any of a variety of known or future devices, including a compact disk drive, tape drive, or diskette drive. These types of memory storage devices typically read from and / or write to a program storage medium (not shown), such as a compact disk. Any of these program storage media, or others now in use or that may be developed in the future, may be considered a computer program product. As will be appreciated, these program storage media typically store computer software programs and / or data. Computer software programs, also called computer control logic, are typically stored in system memory and / or program storage devices used in conjunction with the memory storage devices.

[0161] In some embodiments, a computer program product is described that includes a computer-usable medium having control logic (a computer software program including program code) stored thereon. The control logic, when executed by a processor of a computer, causes the processor to perform the functions described herein. In other embodiments, some functions are implemented primarily in hardware, for example, using hardware state machines. Implementation of hardware state machines to perform the functions described herein will be apparent to one skilled in the relevant art.

[0162] The memory may be any suitable device from which the processor can store and retrieve data, such as a magnetic, optical, or solid-state storage device (including a magnetic or optical disk, tape, RAM, or other suitable device, either fixed or portable). The processor may include a general-purpose digital microprocessor appropriately programmed from a computer-readable medium carrying the necessary program code. The programming may be provided to the processor remotely via a communications channel or may be pre-stored on a computer program product, such as memory, or other portable or fixed computer-readable storage medium using one of these devices connected to the memory. For example, a magnetic or optical disk may carry the programming and be readable by a disk writer / reader. The system of the present invention also includes programming, for example, in the form of a computer program product, algorithms used to implement the methods described above. The programming of the present invention may be recorded on a computer-readable medium, such as any medium that can be directly read and accessed by a computer. Such media include, but are not limited to, magnetic storage media, optical storage media such as CD-ROMs, electrical storage media such as RAM and ROM, portable flash drives, and hybrids of these categories, such as magnetic / optical storage media.

[0163] The processor may have access to a communication channel for communicating with a user at a remote location, meaning that the user does not have direct contact with the system but instead relays input information to the input manager from an external device, such as a computer connected to a wide area network ("WAN"), telephone network, satellite network, or other suitable communication channel, including a mobile phone (i.e., smartphone).

[0164] In some embodiments, a system according to the present disclosure may be configured to include a communications interface. In some embodiments, the communications interface includes a receiver and / or a transmitter for communicating with a network and / or another device. The communications interface may be configured for wired or wireless communications, including, but not limited to, radio frequency (RF) communications (e.g., radio frequency identification (RFID), Zigbee communications protocol, Wi-Fi, infrared, wireless universal serial bus (USB), ultra-wideband (UWB), Bluetooth® communications protocol, and cellular communications such as code division multiple access (CDMA) or global system for mobile communications (GSM).

[0165] In one embodiment, the communication interface is configured to include one or more communication ports (e.g., physical ports or interfaces such as a USB port, a USB-C port, an RS-232 port, or other suitable electrical connection port) to enable data communication between the subject system and other external devices, such as a computer terminal (e.g., in a doctor's office or hospital environment) configured for similar complementary data communication.

[0166] In one embodiment, the communications interface is configured for infrared communications, Bluetooth® communications, or other suitable wireless communications protocols, allowing the subject system to communicate with other devices, such as computer terminals and / or networks, communications-enabled mobile phones, personal digital assistants, or other communications devices that a user may use in conjunction with the subject system.

[0167] In one embodiment, the communication interface is configured to provide a connection for data transfer using Internet Protocol (IP) over a cellular network, Short Message Service (SMS), a wireless connection to a personal computer (PC) on a local area network (LAN) connected to the Internet, or a Wi-Fi connection to the Internet at a Wi-Fi hotspot.

[0168] In one embodiment, the subject system is configured to communicate wirelessly with a server device via a communications interface using common standards such as, for example, 802.11 or Bluetooth® RF protocols, or the IrDA infrared protocol. The server device may be another portable device, such as a smartphone, personal digital assistant (PDA), or notebook computer, or a larger device, such as a desktop computer, appliance, etc. In some embodiments, the server device has a display, such as a liquid crystal display (LCD), and input devices, such as buttons, a keyboard, a mouse, or a touchscreen.

[0169] In some embodiments, the communications interface is configured to automatically or semi-automatically communicate data stored in the subject system (e.g., in the optional data storage unit) with a network or server device using one or more of the communications protocols and / or mechanisms described above.

[0170] An output controller may include a controller for any of a variety of known display devices for presenting information to a user, whether human or machine, local or remote. When a display device provides visual information, this information may typically be logically and / or physically organized as an array of graphical elements. A graphical user interface (GUI) controller may include any of a variety of known or future software programs for providing a graphical input / output interface between the system and a user and for processing user input. The functional elements of a computer may communicate with each other via a system bus. Some of these communications may be realized in alternative embodiments using a network or other type of remote communication. An output manager may provide information generated by a processing module to a remote user, for example, via the Internet, telephone, or satellite network, according to known techniques. Presentation of data by the output manager may be implemented according to various known techniques. As some examples, the data may include SQL, HTML, or XML documents, email or other files, or data in other formats. The data may include an Internet URL address whereby a user may retrieve additional SQL, HTML, XML, or other documents or data from a remote source. The one or more platforms present in the subject system can be any type of known or future-developed computer platform, but they are typically computers of a class commonly referred to as servers. However, they can also be mainframe computers, workstations, or other computer types. They can be connected via known or future types of cables or other communication systems (whether networked or not), including wireless systems. They can be co-located, or they can be physically separate.A variety of operating systems may be employed on any computer platform, possibly depending on the type and / or make of the selected computer platform. Suitable operating systems include Windows NT, Windows XP, Windows 7, Windows 8, Windows 10, iOS, macOS, Linux, Ubuntu, Fedora, OS / 400, i5 / OS, IBM i, Android™, SGI IRIX, Oracle Solaris, and the like.

[0171] FIG. 13 illustrates the general architecture of an exemplary computing device 1300 according to certain embodiments. The general architecture of the computing device 1300 illustrated in FIG. 13 includes an arrangement of computer hardware and software components. However, not all of these typically conventional elements need be shown to provide a useful disclosure. As shown, the computing device 1300 includes a processing unit 1310, a network interface 1320, a computer-readable medium drive 1330, an input / output device interface 1340, a display 1350, and input devices 1360, all of which may communicate with each other via a communications bus. The network interface 1320 may provide a connection to one or more networks or computing systems. Thus, the processing unit 1310 may receive information and instructions from other computing systems or services over a network. The processing unit 1310 also communicates with memory 1370 and may provide output information to the optional display 1350 via the input / output device interface 1340. For example, analysis software (e.g., data analysis software or program such as FlowJo®) stored as executable instructions in non-transitory memory of the analysis system can display flow cytometry event data to a user. Input / output device interface 1340 may also accept input from optional input device(s) 1360, such as a keyboard, mouse, digital pen, microphone, touch screen, gesture recognition system, voice recognition system, gamepad, accelerometer, gyroscope, or other input device.

[0172] Memory 1370 may include computer program instructions (grouped in some embodiments as modules or components) that processing unit 1310 executes to implement one or more embodiments. Memory 1370 typically includes RAM, ROM, and / or other persistent, secondary, or non-transitory computer-readable media. Memory 1370 may store an operating system 1372 that provides computer program instructions used by processing unit 1310 in the overall management and operation of computing device 1300. Data may be stored on data storage device 1390. Memory 1370 may further include computer program instructions and other information for implementing aspects of the present disclosure.

[0173] computer-readable storage medium Aspects of the present disclosure further include non-transitory computer-readable storage media having instructions for implementing the subject methods. The computer-readable storage media may be employed by one or more computers for fully or partially automating systems for implementing the methods described herein. In certain embodiments, instructions according to the methods described herein may be encoded on a computer-readable medium in the form of "programming," and the term "computer-readable medium" as used herein refers to any non-transitory storage medium involved in providing instructions and data to a computer for execution and processing. In some cases, the instructions, when executed by a computer or processor, cause the computer or processor to receive display data in accordance with embodiments of the present invention.

[0174] Examples of suitable non-transitory storage media include floppy disks, hard disks, optical disks, magneto-optical disks, CD-ROMs, CD-Rs, magnetic tape, non-volatile memory cards, ROMs, DVD-ROMs, Blu-ray disks, solid-state disks, flash drives, and network-attached storage (NAS), whether such devices are internal or external to the computer. Files containing information can be "stored" on a computer-readable medium, where "storing" means recording information so that it can be accessed and retrieved at a later date by a computer. The computer-implemented methods described herein can be implemented using programming that can be written in one or more of any number of computer programming languages. Such languages include, for example, Java, Python, Visual Basic, C++, and many others.

[0175] kit Aspects of the present disclosure also include kits. The kits can include, for example, a compensatory control composition as described herein. In certain embodiments, the control composition of the kit can include a positive control particle as described herein. In some embodiments, the control composition of the kit can include a negative control particle as described herein. In some cases, the control composition of the kit can include multiple positive control particles and / or multiple negative control particles.

[0176] The kit may further include an amine-reactive dye and / or a DNA-binding dye for labeling the positive control particles of the compensation control composition. In some embodiments, the kit may further include a buffer. For example, the kit may include a sample buffer, a wash buffer, an assay buffer, or other buffer. The kit may further include additional reagents such as, but not limited to, fluorescent particles and detectable labels (e.g., fluorescent labels, colorimetric labels, chemiluminescent labels, multicolor reagents, avidin-streptavidin association detection reagents, radioactive labels, gold particles, magnetic labels, etc.).

[0177] In certain embodiments, the kit includes a subject compensation control composition and a package configured to hold the composition. The package may be a sealed package, e.g., a water vapor-resistant container, optionally airtight and / or vacuum sealed. In some embodiments, the package may protect its contents (e.g., the subject composition) from light. In these examples, the package may protect from any range or spectrum of light wavelengths, including, for example, visible light, UV light, light within and / or near the excitation spectrum of the encapsulated dye, etc. In certain examples, the package is a sterile package configured to maintain the composition enclosed therein in a sterile environment. "Sterile" means substantially free of microorganisms (e.g., fungi, bacteria, viruses, spore forms, etc.). In some embodiments, the composition and any additional buffers or reagents (e.g., amine-reactive dye and / or DNA-binding dye) are contained in the same package. In other examples, the composition and any additional buffers or reagents are contained in separate packages.

[0178] In addition to the above components, the subject kits may further include instructions for practicing the subject methods. In some embodiments, the instructions are for using the compensation control composition of the kit to determine a compensation value for cell viability data obtained from a flow cytometry analysis. In some embodiments, the instructions are for performing a flow cytometry analysis and using the determined compensation value to perform compensation on the obtained cell viability data. These instructions may be present in a subject kit in a variety of forms, one or more of which may be present in the kit. One form in which these instructions may be present is as information printed on a suitable medium or substrate, such as paper(s) with the information printed on the kit packaging, package insert, etc. Another means would be a computer-readable medium (e.g., as described above) on which the information is recorded or stored. Yet another form in which they may be present is a website address that can be used to access the information remotely via the Internet. The instructions may be present in the kit in any convenient format.

[0179] usefulness The subject compositions and methods find use in applications where cellular analysis of biological samples is desired for use in research, laboratory testing, or treatment. In some embodiments, the subject systems and methods facilitate the analysis of cells obtained from fluid or tissue samples, such as disease specimens, including, but not limited to, cancer. The disclosed compositions and methods also allow for the analysis of cells from biological samples (e.g., organs, tissues, tissue slices, fluids) with increased efficiency and at low cost.

[0180] The subject control compositions and methods find use in applications where it is desired to analyze a sample using an ultraviolet (UV) or violet laser. Embodiments of the subject control compositions and methods find use in applications where it is desired to analyze a sample using two or more fluorescent particles (e.g., two or more dye compositions). For example, the subject compositions and methods find use in applications where it is desired to perform an assay (e.g., a cell viability assay) using an amine-reactive dye and a DNA-binding dye. In some cases, the subject compositions and methods can perform fluorescence compensation for assays including one or more of an amine-reactive dye, a DNA-binding dye, a fluorescently tagged nucleic acid probe, and a fluorescently tagged antibody.

[0181] The subject control compositions and methods find use when it is desirable to perform flow cytometry assays with more precise and / or accurate data. For example, the subject compositions and methods can facilitate accurate data collection and interpretation compared to conventional protocols and control beads that include a positive compensation control with low fluorescence intensity or a negative control sample with high fluorescence intensity. Furthermore, the subject control compositions and methods can facilitate accurate data collection and interpretation compared to conventional protocols and control beads that do not include a fluorescence minus one (FMO) control for assays that include two or more of an amine-reactive dye, a DNA-binding dye, a fluorescently tagged nucleic acid probe, and a fluorescently tagged antibody.

[0182] The subject compositions and methods find use in laboratory and research workflows where a high degree of flexibility is required. For example, the subject compositions and methods can facilitate the ability of laboratories to perform customizable experiments on a wide variety of cell types. Furthermore, the subject compositions and methods can facilitate cost reduction compared to the less stable control beads of the prior art. Embodiments of the subject compositions and methods allow for fluorescence compensation to be performed without depleting cell samples for use as controls. Thus, the subject compositions and methods also find use when it is desirable to analyze as much of a collected cell sample as possible, for example, due to low sample availability, cost savings, or difficulties that may exist in sample collection. [Example]

[0183] As can be seen from the above disclosure, embodiments of the present disclosure have a wide variety of applications. Accordingly, the following examples are presented to provide those skilled in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention, nor are they intended to represent that the following experiments are all or the only experiments performed. While efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), some experimental error and deviation should be accounted for. Unless otherwise specified, temperatures are in degrees Celsius and pressures are near atmospheric. Additionally, common experimental protocol abbreviations may be used (e.g., μl = microliter, min = minute, kcal = kilocalories, mol = mole, etc.).

[0184] The following experiment demonstrates the stability of the cell viability compensation (CVC) beads of the present invention and compares positive and negative CVC beads with conventional compensation beads.

[0185] material and method In accordance with an embodiment of the present invention, an embodiment of a compensation control composition was produced having both positive and negative control beads. The positive control beads were synthesized according to an embodiment of the present invention and included a silica core, five layers of histone protein coating, and five layers of dsDNA molecule coating. For the positive control, the dsDNA coating was first covalently attached to the core. Next, alternating layers of histone and dsDNA coatings were covalently attached to the outermost coating of the positive control beads until there were five layers each. The negative control beads were synthesized according to an embodiment of the present invention and included a silica core, but lacked both an aminated polymer (i.e., histone) coating and a nucleic acid (i.e., dsDNA) coating.

[0186] Figure 3 provides a description of the method used to synthesize the positive control beads. An aminated silica core was obtained, and several of its amine groups were thiolated. Next, coatings of dsDNA molecules modified to contain maleimide groups and histone proteins modified to contain thiol groups were alternately deposited onto the positive control beads, up to five layers each. [Example]

[0187] Dyeing with amine reactive dyes Experiments were performed to compare the fluorescence resulting from staining with amine-reactive dyes between the CVC beads of the present invention (see Materials and Methods above) and two conventional types of beads: ViaComp® (Slingshot) beads and Arc™ (Invitrogen) beads. ViaComp® (Slingshot) beads are composed of hydrogel, while Arc™ (Invitrogen) beads are specially modified polystyrene microspheres. Three different BD Horizon™ Fixable Viability Stains (FVS) amine-reactive dyes were used for staining: BD Horizon™ FVS520 (Figure 4) (peak excitation: 498 nm, peak emission: 521 nm, fluorescence channel: BB515), BD Horizon™ FVS570 (Figure 5) (peak excitation: 547 nm, peak emission: 573 nm, fluorescence channel: BYG584), and BD Horizon™ FVS700 (Figure 6) (peak excitation: 657 nm, peak emission: 700 nm, fluorescence channel: APC-R700). For staining, 50 μl of bead suspension (i.e., both positive and negative beads) was incubated with 1 μl of FVS dye in DMSO buffer for 30 minutes. Data were collected using the same flow cytometer parameter settings.

[0188] The data shown in Figures 4-6 demonstrate that the CVC beads of the present invention exhibit brighter or equivalent staining signals compared to conventional ViaComp® and Arc™ beads. However, preliminary experiments have shown that the fluorescence intensity signal of the stained CVC beads of the present invention can be adjusted or regulated (e.g., increased) by controlling the number of layers of histone coating. Furthermore, unlike CVC beads, the polystyrene of Arc™ beads exhibits significant autofluorescence, resulting in a different spectral signature of the beads after staining with cell viability dyes compared to the stained cell samples. This issue was particularly pronounced for fluorescence emitted by violet and ultraviolet lasers. Such significant autofluorescence can lead to misleading spillover values for dyes, especially those that emit or absorb light in the UV and violet ranges, potentially compromising experimental compensation data.

[0189] The CVC beads of the present invention also demonstrated logistical advantages over Arc™ beads. Unlike the CVC bead embodiment, Arc™ beads are divided into two vials: one containing positive beads and one containing negative control beads. This division increases complexity and manufacturing costs, and also causes inconvenience to users during the experimental process. Furthermore, Arc™ beads do not react with DNA-intercalating dyes and can only be stained with amine-reactive dyes. This limits users' options when designing experiments. [Example]

[0190] Staining with DNA-binding dyes An experiment was conducted to compare the fluorescence of DNA-intercalating dye staining between one embodiment of the CVC beads of the present invention (see Materials and Methods, above) and conventional ViaComp® (Slingshot) hydrogel beads. Two different BD Pharmingen™ DNA-intercalating dyes were used for staining: BD Pharmingen™ 7-AAD (7-amino-actinomycin D) (Figure 7) (fluorescence channel: BB700) and BD Pharmingen™ DAPI (4',6-diamidino-2-phenylindole) (Figure 7) (fluorescence channel: BUV450). For staining, 50 μl of bead suspension (i.e., both positive and negative beads) was incubated with 20 μl of BD Pharmingen™ 7-AAD or 1 μl of BD Pharmingen™ DAPI in solution for 10 minutes. Data were collected using the same flow cytometer parameter settings.

[0191] The data shown in Figure 7 demonstrate that the CVC beads of the present invention exhibit brighter or equivalent staining signals compared to conventional ViaComp® beads. However, preliminary experiments have shown that the fluorescence intensity signal of the dyed CVC beads of the present invention can be adjusted or regulated (e.g., increased) by controlling the number of layers of dsDNA coating. Furthermore, unlike CVC beads, high nonspecific uptake of the DNA-intercalating dye was observed in ViaComp® negative control beads. This nonspecific uptake, likely due to the hydrogel composition of the beads, may result in higher background noise and potentially reduce the accuracy of flow cytometry results obtained using ViaComp® beads. [Example]

[0192] Stability of the control composition An experiment was conducted to test the stability of one embodiment of the CVC beads of the present invention (see "Materials and Methods" above). CVC beads were stored at 37°C for 15 days, and subsets of beads were tested on days 1-3, 7-10, 13, and 15. For testing, beads were stained using BD Pharmingen™ 7-AAD, BD Horizon™ FVS570, or BD Horizon™ FVS700, and the fluorescence intensity of beads stained with each dye was measured. The MFI of beads stained with each dye on each test day can be found in Figure 8.

[0193] The data shown in Figure 8 demonstrates that the CVC beads of the present invention can be stored at relatively high temperatures for several weeks without significant loss of dye staining performance. 10 =3, E a Using a β-kappa-phosphate (β-kappa-phosphate) (=19.4 kcal / mol), CVC beads were predicted to be able to efficiently stain with both amine-reactive and DNA-intercalating dyes even after storage at 4°C for 18 months. Thus, CVC beads demonstrate a significant advantage over conventional beads, such as ViaComp® beads, which have a shelf life of only 6 months at 2-8°C and therefore must be shipped and stored at -20°C. This temperature instability creates inconvenience and additional costs for users, as the beads must be thawed before use and expired beads must be replaced.

[0194] Notwithstanding the appended claims, the present disclosure is also defined by the following clauses. 1. Core, an aminated polymer comprising an amine configured to react with an amine-reactive dye; and Nucleic acids configured to bind to DNA-binding dyes Equipped with The aminated polymer and nucleic acid are covalently associated with the core. Positive control particles. 2. A positive control particle according to clause 1, wherein the core is substantially non-autofluorescent. 3. A positive control particle according to clause 1 or 2, wherein the core has low non-specific binding. 4. A positive control particle according to any preceding clause, wherein the core comprises an inorganic material. 5. A positive control particle according to clause 4, wherein the core comprises silica. 6. A positive control particle according to any one of clauses 1 to 3, wherein the core comprises a polymer. 7. A positive control particle as described in clause 6, wherein the core comprises poly(methyl methacrylate) (PMMA). 8. The positive control particle of clause 6, wherein the core comprises a polyacrylamide hydrogel. 9. Positive control particles according to any of the preceding clauses, wherein the particle diameter is in the range of 0.1 to 150 microns. 10. Positive control particles as described in clause 9, wherein the particle diameter is in the range of 1 to 10 microns. 11. Positive control particles as described in clause 10, wherein the particle diameter is in the range of 5 to 7 microns. 12. A positive control particle according to any preceding clause, wherein an aminated polymer and a nucleic acid are layered onto a core. 13. The positive control particle of clause 12, wherein the particle comprises five or more aminated polymer layers and five or more nucleic acid layers. 14. The positive control particle of any preceding clause, wherein the aminated polymer comprises a polypeptide. 15. The positive control particle of clause 14, wherein the polypeptide is a protein. 16. The positive control particle of clause 15, wherein the protein is a histone. 17. The positive control particles of clause 15, wherein the protein is myelin basic protein. 18. A positive control particle described in any one of clauses 1 to 14, wherein the aminated polymer comprises a polysaccharide. 19. The positive control particle of clause 18, wherein the polysaccharide is a cationic polymer. 20. The positive control particles of clause 19, wherein the cationic polymer is chitosan. 21. A positive control particle according to any of the preceding clauses, wherein the nucleic acid is a double-stranded nucleic acid. 22. Positive control particles according to any of the preceding clauses, wherein the nucleic acid is deoxyribonucleic acid (DNA). 23. A positive control particle according to any of the preceding clauses, wherein the nucleic acid has a size in the range of 450 to 550 base pairs. 24. A positive control particle according to any of the preceding clauses, wherein the nucleic acid is naturally occurring. 25. The positive control particle of clause 24, wherein the nucleic acid comprises salmon DNA. 26. A positive control particle according to any one of clauses 1 to 23, wherein the nucleic acid is synthetic. 27. A positive control particle according to any of the preceding clauses, wherein the aminated polymer and nucleic acid are covalently bound to the surface of the core either directly or via a linker. 28. A positive control particle according to any of the preceding clauses, wherein the particle is a bead. 29. A compensation control composition comprising a plurality of positive control particles according to any of the preceding clauses. 30. The compensatory control composition of clause 29, further comprising a plurality of negative control particles. 31. The compensatory control composition of clause 30, wherein the negative control particles comprise cores of the same material and with the same average diameter as the cores of the positive control particles. 32. The compensatory control composition of clause 30 or 31, wherein polyethylene glycol (PEG) is conjugated to the core of the negative control particles. 33. The compensatory control composition of any of clauses 29-32, wherein the composition is stable at 4°C for 18 months or more. 34. The compensation control composition of clause 1, further comprising an amine-reactive dye. 35. A label compensation control composition comprising a plurality of positive control particles according to any one of clauses 1 to 28, wherein the positive control particles have an amine-reactive dye and / or a DNA-binding dye attached thereto. 36. The label compensation control composition of clause 35, wherein the amine-reactive dye is a cell viability dye. 37. The label compensation control composition of clause 35 or 36, wherein the excitation maximum of the amine-reactive dye is in the range of 350 nm to 1000 nm. 38. A label compensation control composition described in any of clauses 35 to 37, wherein the amine-reactive dye is selected from the group consisting of BD Horizon™ Fixable Viability Stain, Biolegend Zombie™ dye, Thermo Fischer eFluor™ dye, Thermo Fischer LIVE / DEAD™ dye, Proteintech Phantom Dye, and Tombo Biosceinces Ghost Dye™. 39. The label compensation control composition of any of clauses 35-38, wherein the DNA binding dye is a cell viability dye. 40. The label compensation control composition of any of clauses 35 to 39, wherein the DNA binding dye has an excitation maximum in the range of 350 nm to 1000 nm. 41. The label compensation control composition of any of clauses 35 to 40, wherein the DNA binding dye is selected from the group consisting of 7-AAD, DAPI, propidium iodide, Hoechst dye, ethidium bromide, LDS 751, Thermo Fischer Sytox™ dye, Thermo Fischer T-PRO™ dye, Thermo Fischer TOTO™ dye, Thermo Fischer YO-PRO™ dye, Biolegend Helix-NP™ dye, Biotium RedDot™ dye, and Biostatus Limited DRAQ™ dye. 42. The label compensation control composition of any one of clauses 35-41, further comprising a plurality of negative control particles. 43. The label compensation control composition of clause 42, wherein the negative control particles comprise cores of the same material and with the same average diameter as the cores of the positive control particles. 44. The label compensation control composition of clause 43, wherein PEG is conjugated to the core of the negative control particle. 45. A method for determining a compensation value for cell viability data obtained from flow cytometry analysis, comprising: analyzing the labeled compensation control composition of any one of clauses 35 to 44 using a flow cytometer to obtain flow cytometry data; and Calculating compensation values based on flow cytometry data A method comprising: 46. The method of clause 45, further comprising preparing a label compensation control composition. 47. The method of clause 46, wherein preparing comprises contacting the compensation control composition of any of clauses 29-34 with an amine-reactive dye and / or a DNA-binding dye. 48. Further comprising compensation for cell viability data, the compensation comprising: contacting the cell sample with the same dye bound to positive control particles; generating cell viability data by analyzing the cell sample using a flow cytometer; and Correcting cell viability data using the calculated compensation value 48. The method according to any one of clauses 45 to 47, comprising: 49. A method for producing positive control particles, comprising: Preparing positive control particles by covalently associating aminated polymers and nucleic acids with cores A method comprising: 50. The method of clause 49, wherein the aminated polymer and nucleic acid are layered on the surface of the core. 51. A compensation control composition according to any one of clauses 29 to 34. A kit comprising: 52. The kit of clause 51, wherein the kit further comprises an amine-reactive dye. 53. The kit of either clause 51 or 52, wherein the kit further comprises a DNA-binding dye.

[0195] In at least some of the foregoing embodiments, one or more elements used in an embodiment may be used interchangeably in another embodiment unless such substitution is technically feasible. Those skilled in the art will appreciate that various other omissions, additions, and modifications may be made to the methods and structures described above without departing from the scope of the claimed subject matter. All such modifications and variations are intended to fall within the scope of the subject matter defined in the appended claims.

[0196] Those skilled in the art will generally understand that the terms used herein, and particularly in the appended claims (e.g., the body of the appended claims), are generally intended as "open" terms (e.g., the term "comprising" should be interpreted as "including but not limited to," the term "having" should be interpreted as "having at least," the term "including" should be interpreted as "including but not limited to," etc.). Those skilled in the art will further understand that where a specific number of introduced claim recitations is intended, such intention will be expressly set forth in the claim; in the absence of such recitation, no such intention exists. For example, to aid in understanding, the following appended claims may include the use of the introductory phrases "at least one" and "one or more" to introduce claim recitations. However, the use of such phrases should not be construed as meaning that the introduction of a claim recitation by the indefinite article "a" or "an" limits a particular claim containing the introduced claim recitation to embodiments containing only one such recitation, even if the same claim contains the introductory phrases "one or more" or "at least one" and the indefinite article "a" or "an" (e.g., "a" and / or "an" should be interpreted to mean "at least one" or "one or more"). The same applies to the use of definite articles used to introduce claim recitations. Moreover, even when a particular number is explicitly recited in an introduced claim recitation, those skilled in the art will recognize that such a recitation should be interpreted to mean at least the recited number (e.g., the mere recitation of "two recitations" without other modifiers means at least two recitations, or more than two recitations).Furthermore, in instances where a term similar to "at least one of A, B, and C, etc." is used, such a configuration is generally intended in the sense that one of ordinary skill in the art would understand the term (e.g., "a system having at least one of A, B, and C" includes, but is not limited to, systems of A only, B only, C only, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). In instances where a term similar to "at least one of A, B, or C, etc." is used, such a configuration is generally intended in the sense that one of ordinary skill in the art would understand the term (e.g., "a system having at least one of A, B, or C" includes, but is not limited to, systems of A only, B only, C only, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). Furthermore, those skilled in the art will understand that virtually any separating word and / or phrase presenting two or more alternative terms, whether in the description, claims, or drawings, contemplates the possibility of including one of the terms, either term, or both terms. For example, the phrase "A or B" will be understood to include the possibilities of "A" or "B" or "A and B."

[0197] Furthermore, where features or aspects of the disclosure are described in terms of a Markush group, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual members or subgroups of members of the Markush group.

[0198] As one skilled in the art would understand, for all purposes, including providing a written description, all ranges disclosed herein encompass all possible subranges and combinations of subranges. Any ranges described herein are fully described, and it is readily apparent that the same ranges can be divided into at least one half, one third, one quarter, one fifth, one tenth, etc. As a non-limiting example, each range described herein can be easily divided into a lower third, middle third, upper third, etc. As one skilled in the art would understand, all terms such as "up to," "at least," "greater than," "less than," etc., are inclusive of the recited numbers and refer to ranges that can be subsequently divided into subranges as described above. Finally, as one skilled in the art would understand, a range includes each individual member. Thus, for example, a group containing 1 to 3 items refers to groups containing 1, 2, or 3 items. Similarly, a group containing 1 to 5 items refers to groups containing 1, 2, 3, 4, or 5 items, and so on.

[0199] Although the foregoing invention has been described in some detail by way of illustration and example for clarity of understanding, it will be readily apparent to those skilled in the art in light of the teachings of this invention that certain changes and modifications can be made thereto without departing from the spirit or scope of the appended claims.

[0200] Thus, the foregoing merely illustrates the principles of the present invention. It will be appreciated that those skilled in the art will be able to devise various configurations, not explicitly described or shown herein, which embody the principles of the present invention and are within its spirit and scope. Furthermore, all examples and conditional language set forth herein are intended primarily to aid the reader in understanding the principles of the present invention and the concepts the inventors have contributed to furthering the art, and should not be construed as being limited to such specifically recited examples and conditions. Furthermore, all statements herein describing principles, aspects, and embodiments of the present invention, as well as specific examples thereof, are intended to encompass both structural and functional equivalents thereof. Furthermore, such equivalents are intended to include both currently known equivalents and equivalents developed in the future, i.e., any elements developed that perform the same function, regardless of structure. Furthermore, nothing disclosed herein is intended as a dedication to the public, regardless of whether such disclosure is expressly recited in the claims.

[0201] Accordingly, the scope of the present invention is not intended to be limited to the exemplary embodiments shown and described herein. Rather, the scope and spirit of the present invention is embodied by the appended claims. The claims expressly define that 35 U.S.C. §112(f) or 35 U.S.C. §112(6) apply to a claim limitation only if the precise phrase "means for" or the precise phrase "step for" appears in the claim at the beginning of such limitation; if such precise phrases are not used in a claim limitation, 35 U.S.C. §112(f) or 35 U.S.C. §112(6) does not apply.

[0202] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to the filing date of U.S. Provisional Patent Application No. 63 / 627,756, filed January 31, 2024, the disclosure of which is incorporated herein by reference.

Claims

1. core, an aminated polymer comprising an amine configured to react with an amine-reactive dye; and Nucleic acids configured to bind to DNA-binding dyes wherein the aminated polymer and the nucleic acid are covalently associated with the core. Positive control particles.

2. The positive control particle of claim 1 , wherein the core is substantially non-autofluorescent.

3. The positive control particle of claim 1 or 2, wherein the core has low non-specific binding.

4. The positive control particle of any one of claims 1 to 3, wherein the core comprises an inorganic material.

5. The positive control particle according to any one of claims 1 to 4, wherein the aminated polymer and nucleic acid are layered on the core.

6. The positive control particle of any one of claims 1 to 5, wherein the aminated polymer comprises a polypeptide.

7. The positive control particle according to any one of claims 1 to 6, wherein the nucleic acid is a double-stranded nucleic acid.

8. The positive control particle according to any one of claims 1 to 7, wherein the nucleic acid is deoxyribonucleic acid (DNA).

9. The positive control particle according to any one of claims 1 to 8, wherein the nucleic acid has a size in the range of 450 to 550 base pairs.

10. The positive control particle according to any one of claims 1 to 9, wherein the aminated polymer and the nucleic acid are covalently bound to the surface of the core directly or via a linker.

11. The positive control particle according to any one of claims 1 to 10, wherein the particle is a bead.

12. A compensatory control composition comprising a plurality of positive control particles according to any one of claims 1 to 11.

13. 13. The compensatory control composition of claim 12, further comprising a plurality of negative control particles.

14. A label compensation control composition comprising a plurality of positive control particles according to any one of claims 1 to 13, wherein the positive control particles have an amine-reactive dye and / or a DNA-binding dye bound thereto.

15. 1. A method for determining a compensation value for cell viability data obtained from flow cytometry analysis, comprising: analyzing the labeled compensation control composition of claim 14 using a flow cytometer to obtain flow cytometry data; and calculating a compensation value based on said flow cytometry data; A method comprising:

Images